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author:("Zhou, xuelong")
1.  Preliminary analysis of salivary microbiome and their potential roles in oral lichen planus 
Scientific Reports  2016;6:22943.
Several studies have explored the origin and development mechanism of oral lichen planus (OLP) with limited attention to the role of bacteria in the progression of this common oral disease. Here we utilized MiSeq sequencing of 16S rRNA gene amplicons to identify complex oral microbiota associated with OLP from saliva samples of two subtypes (reticular and erosive) of OLP patients and healthy controls. Our analyses indicated that the overall structure of the salivary microbiome was not significantly affected by disease status. However, we did observe evident variations in abundance for several taxonomic groups in OLP. Porphyromonas and Solobacterium showed significantly higher relative abundances, whereas Haemophilus, Corynebacterium, Cellulosimicrobium and Campylobacter showed lower abundances in OLP patients, as compared with healthy controls. In addition, we explored specific microbial co-occurrence patterns in OLP, and revealed significantly fewer linkers of Streptococcus comprising species in erosive OLP. Furthermore, the disease severity and immune dysregulation were also genus-associated, including with Porphyromonas that correlated to disease scores and salivary levels of interleukin (IL)-17 and IL-23. Overall, this study provides a general description of oral microbiome in OLP, and it will be useful for further investigation of their potential roles in the initiation and immune modulation of OLP.
doi:10.1038/srep22943
PMCID: PMC4785528  PMID: 26961389
2.  The Use of Quaternary Ammonium to Combat Dental Caries 
Materials  2015;8(6):3532-3549.
Resin composites and adhesives are increasingly popular in dental restorations, but secondary caries is one of the main reasons for restoration failure. Quaternary ammonium monomers (QAMs) have an anti-microbial effect and are widely used in many fields. Since the concept of the immobilized antibacterial effect was put forward, dental restorations containing QAMs have been studied to reduce secondary caries. Previous studies have been struggling to develop novel anti-caries materials which might have triple benefits: good mechanical properties, antibacterial effects and remineralization potentials. Different kinds of QAMs have been proven to be effective in inhibiting the growth and metabolism of biofilms. Combination of QAMs and other nanoparticles in resin composites and adhesives could enhance their anti-caries capability. Therefore, QAMs are promising to show significant impact on the future of restorative and preventive dentistry.
doi:10.3390/ma8063532
PMCID: PMC4665981  PMID: 26635932
quaternary ammonium monomer; dental caries; anti-biofilms; dental materials
3.  Critical role of Bmpr1a in Mandibular Condyle Growth 
Connective tissue research  2014;55(0 1):73-78.
The importance of Bone Morphogenetic Proteins (BMPs) in the regulation of cell fate, differentiation and proliferation in the growth plate is well-known. However, in secondary cartilages (such as that in the temporomandibular joint) that grow by proliferation of prechondrocytes and differ in their pattern of growth, the role of BMPs is largely unexplored. To examine this question, we ablated Bmpr1a in the condylar cartilage of neonatal mice and assessed the consequences for mandibular condyle growth and organization at intervals over the ensuing four weeks. Bmpr1a deficiency caused significant chondrodysplasia and almost eliminated the chondrocytic phenotype in the TMJ. Expression of Sox9, collagen II, proteoglycan were all greatly reduced, and cell proliferation as detected by BrdU was almost non-existent in the knockout mice. Primary bone spongiosa formation was also disturbed and was accompanied by reduced Osterix expression. These findings strongly suggest that Bmpr1a is critical for the development and growth of the mandibular condyle via its effect on proliferation of prechondroblasts and chondrocyte differentiation.
doi:10.3109/03008207.2014.923858
PMCID: PMC4638146  PMID: 25158185
Bmpr1a; mandibular condyle; endochondral ossification; condylar cartilage
4.  Antibacterial activity and ion release of bonding agent containing amorphous calcium phosphate nanoparticles 
Objectives
Recurrent caries at the margins is a primary reason for restoration failure. The objectives of this study were to develop bonding agent with the double benefits of antibacterial and remineralizing capabilities, to investigate the effects of NACP filler level and solution pH on Ca and P ion release from adhesive, and to examine the antibacterial and dentin bond properties.
Methods
Nanoparticles of amorphous calcium phosphate (NACP) and a quaternary ammonium monomer (dimethylaminododecyl methacrylate, DMADDM) were synthesized. Scotchbond Multi-Purpose (SBMP) primer and adhesive served as control. DMADDM was incorporated into primer and adhesive at 5% by mass. NACP was incorporated into adhesive at filler mass fractions of 10%, 20%, 30% and 40%. A dental plaque microcosm biofilm model was used to test the antibacterial bonding agents. Calcium (Ca) and phosphate (P) ion releases from the cured adhesive samples were measured vs. filler level and solution pH of 7, 5.5 and 4.
Results
Adding 5% DMADDM and 10–40% NACP into bonding agent, and water-aging for 28 days, did not affect dentin bond strength, compared to SBMP control at 1 day (p > 0.1). Adding DMADDM into bonding agent substantially decreased the biofilm metabolic activity and lactic acid production. Total microorganisms, total streptococci, and mutans streptococci were greatly reduced for bonding agents containing DMADDM. Increasing NACP filler level from 10% to 40% in adhesive increased the Ca and P ion release by an order of magnitude. Decreasing solution pH from 7 to 4 increased the ion release from adhesive by 6–10 folds.
Significance
Bonding agents containing antibacterial DMADDM and remineralizer NACP were formulated to have Ca and P ion release, which increased with NACP filler level from 10% to 40% in adhesive. NACP adhesive was “smart” and dramatically increased the ion release at cariogenic pH 4, when these ions would be most-needed to inhibit caries. Therefore, bonding agent containing DMADDM and NACP may be promising to inhibit biofilms and remineralize tooth lesions thereby increasing the restoration longevity.
doi:10.1016/j.dental.2014.05.025
PMCID: PMC4559225  PMID: 24954647
antibacterial bonding agent; nanoparticles of amorphous calcium phosphate; ion release; dental plaque microcosm biofilm; dentin bond strength; caries
5.  Phylogenetic and functional gene structure shifts of the oral microbiomes in periodontitis patients 
The ISME Journal  2014;8(9):1879-1891.
Determining the composition and function of subgingival dental plaque is crucial to understanding human periodontal health and disease, but it is challenging because of the complexity of the interactions between human microbiomes and human body. Here, we examined the phylogenetic and functional gene differences between periodontal and healthy individuals using MiSeq sequencing of 16S rRNA gene amplicons and a specific functional gene array (a combination of GeoChip 4.0 for biogeochemical processes and HuMiChip 1.0 for human microbiomes). Our analyses indicated that the phylogenetic and functional gene structure of the oral microbiomes were distinctly different between periodontal and healthy groups. Also, 16S rRNA gene sequencing analysis indicated that 39 genera were significantly different between healthy and periodontitis groups, and Fusobacterium, Porphyromonas, Treponema, Filifactor, Eubacterium, Tannerella, Hallella, Parvimonas, Peptostreptococcus and Catonella showed higher relative abundances in the periodontitis group. In addition, functional gene array data showed that a lower gene number but higher signal intensity of major genes existed in periodontitis, and a variety of genes involved in virulence factors, amino acid metabolism and glycosaminoglycan and pyrimidine degradation were enriched in periodontitis, suggesting their potential importance in periodontal pathogenesis. However, the genes involved in amino acid synthesis and pyrimidine synthesis exhibited a significantly lower relative abundance compared with healthy group. Overall, this study provides new insights into our understanding of phylogenetic and functional gene structure of subgingival microbial communities of periodontal patients and their importance in pathogenesis of periodontitis.
doi:10.1038/ismej.2014.28
PMCID: PMC4139721  PMID: 24671083
functional gene array; Illumina sequencing; periodontitis; subgingival dental plaque
6.  Cell cycle control, DNA damage repair, and apoptosis-related pathways control pre-ameloblasts differentiation during tooth development 
BMC Genomics  2015;16(1):592.
Background
Ameloblast differentiation is the most critical stepwise process in amelogenesis, and it is controlled by precise molecular events. To better understand the mechanism controlling pre-ameloblasts (PABs) differentiation into secretory ameloblasts (SABs), a more precise identification of molecules and signaling networks will elucidate the mechanisms governing enamel formation and lay a foundation for enamel regeneration.
Results
We analyzed transcriptional profiles of human PABs and SABs. From a total of 28,869 analyzed transcripts, we identified 923 differentially expressed genes (DEGs) with p < 0.05 and Fold-change > 2. Among the DEGs, 647 genes showed elevated expression in PABs compared to SABs. Notably, 38 DEGs displayed greater than eight-fold changes. Comparative analysis revealed that highly expressed genes in PABs were involved in cell cycle control, DNA damage repair and apoptosis, while highly expressed genes in SABs were related to cell adhesion and extracellular matrix. Moreover, coexpression network analysis uncovered two highly conserved sub-networks contributing to differentiation, containing transcription regulators (RUNX2, ETV1 and ETV5), solute carrier family members (SLC15A1 and SLC7A11), enamel matrix protein (MMP20), and a polymodal excitatory ion channel (TRPA1).
Conclusions
By combining comparative analysis and coexpression networks, this study provides novel biomarkers and research targets for ameloblast differentiation and the potential for their application in enamel regeneration.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1783-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s12864-015-1783-y
PMCID: PMC4534026  PMID: 26265206
Ameloblasts; Differentiation; Tooth development; Transcriptional profile
7.  Streptococcus mutans copes with heat stress by multiple transcriptional regulons modulating virulence and energy metabolism 
Scientific Reports  2015;5:12929.
Dental caries is closely associated with the virulence of Streptococcus mutans. The virulence expression of S. mutans is linked to its stress adaptation to the changes in the oral environment. In this work we used whole-genome microarrays to profile the dynamic transcriptomic responses of S. mutans during physiological heat stress. In addition, we evaluated the phenotypic changes, including, eDNA release, initial biofilm formation, extracellular polysaccharides generation, acid production/acid tolerance, and ATP turnover of S. mutans during heat stress. There were distinct patterns observed in the way that S. mutans responded to heat stress that included 66 transcription factors for the expression of functional genes being differentially expressed. Especially, response regulators of two component systems (TCSs), the repressors of heat shock proteins and regulators involved in sugar transporting and metabolism co-ordinated to enhance the cell’s survival and energy generation against heat stress in S. mutans.
doi:10.1038/srep12929
PMCID: PMC4528225  PMID: 26251057
8.  The remaining dentin thickness investigation of the attempt to remove broken instrument from mesiobuccal canals of maxillary first molars with virtual simulation technique 
BMC Oral Health  2015;15:87.
Background
To investigate differences in the estimated minimum remaining dentin thickness (RDT) between periapical radiographs using the paralleling and parallax technique, after simulated removal of broken instrument from the mesiobuccal (MB) canal of maxillary first molar in virtual simulation model. The 3D measurement was taken as the standard for comparison.
Methods
Thirty-six maxillary first molars were scanned by micro-CT and reconstructed as 3-dimensional (3D) model. A virtual fragment of an instrument was created within the MB canal in software. Removal of the broken instrument was simulated in both the 3D and 2D dataset. Then, the models of all specimens were submitted to 2D and 3D measurements for the lowest (RDT) value in each. Differences in the values between the paralleling and parallax radiographic technique and the 3D-RDT value were analyzed with two-way Analysis of Variance. The Intra-class Correlation Coefficient (ICC) was used to assess consistency of the RDT measurements between the two periapical radiographic and techniques and 3D analysis.
Results
There was significant difference between RDT value obtained from the paralleling technique and 3D-RDT. There were no differences between RDT obtained from parallax (angled) technique and 3D-RDT. The ICC of RDT values between paralleling technique and 3D measurement were lower than 0.75. ICC between angled radiographs and 3D technique was close to 0.75. The optimal horizontal angle for the parallax technique was about 21°.
Conclusions
The virtual simulation technique can provide valuable insight into the benefit/risk analysis before removal of a broken instrument. Parallel radiographs overestimate the actual remain dentin thickness in mesiobuccal canals of maxillary first molars, whereas the parallel technique would give a closer estimate to the actual thickness at a projection angle of about 21°.
doi:10.1186/s12903-015-0075-x
PMCID: PMC4517344  PMID: 26215404
Broken instrument; Virtual simulation; Periapical radiography; Remaining dentin thickness
9.  MicroRNA 224 Regulates Ion Transporter Expression in Ameloblasts To Coordinate Enamel Mineralization 
Molecular and Cellular Biology  2015;35(16):2875-2890.
Enamel mineralization is accompanied by the release of protons into the extracellular matrix, which is buffered to regulate the pH value in the local microenvironment. The present study aimed to investigate the role of microRNA 224 (miR-224) as a regulator of SLC4A4 and CFTR, encoding the key buffering ion transporters, in modulating enamel mineralization. miR-224 was significantly downregulated as ameloblasts differentiated, in parallel with upregulation of SLC4A4 and CFTR. Overexpression of miR-224 downregulated SLC4A4 and CFTR expression in cultured human epithelial cells. A microRNA luciferase assay confirmed the specific binding of miR-224 to the 3′ untranslated regions (UTRs) of SLC4A4 and CFTR mRNAs, thereby inhibiting protein translation. miR-224 agomir injection in mouse neonatal incisors resulted in normal enamel length and thickness, but with disturbed organization of the prism structure and deficient crystal growth. Moreover, the enamel Ca/P ratio and microhardness were markedly reduced after miR-224 agomir administration. These results demonstrate that miR-224 plays a pivotal role in fine tuning enamel mineralization by modulating SLC4A4 and CFTR to maintain pH homeostasis and support enamel mineralization.
doi:10.1128/MCB.01266-14
PMCID: PMC4508313  PMID: 26055330
10.  BMP7 and EREG Contribute to the Inductive Potential of Dental Mesenchyme 
Scientific Reports  2015;5:9903.
Odontogenesis is accomplished by reciprocal signaling between the epithelial and mesenchymal compartments. It is generally accepted that the inductive mesenchyme is capable of inducing the odontogenic commitment of both dental and non-dental epithelial cells. However, the duration of this signal in the developing dental mesenchyme and whether adult dental pulp tissue maintains its inductive capability remain unclear. This study investigated the contribution of growth factors to regulating the inductive potential of the dental mesenchyme. Human oral epithelial cells (OEs) were co-cultured with either human dental mesenchymal/papilla cells (FDPCs) or human dental pulp cells (ADPCs) under 2-dimensional or 3-dimensional conditions. Odontogenic-associated genes and proteins were detected by qPCR and immunofluorescence, respectively, and significant differences were observed between the two co-culture systems. The BMP7 and EREG expression levels in FDPCs were significantly higher than in ADPCs, as indicated by human growth factor PCR arrays and immunofluorescence analyses. OEs co-cultured with ADPCs supplemented with BMP7 and EREG expressed ameloblastic differentiation genes. Our study suggests that BMP7 and EREG expression in late bell-stage human dental papilla contributes to the inductive potential of dental mesenchyme. Furthermore, adult dental pulp cells supplemented with these two growth factors re-established the inductive potential of postnatal dental pulp tissue.
doi:10.1038/srep09903
PMCID: PMC4424660  PMID: 25952286
11.  Nanotechnology strategies for antibacterial and remineralizing composites and adhesives to tackle dental caries 
Nanomedicine (London, England)  2015;10(4):627-641.
Dental caries is the most widespread disease and an economic burden. Nanotechnology is promising to inhibit caries by controlling biofilm acids and enhancing remineralization. Nanoparticles of silver were incorporated into composites/adhesives, along with quaternary ammonium methacrylates (QAMs), to combat biofilms. Nanoparticles of amorphous calcium phosphate (NACP) released calcium/phosphate ions, remineralized tooth-lesions and neutralized acids. By combining NAg/QAM/NACP, a new class of composites and adhesives with antibacterial and remineralization double benefits was developed. Various other nanoparticles including metal and oxide nanoparticles such as ZnO and TiO2, as well as polyethylenimine nanoparticles and their antibacterial capabilities in dental resins were also reviewed. These nanoparticles are promising for incorporation into dental composites/cements/sealants/bases/liners/adhesives. Therefore, nanotechnology has potential to significantly improve restorative and preventive dentistry.
doi:10.2217/nnm.14.191
PMCID: PMC4347904  PMID: 25723095
amorphous calcium phosphate nanoparticles; dental caries; oral biofilms; quaternary ammonium methacrylate; silver nanoparticles; tooth lesion remineralization
12.  Bone tissue engineering via nanostructured calcium phosphate biomaterials and stem cells 
Bone Research  2014;2:14017-.
Tissue engineering is promising to meet the increasing need for bone regeneration. Nanostructured calcium phosphate (CaP) biomaterials/scaffolds are of special interest as they share chemical/crystallographic similarities to inorganic components of bone. Three applications of nano-CaP are discussed in this review: nanostructured calcium phosphate cement (CPC); nano-CaP composites; and nano-CaP coatings. The interactions between stem cells and nano-CaP are highlighted, including cell attachment, orientation/morphology, differentiation and in vivo bone regeneration. Several trends can be seen: (i) nano-CaP biomaterials support stem cell attachment/proliferation and induce osteogenic differentiation, in some cases even without osteogenic supplements; (ii) the influence of nano-CaP surface patterns on cell alignment is not prominent due to non-uniform distribution of nano-crystals; (iii) nano-CaP can achieve better bone regeneration than conventional CaP biomaterials; (iv) combining stem cells with nano-CaP accelerates bone regeneration, the effect of which can be further enhanced by growth factors; and (v) cell microencapsulation in nano-CaP scaffolds is promising for bone tissue engineering. These understandings would help researchers to further uncover the underlying mechanisms and interactions in nano-CaP stem cell constructs in vitro and in vivo, tailor nano-CaP composite construct design and stem cell type selection to enhance cell function and bone regeneration, and translate laboratory findings to clinical treatments.
doi:10.1038/boneres.2014.17
PMCID: PMC4472121  PMID: 26273526
13.  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.
doi:10.1371/journal.pone.0102116
PMCID: PMC4099009  PMID: 25025462
14.  Regenerative Endodontics: Barriers and Strategies for Clinical Translation 
Dental clinics of North America  2012;56(3):639-649.
SYNOPSIS
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.
doi:10.1016/j.cden.2012.05.005
PMCID: PMC4093795  PMID: 22835543
regenerative; endodontics; pulp; dentin; regeneration; stem cells; tissue engineering
15.  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.
doi:10.1128/JB.00995-13
PMCID: PMC3911236  PMID: 24163340
16.  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.
doi:10.3390/ijms150712791
PMCID: PMC4139875  PMID: 25046750
antibacterial adhesive; DMADDM; S. mutans biofilm; microtensile
17.  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.
doi:10.1371/journal.pone.0097824
PMCID: PMC4023953  PMID: 24836538
18.  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.
doi:10.4248/BR201303005
PMCID: PMC3994128  PMID: 24761229
Rgs10; immune cell; AAV-mediated RNAi knockdown; gene therapy; periodontal disease; gingival inflammation; bone resorption
19.  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.
doi:10.1371/journal.pone.0090546
PMCID: PMC3942451  PMID: 24595026
20.  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.
doi:10.1155/2014/463826
PMCID: PMC3913516  PMID: 24526893
21.  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.
doi:10.1038/nm.3143
PMCID: PMC3676689  PMID: 23685840
22.  Hyperosmotic response of streptococcus mutans: from microscopic physiology to transcriptomic profile 
BMC Microbiology  2013;13:275.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2180-13-275
PMCID: PMC4219374  PMID: 24289739
Streptococcus mutans; Hyperosmotic condition; Transcriptional profile; Biofilm dispersal; Environmental fitness; Dental plaque
23.  Phenotypic characterization of the foldase homologue PrsA in Streptococcus mutans 
Molecular oral microbiology  2012;28(2):10.1111/omi.12014.
SUMMARY
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.
doi:10.1111/omi.12014
PMCID: PMC3819222  PMID: 23241367
foldase protein PrsA; protein secretion; Streptococcus mutans
24.  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.
doi:10.4248/BR201303005
PMCID: PMC3994128  PMID: 24761229
Rgs10; immune cell; AAV-mediated RNAi knockdown; gene therapy; periodontal disease; gingival inflammation; bone resorption
25.  Dental plaque microcosm biofilm behavior on calcium phosphate nanocomposite with quaternary ammonium 
Dental Materials  2012;28(8):853-862.
Objectives
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.
Methods
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.
Results
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.
Significance
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.
doi:10.1016/j.dental.2012.04.024
PMCID: PMC3393817  PMID: 22578992
Antibacterial nanocomposite; amorphous calcium phosphate nanoparticles; quaternary ammonium; dental plaque microcosm biofilm; stress-bearing; dental caries

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