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1.  Heritability of Periodontal Bone Loss in Mice 
Journal of periodontal research  2015;50(6):730-736.
Periodontitis (PD) is an inflammatory disease of the periodontal tissues that compromises tooth support and can lead to tooth loss. Although bacterial biofilm is central in disease pathogenesis, host response plays an important role in the progression and severity of PD. Indeed, clinical genetic studies indicate that PD is 50% heritable. In this study, we hypothesized that LPS injections lead to a strain-dependent periodontal bone loss pattern. We utilized five inbred mouse strains that derive the recombinant strains of the hybrid mouse diversity panel (HMDP). Mice received P. gingivalis-LPS injections for six weeks. Micro-CT analysis demonstrated a statistically significant strain-dependent bone loss. The most susceptible strain, C57BL/6J, had a 5-fold higher LPS-induced bone loss compared to the most resistant strain, A/J. More importantly, periodontal bone loss revealed 49% heritability, which closely mimics PD heritability for patients. To further evaluate functional differences that underlie periodontal bone loss, osteoclast numbers of C57BL/6J and A/J mice were measured in vivo and in vitro. In vitro analysis of osteoclastogenic potential showed a higher number of osteoclasts in C57BL/6J compared to A/J mice. In vivo LPS-injections statistically significantly increased osteoclasts numbers in both groups. Importantly, the number of osteoclasts was higher in C57BL/6J vs. A/J mice. These data support a significant role of the genetic framework in LPS-induced periodontal bone loss and the feasibility of utilizing the HMDP to determine the genetic factors that affect periodontal bone loss. Expanding these studies will contribute in predicting patients genetically predisposed to PD and in identifying the biological basis of disease susceptibility.
doi:10.1111/jre.12258
PMCID: PMC4499504  PMID: 25581386
Alveolar bone; animal model; genome; lipopolysaccharide
2.  Differentiating Zones at Periodontal Ligament-Bone and Periodontal Ligament-Cementum Entheses 
Journal of periodontal research  2015;50(6):870-880.
Background and Objective
The structural and functional integrity of bone-periodontal ligament (PDL)-cementum complex stems from the load-bearing attachment sites (entheses) between soft (PDL) and hard (bone, cementum) tissues. These attachment sites are responsible for maintenance of a bone-PDL-cementum complex biomechanical function. The objective was to investigate changes in spatiotemporal expression of key biomolecules in developing and functionally active entheses.
Material and Methods
Multilabeling technique was performed on hemimandibles of 3 week and 3 month-old scleraxis (scx)-GFP transgenic mice for CD146, CD31, NG2, osterix (OSX), and bone sialoprotein (BSP). Regions of dominant stretch within the PDL were evaluated by identifying directionality of collagen fibrils, PDL fibroblasts, and PDL cell cytoskeleton.
Results
CD146+ cells adjacent to CD31+ vasculature were identified at PDL-bone enthesis. NG2+ cells were located at coronal bone-PDL and apical cementum-PDL entheses in the 3 weeks-old group, but at 3 months, NG2 was positive at the entheses of the apical region and alveolar crest. NG2 and OSX were colocalized at the osteoid and cementoid regions of the PDL-bone and PDL-cementum entheses. BSP was prominent at the apical region of 3 week old mice. The directionality of collagen fibers, fibroblasts, and their cytoskeleton overlapped, except in the apical region of 3 weeks.
Conclusion
Colocalization of biomolecules at zones of the periodontal ligament adjacent to attachment sites may be essential for the formation of precementum and osteoid interfaces at a load-bearing bone-PDL-tooth fibrous joint. Biophysical cues resulting from development and function can regulate recruitment and differentiation of stem cells potentially from vascular origin toward osteo- and cemento-blastic lineages at the PDL-bone and PDL-cementum entheses. Investigating the coupled effect of biophysical and biochemical stimuli leading to cell differentiation at the functional attachment sites is critical for developing regeneration strategies to enable functional reconstruction of the periodontal complex.
doi:10.1111/jre.12281
PMCID: PMC4663098  PMID: 26031604
tissue entheses; periodontal ligament; tooth-bone biomechanics
3.  Ligature-Induced Peri-Implantitis in Mice 
Journal of periodontal research  2014;50(4):519-524.
Introduction
Peri-implantitis has a prevalence of 11-47%, involves destruction of peri-implant bone, and may lead to implant loss. A detailed understanding of the pathogenesis of peri-implantitis is lacking. The objective of this study was to develop a murine model of experimental peri-implantitis.
Materials and Methods
Machined, smooth surface screw-shaped titanium implants were placed in the healed alveolar bone of the left maxillary molars of C57BL/6J male mice, eight weeks after tooth extraction. Peri-implantitis was induced by securing silk ligatures around the head of the implant fixtures. Implant survival and peri-implant bone levels were analyzed by micro-computerized tomography (micro-CT) scans and histology twelve weeks after ligature placement.
Results
Implant survival was 60% (6/10) for implants with ligatures and 100% (8/8) for controls. Micro-CT revealed significantly greater bone loss around the implants that received ligatures and that survived as compared to controls. The radiographic findings were confirmed via histology and toluidine blue staining.
Conclusions
This study describes a murine model of experimental peri-implantitis around screw-shaped titanium implants placed in the edentulous alveolar bone. This model should be a useful tool to dissect pathogenic mechanisms of peri-implantitis and evaluate potential treatment interventions.
doi:10.1111/jre.12234
PMCID: PMC4368501  PMID: 25244403
peri-implantitis; dental implant; animal model; bone loss
4.  Increased and Correlated Expression of CTGF and TGFβ1 in Surgically Removed Periodontal Tissues with Chronic Periodontitis 
Journal of periodontal research  2014;50(3):315-319.
Background and Objective
Both gingival tissue destruction and regeneration are associated with chronic periodontitis although the former overwhelms the latter. Studies have shown that transforming growth factor beta (TGFβ)1, a growth factor largely involved in tissue regeneration and remodeling, is upregulated in chronic periodontitis. However, the gingival expression of connective tissue growth factor (CTGF or CCN2), a TGFβ1-upregulated gene, in patients with periodontitis remains undetermined. Although both CTGF/CCN2 and TGFβ1 increase extracellular matrix, they have many different biological functions. Therefore, it is important to delineate the impact of periodontitis on gingival CTGF/CCN2 expression.
Material and Methods
Periodontal tissue specimens were collected from 7 individuals without periodontitis (group 1) and 14 with periodontitis (group 2). The CTGF and TGFβ1 mRNA were quantified using real-time polymerase chain reaction.
Results
Analysis using the nonparametric Mann-Whitney test showed that both CTGF/CCN2 and TGFβ1 mRNA expression levels were significantly increased in individuals with periodontitis as compared to individuals without periodontitis. Further, analysis using the nonparametric correlation (Spearman r) test showed a positive correlation between TGFβ1 and CTGF/CCN2 mRNA.
Conclusion
The gingival CTGF/CCN2 and TGFβ1 mRNA expression in individuals with periodontitis are upregulated and correlated.
doi:10.1111/jre.12208
PMCID: PMC4289131  PMID: 25040058
Periodontitis; growth factors; CTGF; TGFβ1; gene expression
5.  Subgingival microbial profiles of Sudanese patients with aggressive periodontitis 
Journal of periodontal research  2014;50(5):674-682.
Background and Objective
Aggressive periodontitis (AgP) is prevalent and shows a rapid course in African individuals. Although a strong focus has been placed on Aggregatibacter actinomycetemcomitans, new methods support the existence of a complex subgingival microflora in AgP. The purpose of the present study was to map the subgingival microbiota as well as explore the presence of A. actinomycetemcomitans and the JP2 clone in a group of Sudanese individuals with AgP, using different analytical methods.
Material and Methods
A study population consisting of 19 patients with AgP was recruited from patients seeking treatment at University of Science and Technology (UST) in Khartoum. Fifteen healthy subjects were included as controls. Plaque samples were analyzed for 272 taxa using human oral microbe identification microarrays and for 26 periodontal taxa using DNA-DNA hybridization checkerboard. Conventional polymerase chain reaction (PCR) was applied for the detection of A. actinomycetemcomitans and the JP2 clone in plaque. Saliva from patients with AgP was analyzed using quantitative PCR (qPCR) for the detection of A. actinomycetemcomitans.
Results
Eubacterium yurii was detected more frequently in patients with AgP than in controls, and E. nodatum was found in patients with AgP only. A. actinomycetemcomitans was found in plaque samples of two (12%) patients by human oral microbe identification microarrays and in five (29%) patients with AgP by conventional PCR, as well as in six (32%) of the AgP saliva samples by qPCR. The JP2 clone was identified in only one patient.
Conclusion
The classical periodontal pathogens were not present in high amounts in AgP in the population studied here. Species of Eubacterium, which are not typically associated with AgP, were often detected in individuals with disease. Using laboratory methods with different sensitivities and detection levels allowed identification of variances in microbial communities. The findings reported in this study provide a basis for the further understanding of AgP.
doi:10.1111/jre.12250
PMCID: PMC4646740  PMID: 25487558
human oral microbe identification microarrays; PCR; plaque; saliva
6.  Association of IL-1 gene variations with moderate to severe chronic periodontitis in multiple ethnicities 
Background and Objective
Genetic markers associated with disease are often non-functional and generally tag one or more functional “causative” variants in linkage disequilibrium. Markers may not show tight linkage to the causative variants across multiple ethnicities due to evolutionary divergence, and therefore may not be informative across different population groups. Validated markers of disease suggest causative variants exist in the gene and, if the causative variants can be identified, it is reasonable to hypothesize that such variants will be informative across diverse populations. The aim of this study was to test that hypothesis using functional interleukin-1 gene variations across multiple ethnic populations to replace the non-functional markers originally associated with chronic adult periodontitis in Caucasians.
Material and Methods
Adult chronic periodontitis cases and controls from 4 ethnic groups (Caucasians, African Americans, Hispanics and Asians) were recruited in the U.S., Chile and China. Genotypes of IL1B gene SNPs, including 3 functional SNPs (rs16944, rs1143623, rs4848306) in the promoter and 1 intronic SNP (rs1143633), were determined using single base extension method or TaqMan 5′ nuclease assay. Logistic regression and other statistical analyses were used to examine the association between moderate to severe periodontitis and IL1B gene variations, including SNPs, haplotypes, and composite genotypes. Genotype patterns associated with disease in the discovery study were then evaluated in independent validation studies.
Results
Significant associations were identified in the discovery study, consisting of Caucasians and African Americans, between moderate to severe adult chronic periodontitis and functional variations in the IL1B gene, including a pattern of 4 IL1B SNPs (OR=1.87, P-value<0.0001). The association between the disease and this IL1B composite genotype pattern was validated in two additional studies consisting of Hispanics (OR=1.95, P-value=0.04) or Asians (OR=3.27, P-value=0.01). A meta-analysis of the 3 populations supported the association between the IL-1 genotype pattern and moderate to severe periodontitis (OR 1.95; P-value <0.001). Our analysis also demonstrated that IL1B gene variations had added value to conventional risk factors in predicting chronic periodontitis.
Conclusion
This study validated the influence of IL-1 genetic factors on the severity of chronic periodontitis in four different ethnicities.
doi:10.1111/jre.12181
PMCID: PMC4183738  PMID: 24690098
Interleukin-1; periodontitis; genetic risk; multiple ethnicities
7.  Aging and contribution of MyD88 and TRIF in expression of TLR pathway associated genes to Porphyromonas gingivalis 
Journal of periodontal research  2014;50(1):89-102.
BACKGROUND AND OBJECTIVE
Periodontal disease is a highly complex chronic inflammatory disease of the oral cavity. Multiple factors influence periodontal disease including socioeconomic status, genetics, age, however, inflammation elicited by the presence of specific bacteria in the subgingival space is thought to drive the majority of soft and hard tissue destruction. Porphyromonas gingivalis is closely associated with periodontal disease. Toll-like receptors (TLRs) and their intracellular signaling pathways play roles in host responses to P. gingivalis. The focus of current study was to use microarray analysis to define the contributions that TLR adaptor molecules MyD88 and TRIF, and aging have on TLR pathway associated mRNA expression in response to P. gingivalis.
MATERIALS AND METHODS
Bone marrow derived macrophages (BMØ) from wild type (Wt), MyD88-KO and TrifLps2 mice at 2-months and 12-months of age were cultured with P. gingivalis. Expression of genes in BMØ cultured with P. gingivalis was determined in comparison to medium alone control.
RESULTS
Using a two-fold cut-off in mRNA expression criteria, differential expression of 32 genes was observed when Wt BMØ from 2-month old mice were cultured with P. gingivalis compared with medium alone control. When compared with 2-month old Wt, 21 and 12 genes were differentially expressed (P<0.05) as a result of MyD88 or TRIF mutations respectively. The expression of 5 genes was significantly (P<0.05) reduced in the 12-month group compared to the 2-month group in Wt BMØ following culture with P. gingivalis. Age also influenced expression of genes in MyD88-KO and TrifLps2 mice challenged with P. gingivalis.
CONCLUSION
Our results indicate that P. gingivalis induces differential expression of TLR pathway associated genes, and both MyD88, and TRIF play roles in the expression of these genes. Age also played a role in the expression of TLR-associated genes following stimulation of BMØ with P. gingivalis.
doi:10.1111/jre.12185
PMCID: PMC4242805  PMID: 24862405
MRNA expression; Innate immunity; Macrophage; MyD88; Porphyromonas gingivalis; TLRs; TRIF
8.  Porphyromonas gingivalis and related bacteria: from colonial pigmentation to the type IX secretion system and gliding motility 
Porphyromonas gingivalis is a gram-negative, non-motile, anaerobic bacterium implicated as a major pathogen in periodontal disease. P. gingivalis grows as black-pigmented colonies on blood agar, and many bacteriologists have shown interest in this property. Studies of colonial pigmentation have revealed a number of important findings, including an association with the highly active extracellular and surface proteinases called gingipains that are found in P. gingivalis. The Por secretion system, a novel type IX secretion system (T9SS), has been implicated in gingipain secretion in studies using non-pigmented mutants. In addition, many potent virulence proteins, including the metallocarboxypeptidase CPG70, 35 kDa hemin-binding protein HBP35, peptidylarginine deiminase PAD and Lys-specific serine endopeptidase PepK, are secreted through the T9SS. These findings have not been limited to P. gingivalis but have been extended to other bacteria belonging to the phylum Bacteroidetes. Many Bacteroidetes species possess the T9SS, which is associated with gliding motility for some of these bacteria.
doi:10.1111/jre.12255
PMCID: PMC4674972  PMID: 25546073
gliding motility; Porphyromonas gingivalis; protease; protein secretion system; review; virulence factors
9.  Topical treatment with probiotic Lactobacillus brevis CD2 inhibits experimental periodontal inflammation and bone loss 
Journal of periodontal research  2014;49(6):785-791.
Background and Objective
An increasing body of evidence suggests that the use of probiotic bacteria is a promising intervention approach for the treatment of inflammatory diseases with polymicrobial etiology. The objective of this study was to determine whether Lactobacillus brevis CD2 could inhibit periodontal inflammation and bone loss in experimental periodontitis.
Materials and Methods
Periodontitis was induced by placing a silk ligature around the second maxillary molar of mice treated with L. brevis CD2 (8×105 CFU in 1-mm2 lyopatch) or placebo, which were placed between the gingiva and the buccal mucosa in the vicinity of the ligated teeth. The mice were euthanized after 5 days and bone loss was measured morphometrically, gingival expression of proinflammatory cytokines was determined by quantitative real-time PCR, and CFU counts of periodontitis-associated bacteria were determined after aerobic and anaerobic culture. To determine the role of arginine deiminase released by L. brevis CD2, soluble extracts with or without formamidine (arginine deiminase inhibitor) were tested in in vitro cellular activation assays.
Results
Mice topically treated with L. brevis CD2 displayed significantly decreased bone loss and lower expression of TNF, IL-1β, IL-6, and IL-17A as compared to placebo-treated mice. Moreover, L. brevis CD2-treated mice displayed lower counts of anaerobic bacteria but higher counts of aerobic bacteria than placebo-treated mice. In in vitro assays, the anti-inflammatory effects of soluble L. brevis CD2 extracts were heavily dependent on the presence of functional arginine deiminase, an enzyme that can inhibit nitric oxide synthesis.
Conclusion
These data provide proof-of-concept that the probiotic L. brevis CD2 can inhibit periodontitis through modulatory effects on the host response and the periodontal microbiota.
doi:10.1111/jre.12164
PMCID: PMC4119090  PMID: 24483135
Lactobacillus brevis CD2; probiotics; arginine deiminase; inflammation; periodontitis
10.  Wnt signaling regulates homeostasis of the periodontal ligament 
Journal of periodontal research  2014;49(6):751-759.
Background and Objective
In health, the periodontal ligament maintains a constant width throughout an organism’s lifetime. The molecular signals responsible for maintaining homeostatic control over the periodontal ligament are unknown. The purpose of this study was to investigate the role of Wnt signaling in this process by removing an essential chaperone protein, Wntless (Wls) from odontoblasts and cementoblasts, and observing the effects of Wnt depletion on cells of the periodontal complex.
Material and Methods
The Wnt responsive status of the periodontal complex was assessed using two strains of Wnt reporter mice, Axin2LacZ/+ mice and Lgr5LacZ/+. The function of this endogenous Wnt signal was evaluated by conditionally eliminating the Wntless (Wls) gene using an Osteocalcin Cre driver. The resulting OCN-Cre;Wlsfl/fl mice were examined using micro-CT and histology, immunohistochemical analyses for Osteopontin, Runx2 and Fibromodulin, in situ hybridization for Osterix, and alkaline phosphatase activity.
Results
The adult periodontal ligament is Wnt responsive. Elimination of Wnt signaling in the periodontal complex of OCN-Cre;Wlsfl/fl mice results in a wider periodontal ligament space. This pathologically increased periodontal width is due to a reduction in the expression of osteogenic genes and proteins, which results in thinner alveolar bone. A concomitant increase in fibrous tissue occupying the periodontal space was observed along with a disruption in the orientation of the periodontal ligament.
Conclusion
The periodontal ligament is a Wnt dependent tissue. Cells in the periodontal complex are Wnt responsive and eliminating an essential component of the Wnt signaling network leads to a pathological widening of the periodontal ligament space. Osteogenic stimuli are reduced and a disorganized fibrillary matrix results from depletion of Wnt signaling. Collectively, these data underscore the importance of Wnt signaling in homeostasis of the periodontal ligament.
doi:10.1111/jre.12158
PMCID: PMC4528390  PMID: 24410666
Wntless; periodontal ligament; width; osteogenic factor
11.  Smoking Related Cotinine Levels and Host Responses in Chronic Periodontitis 
Journal of periodontal research  2013;49(5):642-651.
Smoking has been reported to increase the risk of periodontal disease by disrupting the balance of immune responses and tissue repair processes; however, this risk varies among smokers. Cotinine levels in saliva are routinely used to measure the level of smoking, and reflect the quantity of nicotine, and other smoking related xenobiotics that challenge host systems. This study delineated characteristics of inflammatory mediators in saliva and serum antibody responses to both periodontal pathogens and commensal bacteria in smokers as they related to cotinine levels. This case-control study (n=279) examined salivary inflammatory mediator responses (IL-1ß, IL-10, PGE2, MPO, PAI-1) and serum IgG antibody responses to 3 periodontal pathogens (A. actinomycetemcomitans, P. gingivalis, T. denticola) and 5 commensal oral microorganisms (V. parvula, S. sanguis, P. loescheii, A. naeslundii, C. ochracea). The patients were stratified into health (n=30), gingivitis (n=55) and periodontitis (n=184); cotinine levels correlated with reported smoking habits in health, less so with gingivitis, and were not correlated in periodontitis. Of the inflammatory mediators/acute phase proteins, only IL-1ß levels were positively associated (p<0.001) with the pack years and cotinine levels. As might be predicted, patients with periodontitis smoked more (p<0.001) and had higher levels of cotinine. IL-1ß and antibody to Aa, Pg, and Td were significantly higher in the periodontitis patients than either gingivitis or healthy patients. Generally antibody to the pathogens and commensals was lower with decreased cotinine levels. Smoking exacerbated differences in both inflammatory mediators and antibody in periodontal disease compared to healthy subjects.
doi:10.1111/jre.12146
PMCID: PMC4035475  PMID: 24283398
smoking; cotinine; periodontitis; inflammation
12.  Involvement of toll-like receptor 4 in alveolar bone loss and glucose homeostasis in experimental periodontitis 
Background and Objective
There is general agreement that certain fatty acids and lipopolysaccharides (LPS) promote inflammation through toll-like receptor 4 (TLR4), and that inflammation promotes insulin resistance. We therefore hypothesized that mice with periodontitis and a TLR4 loss-of-function (LOF) mutation fed a high-fat (HF) diet would develop improved glucose homeostasis compared with wild-type (WT) animals with periodontitis fed a HF diet.
Material and Methods
Wild-type and TLR4 mutant mice fed a HF diet were divided into four groups (n = 6/group): WT; WT with periodontitis (WT/P); mutant (Mut); and mutant with periodontitis (Mut/P). Periodontitis was induced by placing LPS soaked ligatures around maxillary second molars. Fasting insulin and glucose levels were measured weekly for 10 wk. Glucose tolerance was evaluated at baseline (week 1) and at 9 wk. Insulin signaling (phosphorylation of Akt) and tumor necrosis factor-α (TNF-α) mRNA levels in liver were determined when the mice were killed at week 10.
Results
Mut/P mice developed less alveolar bone loss compared with WT/P mice (p < 0.05). Fasting glucose levels were improved after 8 wk of feeding a HF diet (weeks 9 and 10) in Mut/P mice compared with Mut, WT and WT/P mice (p < 0.05). Glucose tolerance was impaired in all groups compared with baseline (p < 0.05), except for the Mut/P group. Insulin signaling was improved (p < 0.05), and expression of TNF-α was decreased (p < 0.05) in the liver of Mut/P mice compared with the liver of WT/P mice.
Conclusion
The TLR4 LOF mutation partially protects against alveolar bone loss and improves glucose homeostasis in mice with periodontitis fed a HF diet.
doi:10.1111/j.1600-0765.2010.01304.x
PMCID: PMC4569008  PMID: 20860587
diabetes; inflammation; periodontal disease; alveolar bone
13.  Porphyromonas gingivalis Promotes Monocyte Migration by Activating MMP-9 
Journal of periodontal research  2011;47(2):236-242.
Objectives
The migration of monocytes into local environment is crucial for its maturation into macrophages or osteoclasts in the pathogenesis of periodontal disease. The objective of this study was to investigate the role and mechanisms mediated by Porphyromonas gingivalis (P. gingivalis) in promoting the migration of monocytes through regulating matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 expression.
Methods
Human THP1 monocytes were treated with culture supernatant derived from P. gingivalis (ATCC 33277) for 24 hours. Zymography, Western blot analysis and quantitative polymerase chain reactions were performed to analyze protein and mRNA levels of MMP-9. Protein and mRNA levels of TIMP-1 from monocytes treated with or without P. gingivalis were determined as well. Transwell migration assay was carried out to analyze the effect of P. gingivalis on the migration of human peripheral blood CD14+ monocytes. MMP inhibitor (GM6001) and proteinase inhibitor (Leupeptin) were used to determine the role of MMP-9 in P. gingivalis supernatant- and Lipopolysaccharide (LPS)-induced monocyte migration.
Results
In zymography and Western blot, an 82-kDa band of active MMP-9 emerged in P. gingivalis-treated monocyte culture media in a dose-dependent manner in addition to the MMP-9 proenzyme (92 kDa) band expressed in control cell culture media. P. gingivalis supernatant increased both the protein and mRNA levels of MMP-9 and TIMP-1. P. gingivalis supernatant, but not its LPS, increased the migratory ability of CD14+ monocytes. The increased migratory ability of P. gingivalis-treated monocytes was partially inhibited by leupeptin (200 µg/ml) and completely antagonized by the MMP inhibitor GM6001 (100 nM). LPS of P. gingivalis increased protein and mRNA levels of MMP-9 in monocytes, but had no effect on the migratory ability or MMP-9 activation.
Conclusion
P. gingivalis supernatant increased the migratory ability of monocytes, in part, by increasing activation and expression of MMP-9.
doi:10.1111/j.1600-0765.2011.01427.x
PMCID: PMC4527323  PMID: 22035412
Porphyromonas gingivalis; monocytes; migration; MMP-9
14.  Eruptive and Functional Changes in Periodontal Ligament Fibroblast Orientation in CD44 Wild-type vs. Knockout Mice 
Journal of periodontal research  2013;49(3):355-362.
Background and Objective
Periodontal ligament (PDL) fibroblasts establish the principal fibers of the ligament during tooth eruption, and maintain these fibers during occlusion. PDL development and occlusal adaptation includes changes in the orientation of PDL fibroblasts, however, the mechanism for these changes in orientation is unclear. The objective of this study was to compare the periodontal ligament fibroblast orientation in different stages corresponding with first molar eruption and occlusion in CD44 wild-type (WT) and knockout (KO) mice.
Materials and Methods
CD44 WT and KO mice were raised to 6 postnatal stages corresponding with first molar (M1) eruption (8, 11, 14, 18 days dpn) and occlusion (26 and 41 days dpn). Coronal sections of the first mandibular molar (M1) were prepared and the orientation of fibroblasts in the cervical root region was measured. Angle measurements were compared across developmental stages and between strains using Watson-Williams F-test (Oriana software) and ANCOVA.
Results
PDL fibroblast orientation increased significantly in CD44 WT (9–87°) and KO mice (14–93°; p ≤ 0.05) between intraosseous eruption (Day 11), mucosal penetration (Day 14), and preocclusal eruption (Day 18), however, the PDL fibroblast orientation did not change significantly with the onset of occlusion (Day 26) or continued function (Day 41). Within each strain, the variance in fibroblast orientation during preocclusal eruption (Day 18) was significantly higher than the variance of all other timepoints (p < 0.0005). CD44 WT and KO mice showed a similar pattern of PDL development and eruption with a significant difference in CD44 WT vs. KO fibroblast orientations only during early function (Day 26, 92° vs 116°; p = 0.05).
Conclusions
The development of PDL fibroblast orientation is highly similar between CD44 WT and KO mice. Between early (Day 11) and late (Day 18) eruptive stages PDL fibroblast orientation increases, corresponding with the upward movement of M1. The PDL fibroblast orientation established in preocclusal eruption (Day 18) is maintained during early (Day 26) and late (Day 41) stages of occlusal function, suggesting that PDL cells adapt to mechanical loads in the oral cavity prior to M1 occlusion.
doi:10.1111/jre.12113
PMCID: PMC4527325  PMID: 23808836
cellular receptor; fibroblast; periodontal ligament; in vivo model; morphology; occlusion
15.  Simvastatin Inhibits Lipopolysaccharide-Induced Osteoclastogenesis and Reduces Alveolar Bone Loss in Experimental Periodontal Disease 
Journal of periodontal research  2013;49(4):518-526.
Background and Objective
Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase and have anti-inflammatory effects independent of cholesterol lowering. Recent clinical studies have indicated that statin intake has beneficial effect on periodontal disease. However, the underlying mechanisms have not been well understood. In the current study, we employed a rat model with lipopolysaccharide (LPS)-induced periodontal disease and determined the effect of simvastatin, a commonly prescribed statin, on osteoclastogenesis, gingival inflammation and alveolar bone loss.
Material and Methods
Sprague-Dawley rats were injected with A. actinomycetemcomitans LPS in periodontal tissue 3 times per week for 8 weeks and part of the rats with LPS injection were also given simvastatin via gavage. After the treatments, the rat maxillae were scanned by microcomputed tomography and the images were analyzed to determine alveolar bone loss. To explore the underlying mechanisms, the effect of simvastatin on osteoclastogenesis and gingival expression of pro-inflammatory cytokines were also determined by tartate-resistant acid phosphatase staining and real-time polymerase chain reaction assays, respectively.
Results
Results showed that LPS treatment markedly increased bone loss, but administration of simvastatin significantly alleviated the bone loss. Results also showed that LPS treatment stimulated osteoclastogenesis and the expression of inflammatory cytokines, but simvastatin significantly modulate the stimulatory effect of LPS on osteoclastogenesis and cytokine expression.
Conclusion
This study demonstrated that simvastatin treatment inhibits LPS-induced osteoclastogenesis and gingival inflammation and reduces alveolar bone loss, indicating that the intake of simvastatin may hinder the progression of periodontal disease.
doi:10.1111/jre.12132
PMCID: PMC3979522  PMID: 24117880
Simvastatin; periodontal disease; lipopolysaccharide; inflammation; alveolar bone
16.  Moesin-induced signaling in response to lipopolysaccharide in macrophages 
Journal of periodontal research  2010;45(5):589-601.
Background and Objective
Many physiological and pathophysiological conditions are attributable in part to cytoskeletal regulation of cellular responses to signals. Moesin (membrane-organizing extension spike protein), an ERM (ezrin, radixin and moesin) family member, is involved in lipopolysaccharide (LPS)-mediated events in mononuclear phagocytes; however, its role in signaling is not fully understood. The aim of this study was to investigate the LPS-induced moesin signaling pathways in macrophages.
Material and Methods
Macrophages were stimulated with 500 ng/mL LPS in macrophage serum-free medium. For blocking experiments, cells were pre-incubated with anti-moesin antibody. Moesin total protein and phosphorylation were studied with western blotting. Moesin mRNA was assessed using quantitative real-time PCR. To explore binding of moesin to LPS, native polyacrylamide gel electrophoresis (PAGE) gel shift assay was performed. Moesin immunoprecipitation with CD14, MD-2 and Toll-like receptor 4 (TLR4) and co-immunoprecipitation of MyD88–interleukin-1 receptor-associated kinase (IRAK) and IRAK–tumor necrosis factor receptor-activated factor 6 (TRAF6) were analyzed. Phosphorylation of IRAK and activities of MAPK, nuclear factor κB (NF-κB) and IκBα were studied. Tumor necrosis factor α, interleukin-1β and interferon β were measured by ELISA.
Results
Moesin was identified as part of a protein cluster that facilitates LPS recognition and results in the expression of proinflammatory cytokines. Lipopolysaccharide stimulates moesin expression and phosphorylation by binding directly to the moesin carboxyl-terminus. Moesin is temporally associated with TLR4 and MD-2 after LPS stimulation, while CD14 is continuously bound to moesin. Lipopolysaccharide-induced signaling is transferred downstream to p38, p44/42 MAPK and NF-κB activation. Blockage of moesin function interrupts the LPS response through an inhibition of MyD88, IRAK and TRAF6, negatively affecting subsequent activation of the MAP kinases (p38 and ERK), NF-κB activation and translocation to the nucleus.
Conclusion
These results suggest an important role for moesin in the innate immune response and TLR4-mediated pattern recognition in periodontal disease.
doi:10.1111/j.1600-0765.2010.01271.x
PMCID: PMC4502922  PMID: 20546116
macrophage; lipopolysaccharide; signal transduction; moesin
17.  Evaluation of periodontitis in hospital outpatients with major depressive disorder 
Background and Objective
Major depressive disorder (MDD) has been associated with alterations in the neuroendocrine system and immune function and may be associated with an increased susceptibility to cardiovascular disease, cancer and autoimmune/inflammatory disease. This study was conducted to investigate the relationship between periodontitis and MDD in a convenience sample of hospital outpatients.
Material and Methods
The sample consisted of 72 physically healthy subjects (36 outpatients with MDD and 36 age-matched controls [± 3 years]). Patients with bipolar disorder, eating disorders and psychotic disorders were excluded. Probing pocket depth and clinical attachment level were recorded at six sites per tooth. Depression was assessed by means of Structured Clinical Interview for DSM-IV.
Results
Extent of clinical attachment level and probing pocket depth were not different between controls and subjects with depression for the following thresholds: ≥ 3 mm (Mann-Whitney, p = 0.927 and 0.756); ≥ 4 mm (Mann-Whitney, p = 0.656 and 0.373); ≥ 5 mm (Mann-Whitney, p = 0.518 and 0.870);, and ≥ 6 mm (Mann-Whitney, p = 0.994 and 0.879). Depression parameters were not associated with clinical attachment level ≥ 5 mm in this sample. Smoking was associated with loss of attachment ≥ 5 mm in the multi-variable logistic regression model (odds ratio = 6.99, 95% confidence interval = 2.00–24.43).
Conclusions
In this sample, periodontal clinical parameters were not different between patients with MDD and control subjects. There was no association between depression and periodontitis.
doi:10.1111/jre.12082
PMCID: PMC4479258  PMID: 23586804
depression; major depressive disorder; periodontitis; risk factor
18.  Streptococcus cristatus ArcA Interferes with Porphyromonas gingivalis Pathogenicity in Mice 
Journal of periodontal research  2012;47(5):578-583.
Background and Objective
Porphyromonas gingivalis has been implicated as one of the major pathogens in chronic periodontitis, an infectious disease affecting the majority of the adult population. We have previously demonstrated that a surface protein, arginine deiminase (ArcA), of Streptococcus cristatus represses production of P. gingivalis long fimbriae and interrupts the formation of P. gingivalis biofilms in vitro. Our in vivo studies have also shown that the distribution of P. gingivalis and S. cristatus in human subgingival plaque is negatively correlated. The objective of this study is to determine if S. cristatus ArcA inhibits P. gingivalis colonization and attenuates its subsequent pathogenesis in alveolar bone loss in the murine oral cavity.
Material and Methods
A wild-type strain of S. cristatus, CC5A and its arcA knock-out mutant ArcAE were used as initial colonizers in the oral cavity of BALB/cByJ mice. Colonization of P. gingivalis on the existing S. cristatus biofilms was assessed by qPCR and P. gingivalis-induced alveolar bone loss was measured 6 weeks after P. gingivalis infection.
Results
The presence of S. cristatus CC5A, but not its arcA mutant, attenuated P. gingivalis colonization in the murine oral cavity. In addition, P. gingivalis-induced alveolar bone loss was significantly less in mice initially infected with S. cristatus CC5A than in those infected with the arcA mutant.
Conclusions
This study provides direct evidence that S. cristatus ArcA has an inhibitory effect on P. gingivalis colonization, which may in turn attenuate the pathogenecity of P. gingivalis.
doi:10.1111/j.1600-0765.2012.01469.x
PMCID: PMC4401471  PMID: 22448761
Porphyromonas gingivalis; Streptococcus cristatus ArcA; mouse; pathogenesis
19.  Periodontitis in Pregnant Baboons: Systemic Inflammation and Adaptive Immune Responses and Pregnancy Outcomes in a Baboon Model 
Journal of periodontal research  2013;49(2):226-236.
Chronic periodontal infections have been suggested to contribute to the risk of adverse pregnancy outcomes. This study describes the relationship of patterns of systemic inflammatory mediators and IgG antibody to 20 oral bacteria in pregnant female baboons (Papio anubis) coupled with clinical features of ligature-induced periodontitis, as risk indicators for adverse pregnancy outcomes. Animals showing a preterm delivery and/or low birth weight newborns, as well as those pregnancies resulting in spontaneous abortion, stillbirth, or fetal demise were tabulated as adverse pregnancy outcomes. A significantly greater frequency of the periodontitis group neonates had a low birth weight (18.1%; p=0.008) and decreased gestational age (9.8%). Spontaneous abortion/stillbirth/fetal demise were increased in the periodontitis (8.7%) versus the control group (3.8%) (p=0.054). The baseline oral clinical presentation of the experimental animals did not relate to the adverse pregnancy outcomes. Animals with the greatest extent/severity of periodontitis progression during the initial ½ of gestation (ie. to mid-pregnancy) had the greatest risk for adverse pregnancy outcomes. Baseline biological parameters indicating historical responses of the animals to periodontal challenge demonstrated individual variation in selected mediators, some of which became more differential during ligature-induced periodontitis. The relationship of clinical parameters to systemic inflammatory responses was consistent with a temporal contribution to adverse pregnancy outcomes in a subset of the animals. These results support a link between periodontitis and adverse pregnancy outcomes in the baboons and provide a prospective experimental model for delineating the biologic parameters that contribute to a causal relationship between chronic oral infections and birth events.
doi:10.1111/jre.12099
PMCID: PMC3969847  PMID: 23710643
baboons; pregnancy; periodontitis; inflammation
20.  [No title available] 
PMCID: PMC3732561  PMID: 23488730
21.  [No title available] 
PMCID: PMC3758777  PMID: 23662917
22.  Establishment and characterization of a telomerase immortalized human gingival epithelial cell line 
Journal of periodontal research  2013;48(6):10.1111/jre.12059.
Background and Objective
Gingival keratinocytes are used in model systems to investigate the interaction between periodontal bacteria and the epithelium in the initial stages of the periodontal disease process. Primary gingival epithelial cells (GECs) have a finite lifespan in culture before they enter senescence and cease to replicate, while epithelial cells immortalized with viral proteins can exhibit chromosomal rearrangements. The aim of this study was to generate a telomerase-immortalized human gingival epithelial cell line and compare its in vitro behavior to that of human GECs.
Material and Methods
Human primary gingival epithelial cells were immortalized with a bmi1/hTERT combination to prevent cell cycle triggers of senescence and telomere shortening. The resultant cell-line, Telomerase Immortalized Gingival Keratinocytes (TIGKs), were compared to GECs for cell morphology, karyotype, growth and cytokeratin expression, and further characterized for replicative lifespan, expression of toll-like receptors (TLRs) and invasion by P. gingivalis.
Results
TIGKs showed morphologies, karyotype, proliferation rates and expression of characteristic cytokeratin proteins comparable to GECs. TIGKs underwent 36 passages without signs of senescence and expressed transcripts for TLRs 1-6, 8 and 9. A subpopulation of cells underwent stratification after extended time in culture. The cytokeratin profiles of TIGK monolayers were consistent with basal cells. When allowed to stratify, cytokeratin profiles of TIGKs were consistent with suprabasal cells of the junctional epithelium. Further, TIGKs were comparable to GECs in previously reported levels and kinetics of invasion by wild type Porphyromonas gingivalis and an invasion defective ΔserB mutant.
Conclusion
Results confirm bmi1/hTERT-immortalization of primary gingival epithelial cells generated a robust cell-line with similar characteristics to the parental cell type. TIGKs represent a valuable model system for the study of oral bacteria interactions with host gingival cells.
doi:10.1111/jre.12059
PMCID: PMC3709015  PMID: 23441958
Telomerase; Immortalization; hTERT; bmi1; Gingival Epithelial Cells; Porphyromonas gingivalis
23.  Neutrophils in chronic and aggressive periodontitis in interaction with Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans 
Journal of periodontal research  2009;44(3):368-377.
AIM
The aim of this in vitro study was to study phagocytosis and killing of Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans Y4 by peripheral blood PMNs taken from aggressive and chronic periodontitis patients. Also, the release of reactive oxygen species (ROS) and human neutrophil elastase (HNE) upon the interaction of PMNs with bacteria was measured.
METHODS
Peripheral blood PMNs obtained from 12 chronic periodontitis patients, 6 aggressive periodontitis patients and 12 healthy controls were exposed to Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans following opsonisation of the bacteria using the patient’s own serum. Phagocytosis and killing of the bacteria as well as the extracellular HNE activity were quantified for up to 2 hours. The total amount and the extracellular release of ROS were measured by luminol and isoluminol dependent chemiluminescence.
RESULTS
PMNs from chronic (62.16 ± 19.39 %) and aggressive periodontitis (43.26 ± 26.63 %) patients phagocytosed more P. gingivalis than the healthy controls (24.43 ± 19.87 %) at 30 mins after exposure to the bacteria (p < 0.05). PMNs from subjects with chronic and aggressive periodontitis released significantly more ROS and demonstrated greater HNE activity in the absence of any stimulus than PMNs from healthy controls (p < 0.05). The total release of ROS increased in chronic periodontitis PMNs and in the control group PMNs after interaction with P. gingivalis. The interaction with A. actinomycetemcomitans resulted in greater total ROS release in chronic periodontitis PMNs and in the control group PMNs than P. gingivalis.
CONCLUSION
PMNs in aggressive and chronic periodontitis are hyperactive even without any particular stimulus. The extracellular release of ROS and neutrophil elastase by PMNs may not only affect bacterial virulence and/or viability, but also contribute to damage of the surrounding periodontal tissues.
doi:10.1111/j.1600-0765.2008.01113.x
PMCID: PMC4180098  PMID: 19210340
PMN; phagocytosis; ROS; aggressive periodontitis; chronic periodontitis; Aggregatibacter actinomycetemcomitans; Porphyromonas gingivalis
24.  Cleavage of IgG1 in GCF is associated with presence of Porphyromonas gingivalis 
Journal of periodontal research  2012;48(4):458-465.
Background and Objectives
Immunoglobulin (Ig) G1 plays an important role in the adaptive immune response. Kgp, a lysine-specific cysteine protease from Porphyromonas gingivalis, specifically hydrolyses IgG1 heavy chains. The purpose of this study was to examine whether cleavage of IgG1 occurs in gingival crevicular fluid (GCF) in vivo, and whether there is any association with the presence of P. gingivalis and other periodontopathogens.
Material and methods
GCF was obtained from nine patients with aggressive periodontitis, nine with chronic periodontitis, and five periodontally-healthy individuals. The bacterial loads of P. gingivalis, Aggregatibacter actinomycetemcomitans, Treponema denticola, Prevotella intermedia, and Tannerella forsythia were analysed by real-time PCR, and the presence and cleavage of IgG1 and IgG2 were determined using Western blotting. Kgp levels were measured by ELISA.
Results
Cleaved IgG1 was identified in the GCF from 67% of patients with aggressive periodontitis and in 44% of patients with chronic periodontitis. By contrast, no cleaved IgG1 was detectable in the healthy controls. No degradation of IgG2 was detected in any of the samples, regardless of health status. P. gingivalis was found in high numbers in all samples in which cleavage of IgG1 was detected (p < 0.001 compared with samples with no IgG cleavage). Furthermore, high numbers of T. forsythia and P. intermedia were also present in these samples. The level of Kgp in the GCF correlated with the load of P. gingivalis (r = 0.425, p < 0.01). The presence of Kgp (range 0.07–10.98 ng/ml) was associated with proteolytic fragments of IgG1 (p < 0.001). However, cleaved IgG1 was also detected in samples with no detectable Kgp.
Conclusion
In patients with periodontitis cleavage of IgG1 occurs in vivo and may suppress antibody-dependent antibacterial activity in subgingival biofilms especially those colonized by P. gingivalis.
doi:10.1111/jre.12027
PMCID: PMC3566341  PMID: 23116446
IgG; GCF; periodontitis Porphyromonas gingivalis; gingipains
25.  Systemic endotoxin levels in chronic indolent periodontal infections 
Background and Objective:
Periodontal disease has been linked with an increased risk of various systemic diseases. A plausible biologic explanation for this link includes the opportunity for oral pathogens to translocate to the circulation as a result of breakdown in integrity of the oral epithelium. This study refined a methodology used to detect endotoxin activity in the serum of subjects with indolent periodontal infections.
Material and Methods:
The QCL® Kinetic Chromogenic Assay (Cambrex) is a kinetic measure of endotoxin activity. Sera from 211 pregnant women with periodontitis enrolled in the Obstetrics and Periodontal Therapy Trial were used to develop the assay further and to evaluate the detection of endotoxin activity that might accompany a low-level bacteremia in chronic periodontitis.
Results:
We optimized the system to increase the sensitivity and reproducibility of the assay. The refined system was able to detect endotoxin activity in serum at > 0.0125 EU/mL. At baseline (13–16 wk of gestation), 35.5% of the women were positive for endotoxin activity (1.62 ± 2.21; range: 0.38–15 EU/mL).
Conclusion:
This report describes a sensitive measure of endotoxin activity in serum. The procedure allowed us to document levels of this microbial virulence factor in serum of individuals with indolent infections such as periodontal disease.
doi:10.1111/j.1600-0765.2008.01169.x
PMCID: PMC4102883  PMID: 20465752
periodontitis; endotoxin; serum; pregnancy; indolent infection

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