An acute injection of Porphyromonas gingivalis lipopolysaccharide modulates the OPG/RANKL system and interleukin‐6 in an ovariectomized mouse model

Background/aims: In the present study, we attempted to develop a simulated model to explore the causal effects of periodontal pathogens on skeletal homeostasis in postmenopausal osteoporosis. Methods: Fifty‐three female adult ICR mice were randomly assigned to an experimental group (ovariectomized) or a control group. A single injection of Porphyromonas gingivalis lipopolysaccharide (P. gingivalis‐LPS, ATCC 33277) or Escherichia coli lipopolysaccharide (E. coli‐LPS) was administered intraperitoneally 4 weeks after an ovariectomy. Concentrations of interleukin‐6 (IL‐6), osteoprotegerin (OPG), and the receptor activator of nuclear factor‐κB ligand (RANKL) in serum were subsequently analyzed using an enzyme‐linked immunosorbent assay (ELISA). Results: Under stimulation with P. gingivalis‐LPS or E. coli‐LPS, the concentration of OPG rose in both groups. The serum level of RANKL showed a decreasing trend 24 h after the injection in both groups. After injection of P. gingivalis‐LPS in both the experimental and control animals, the OPG : RANKL ratio increased 24 h after the booster (22.26–620.99, P < 0.05). The serum level of IL‐6 in the experimental group significantly increased 1–6 h after administration of E. coli‐LPS and 1–3 h after administration of P. gingivalis‐LPS (P < 0.05). Conclusions: A single booster injection of P. gingivalis‐LPS induced short‐term changes in OPG, RANKL, and IL‐6 serum levels in this ovariectomized mouse model.     

牙龈卟啉单胞菌诱导的耐受对小鼠牙槽骨吸收的影响

目的:研究牙龈卟啉单胞菌(P.gingivalis)诱导的耐受对小鼠骨吸收相关因子白细胞介素-1β(IL-1β)、骨保护素(OPG)/NF-κB受体激活蛋白配体(RANKL)表达水平,以及牙槽骨吸收的影响。方法:采用1×10~9CFU P.gingivalis口内涂布给菌5 d,磷酸盐缓冲液(PBS)洗脱后,再次口内涂布P.gingivalis给菌2 d,构建Balb/c小鼠牙周组织耐受模型。10 d后收集新鲜牙龈组织,采用western bolt检测牙龈组织中IL-1β、OPG和RANKL表达水平的变化。6周后,收集上颌骨,采用亚甲基蓝染色法检测牙槽骨吸收水平的改变。结果:耐受组小鼠牙龈组织中IL-1β表达水平较非耐受组降低,OPG/RANKL比值较非耐受组升高。亚甲基蓝染色结果显示,耐受组小鼠釉牙骨质界至牙槽嵴顶(ABC-CEJ)的距离小于非耐受组(P<0.01),说明耐受组小鼠牙槽骨吸收较非耐受组降低。结论:P.gingivalis诱导小鼠牙周组织耐受后,小鼠牙龈组织中骨吸收相关因子IL-1β分泌降低,OPG/RANKL比值升高,抑制了牙槽骨吸收。     

Association among interleukin-6 gene polymorphism, diabetes and periodontitis in a Chinese population

Objectives:  Diabetics significantly increase risk for periodontitis. Interleukin-6 (IL-6) gene polymorphism may play certain roles in the progression of periodontitis with diabetes. The purpose of this study was to assess the association among IL-6 gene polymorphisms, type 2 diabetes mellitus (T2DM) and chronic periodontitis (CP) in a Chinese population.
Material and methods:  DNA was obtained from 159 patients with CP, 88 patients with T2DM, 110 patients with CP&T2DM and 135 control subjects. The -174/-572/-597 polymorphisms of IL-6 gene were investigated by restriction fragment length polymorphism of polymerase chain reaction products. The results were further confirmed by sequencing. Significance was set at P  < 0.008 after Bonferroni correction.
Results:  Among four groups, CP&T2DM group showed the lowest IL-6-572 CC genotype and C-allele frequencies (54.5% and 74.1%). In this regard, there were significant differences between CP&T2DM group and the control group [ P  = 0.006, odds ratio (OR)  =  0.475, 95% CI: 0.279–0.808 and P  = 0.002, OR = 0.502, 95% CI: 0.319–0.788 respectively]. Logistic regression with adjustment for age, gender, body mass index, smoking and stress showed no significant difference in terms of IL-6-572 genotypes ( P  = 0.058, OR= 0.523, 95% CI: 0.268–1.022).
Conclusions:  The IL-6-572 genotype and allele distributions are unique to subjects with CP&T2DM in a Chinese population.     

Lack of Toll-like receptor 4 decreases lipopolysaccharide-induced bone resorption in C3H/HeJ mice in vivo

Introduction:  Few in vivo studies have demonstrated whether Toll-like receptor 4 (TLR4) is indispensable for lipopolysaccharide (LPS)-induced bone resorption and little is known about the receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) expression induced by LPS under conditions of lack of TLR4.
Methods:  We compared bone resorption histomorphometrically in C3H/HeN and C3H/HeJ mice that were repeatedly injected with Actinobacillus actionmycetemcomitans LPS into their gingiva every 48 h. RANKL-, interleukin-1β- and OPG-positive cells in the connective tissue were also compared immunohistochemically.
Results:  Bone resorption in C3H/HeJ mice in the fourth, seventh, and tenth injection groups was significantly less than that C3H/HeN mice ( P  < 0.05). The number of RANKL-positive cells in C3H/HeJ mice in the 10th injection group was significantly smaller than that in C3H/HeN mice ( P  < 0.05). The numbers of interleukin-1β-positive cells in C3H/HeJ mice in the seventh and tenth injection groups were significantly decreased compared with those in C3H/HeN mice ( P  < 0.05). The numbers of OPG-positive cells in C3H/HeN and C3H/HeJ mice gradually increased, but there was no significant difference between the two strains of mice.
Conclusion:  TLR4 is indispensable for LPS-induced bone resorption in vivo .     

Effects of smoking on the ex vivo cytokine production in periodontitis

Background and Objective:  Smoking is associated with increased severity of periodontitis. The underlying mechanisms of this phenomenon are not well understood. The purpose of the present study was to compare the monocyte-derived T cell directing (Th1/Th2) response and pro-inflammatory cytokine production in ex vivo whole blood cell cultures (WBCC) of smoking and non-smoking chronic periodontitis patients.
Material and Methods:  Venous blood was collected from 29 periodontitis patients (18 non-smokers and 11 smokers) receiving supportive periodontal treatment, and diluted 10-fold for WBCC. The WBCC were stimulated for 18 h with Neisseria meningitidis lipo-oligosaccharide (LOS) or Porphyromonas gingivalis sonic extract (Pg-SE). The production of the T cell directing cytokines interleukin (IL)-12 p40 and IL-10, as well as the pro-inflammatory cytokines IL-1β, IL-6 and IL-8, was measured in the culture supernatants.
Results:  After LOS stimulation of WBCC, smokers showed a lower IL-12 p40/IL-10 ratio than non-smokers ( P <  0.05). Interleukin-1β production was significantly lower in smokers compared with non-smokers after stimulation with either LOS or Pg-SE ( P <  0.05). Interleukin-6 and IL-8 production was similar in WBCC from both smokers and non-smokers, for both LOS and Pg-SE.
Conclusion:  A more pronounced Th2 response in smoking periodontitis patients may be related to increased severity of the disease.     

Local osteoprotegerin gene transfer to periodontal tissue inhibits lipopolysaccharide-induced alveolar bone resorption

Background and Objective:  Osteoclastogenesis is primarily activated by receptor activator of nuclear factor κB ligand (RANKL) and is inhibited by osteoprotegerin (OPG). A previous study demonstrated that local OPG gene transfer to periodontal tissue inhibited RANKL-mediated osteoclastogenesis and experimental tooth movement. In the present study, we tested the hypothesis that local OPG gene transfer to the periodontium can neutralize RANKL activity induced by lipopolysaccharide injection, thereby inhibiting osteoclastogenesis and diminishing alveolar bone resorption in experimental periodontal disease.
Material and methods:  Seven-week-old male Wistar rats received an injection of lipopolysaccharide or phosphate-buffered saline in the palatal gingiva of the upper first molars on both the right and left sides. An inactivated haemagglutinating virus of Japan (HVJ) envelope vector containing a mouse OPG expression plasmid [pcDNA3.1(+)-mOPG] or mock vector was injected periodically into the palatal periodontal tissue of the upper first molars.
Results:  Lipopolysaccharide injection induced severe periodontal bone resorption. Local OPG gene transfer induced OPG production, and osteoclastogenesis was inhibited. Local OPG gene transfer significantly decreased alveolar bone resorption.
Conclusion:  Osteoprotegerin gene transfer to periodontal tissue inhibited osteoclastogenesis and alveolar bone resorption in lipopolysaccharide-induced experimental periodontal disease.     

Human gingival fibroblasts release high-mobility group box-1 protein through active and passive pathways

Introduction:  The nuclear protein high-mobility group box-1 (HMGB1) acts as a late mediator of inflammation when secreted in the extracellular milieu. In this study, we examined the effect of lipopolysaccharides from periodontal pathogens and apoptotic and necrotic cell death on HMGB1 production in human gingival fibroblasts (HGF).
Methods:  HGF from healthy periodontal tissue were cultured and stimulated with lipopolysaccharides (LPS) from Aggregatibacter actinomycetemcomitans , Porphyromonas gingivalis , and Escherichia coli . We also initiated apoptotic and necrotic cell deaths in HGF. The HMGB1 released in the supernatants from stimulated or dying cells was measured. Immunocytochemical staining against HMGB1 was performed in LPS-stimulated HGF.
Results:  A significantly higher amount of HMGB1 was detected from necrotic and apoptotic HGF. LPS from A. actinomycetemcomitans , P. gingivalis , and E. coli significantly induced the production of HMGB1 in a time-dependent manner. After 6 h of LPS stimulation, HMGB1 was present in the cytoplasm of cells whereas its location was mainly nuclear after 24 h.
Conclusions:  LPS from two major periodontal pathogens, A. actinomycetemcomitans and P. gingivalis , induced HMGB1 secretion from HGF. Apoptotic and necrotic cell deaths resulted in the enhancement of HMGB1. Our results suggest that HGF can be a source of HMGB1 by both active secretion and passive release, and that HMGB1 from HGF may contribute to periodontal tissue destruction.     

The association of osteoprotegerin gene polymorphisms with periodontitis

Background:  It has been demonstrated that genetic variation accounts for approximately half of the variance in periodontitis. The reported association of polymorphisms in the osteoprotegerin (OPG) gene with osteoporosis suggests that the OPG gene may also influence the genetic risk for periodontitis.
Subjects and methods:  We investigated the distribution of OPG gene polymorphisms in 49 patients with aggressive ( n  =   14) or chronic ( n  =   35) periodontitis and 49 control subjects without periodontitis, using polymerase chain reaction (PCR)–restriction fragment length polymorphism and PCR–single strand conformation polymorphism followed by direct sequencing.
Results:  A total of seven known polymorphisms and one new mutation, G373A, were identified. The T950 and G1181 alleles were more common in patients with periodontitis ( P  =   0.028 and P  =   0.047, respectively) than in control subjects. Especially, G1181 allele was associated with patients with aggressive periodontitis.
Conclusion:  The TG haplotype of T950C and G1181C polymorphisms in the OPG gene may be useful genetic markers for the prediction of periodontitis. Further studies in a larger population are required to determine whether these alleles directly contribute to periodontitis susceptibility.     

牙龈卟啉单胞菌对纯钛表面成骨细胞表达OPG和RANKL mRNA的影响

目的:在转录水平上观察牙龈卟啉单胞菌对成骨细胞表达OPG及RANKL mRNA的影响.方法:用浓度分别为106、18CFU/mL的牙龈卟啉单胞菌刺激接种于纯钛表面的成骨细胞,24 h后提取各组成骨细胞总RNA并应用逆转录-聚合酶链式反应和mRNA比色定量的方法检测OPG及RANKL 基因表达.结果:MG-63成骨细胞基础表达较强的OPG mRNA及少量的RANKL mRNA,牙龈卟啉单胞菌以浓度依赖的方式增强RANKL mRNA的表达,减弱OPG mRNA的表达.结论:牙龈卟啉单胞菌通过上调RANKL/OPG的比率可间接的调节破骨细胞的活性从而影响支抗种植体周的骨代谢.     

The host cytokine response to Porphyromonas gingivalis is modified by gingipains

Background/aims:  Clinical studies indicate that primary proinflammatory cytokines, such as interleukin-1β (IL-1β) are elevated in the gingival crevice around teeth with periodontitis but the secondary cytokines and chemokines, IL-6 and IL-8, are not. The human gingival epithelial cells (HGECs) lining the gingival sulcus respond to perturbation by microbes of dental plaque by releasing a wide range of cytokines. Porphyromonas gingivalis , a putative periodontal pathogen, possesses numerous virulence factors some of which directly impact on the host response. In the present study, we sought to determine how P. gingivalis influences the inflammatory cytokine responses.
Methods:  HGECs were challenged with P. gingivalis and other putative periodontal pathogens, and the resultant production of IL-1β, IL-6, and IL-8 was assayed by enzyme-linked immunosorbent assay (ELISA). Culture supernatants and recombinant human cytokines were challenged with live P. gingivalis wild-type and gingipain-deficient strains and the resultant cytokine profile was assessed by ELISA and Western blot.
Results:  We show here that primary HGECs challenged with live P. gingivalis result in high levels of IL-1β but not the related secondary cytokines IL-6 and IL-8. We further demonstrate that cytokine response differences are the result of the action of P. gingivalis proteases, with lysine gingipain being the most effective.
Conclusion:  We conclude that P. gingivalis , through lysine gingipain, can subvert the protective host proinflammatory response by direct cytokine degradation. Changes in the crevicular cytokine profile have consequences in periodontal disease pathogenesis that should be considered in the development of diagnostic and therapeutic modalities.     

Treatment with levamisole and colchicine can result in a significant reduction of IL-6, IL-8 or TNF-α level in patients with mucocutaneous type of Behcet's disease

Background:  Mucocutaneous type of Behcet's disease (MCBD) is a multisystemic inflammatory disease with oral and genital ulcers with or without skin lesions.
Methods:  A solid phase, two-site sequential chemiluminescent immunometric assay was used to measure serum levels of interleukin (IL)-6, IL-8 and tumour necrosis factor (TNF)-α in 54 normal control subjects and in 64 MCBD patients before and after treatment with levamisole plus colchicine.
Results:  We found that 67%, 83% or 67% of MCBD patients had a serum IL-6, IL-8 or TNF-α level greater than the upper normal limit of 4.7, 8.7 or 7.4 pg/ml, respectively. The mean serum level of IL-6 (9.9 ± 2.4 pg/ml, P  < 0.005), IL-8 (107.5 ± 21.4 pg/ml, P  < 0.001) or TNF-α (22.5 ± 4.1 pg/ml, P  < 0.001) in 64 MCBD patients was significantly higher than that (2.1 ± 0.2, 5.7 ± 0.2 or 3.8 ± 0.2 pg/ml for IL-6, IL-8 or TNF-α level, respectively) in normal control subjects. In 43 MCBD patients with all the serum IL-6, IL-8 and TNF-α levels higher than their upper normal limits, treatment with levamisole plus colchicine for a period of 0.5–11.5 (mean, 3.2 ± 2.4) months could significantly reduce the mean serum IL-6, IL-8 and TNF-α levels from 9.0 ± 1.7 to 1.6 ± 0.2 pg/ml ( P  < 0.001), 134.6 ± 28.2–6.0 ± 0.4 pg/ml ( P  < 0.001) and 25.7 ± 5.6–3.5 ± 0.4 pg/ml ( P  < 0.001), respectively.
Conclusions:  Treatment with levamisole and colchicine can result in a significant reduction of serum IL-6, IL-8 or TNF-α level in MCBD patients.     

The upregulation of receptor activator NF-κB ligand expression by interleukin-1α and Porphyromonas endodontalis in human osteoblastic cells

Aim  To investigate the receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL) in osteoblastic cells stimulated with inflammatory mediators.
Methodology  The expression of RANKL in human osteoblastic cell line U2OS stimulated by pro-inflammatory cytokine interleukin (IL)-1α and black-pigmented bacteria Porphyromonas endodontalis was investigated by Western blot and enzyme-linked immunosorbent assay (ELISA). The significance of the results obtained from control and treated groups was statistically analysed by the paired Student's t -test.
Results  IL-1α was found to upregulate RANKL production in U2OS cells ( P  < 0.05). Investigations of the time dependence of RANKL expression in IL-1α-treated cells revealed a rapid accumulation of RANKL protein after 1 h of exposure; it remained elevated throughout the 24-h incubation period shown by Western blot and ELISA. In addition, P. endodontalis also increased RANKL expression in U2OS cells after 4-h incubation period demonstrated by Western blot and ELISA ( P  < 0.05).
Conclusions  IL-1α and P. endodontalis may be involved in developing apical periodontitis through the stimulation of RANKL production.     

Differential cytokine expressions affect the severity of peri-implant disease

Objective: This study assessed gene expression by quantitative polymerase chain reaction of inflammatory- [interleukin (IL)-12, tumor necrosis factor-α (TNF-α), IL-4, and IL-10] and osteoclastogenesis-related factors [receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG)] in sites exhibiting different severities of peri-implant disease.
Material and methods: Peri-implant soft tissue biopsies ( n =48) were harvested from healthy implant (HI), mucositis (MC), initial peri-implantitis (IP) and severe peri-implantitis (SP) sites.
Results: IL-12 and TNF-α mRNA levels were higher in SP, followed by IP and MC ( P <0.05). IL-4 was higher in HI, followed by MC, SP and IP ( P <0.05). IL-10 was the lowest in HI, while no differences were detected among the diseased groups ( P >0.05). OPG mRNA levels were higher in HI, followed by IP, SP and MC, whereas RANKL was increased as the peri-implantitis severity increased ( P <0.05). The highest OPG/RANKL ratio was observed in HI and the lowest in SP ( P <0.01).
Conclusion: These findings suggest that expressions of inflammatory- and osteoclastogenesis-related factors may play an important role in the onset and severity of the peri-implant diseases.     

Inflammatory response to chlorhexidine, minocycline HCl and doxycycline HCl in an in vivo mouse model

Aim: To examine the effect of locally delivered antimicrobial drugs on the inflammatory response in an in vivo mouse chamber model.
Material and Methods: Two weeks following chamber implantation, 24 BALB/c mice, in the experimental group, were given an intra-chamber challenge of heat-killed Porphyromonas gingivalis , followed immediately by injection of the specific antimicrobial drug: 2000  μ g/ml chlorhexidine (CHX); 1500  μ g/ml minocycline HCl;and 1500  μ g/ml doxycycline HCl (concentrations achieved in the periodontal pocket with commercial controlled-release delivery systems). A second group of 24 animals received only the antimicrobial treatment without P. gingivalis challenge. Intra-chamber exudates were sampled at 2 and 24 h following the challenge, and leucocytes, TNF α , IFN γ and IL-10 were evaluated.
Results: At 2 h, minocycline HCl induced high levels of IL-10, TNF α and IFN γ , while CHX reduced the levels of TNF α and IFN γ . By 24 h, these responses were attenuated. Following bacterial challenge, the antibacterial agents attenuated the inflammatory process, each in its own fashion.
Conclusions: Antibacterial agents applied locally have the ability to induce an inflammatory response. They also modify the inflammatory response to P. gingivalis independent of their antimicrobial effect. CHX and doxycycline HCl appear to have the most marked anti-inflammatory effect.     

LPS刺激小鼠骨细胞RANKL/OPG和IL-6表达的实验研究

目的: 探讨LPS对体外培养的小鼠MLO-Y4细胞RANKL/OPG和IL-6表达的影响。方法: 以5 mg/L的LPS刺激细胞,用CCK-8法于(12、24、48 h)后检测细胞的增殖;以不同浓度(1、10、100、500、1000 μg/L)的LPS刺激细胞,分别在作用4 h和1.5 h后用RT-PCR检测细胞对RANKL/OPG和IL-6的相对表达;以100 μg/L的LPS刺激细胞,分别在作用(0.5、1、2、4、8 h)和(0.5、1、1.5、2、4 h)两种不同时间后用RT-PCR检测细胞对RANKL/OPG和IL-6的相对表达。结果: LPS对MLO-Y4细胞增殖无影响;100 μg/L的LPS能显著上调细胞对RANKL和IL-6的相对表达,与(500, 1000 μg/L)LPS的上调结果无统计学差别;除0.5 h外其余时间点LPS均上调细胞对RANKL和IL-6的相对表达,并分别在4 h和1.5 h达到峰值;所有样本LPS对细胞OPG的相对表达均无影响。结论: 一定浓度的 LPS上调了MLO-Y4 细胞对RANKL和IL-6的表达,而对OPG的表达无影响。     

Diabetes modulates gene expression in the gingival tissues of patients with chronic periodontitis

AIM: This study evaluated whether diabetes modulates gene expression [interleukin (IL)-1beta, IL-1ra, IL-6, IL-8, IL-10; tumor necrosis factor (TNF)-alpha; interferon (IFN)-gamma, receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG)] in sites with periodontitis. MATERIALS AND METHODS: Gingival biopsies were harvested and divided into three groups--Control group: systemically and periodontally healthy subjects (n = 10); Periodontitis group: systemically healthy subjects diagnosed with chronic periodontitis (n = 20); Diabetes group: type 1 diabetic subjects, diagnosed with chronic periodontitis (n = 20). Total RNA was obtained and analyzed by quantitative polymerase chain reaction. RESULTS: Data analysis demonstrated that, except for OPG, mRNA levels for all factors were increased by inflammation (P < 0.001). Interleukin-1beta, IL-1ra, IL-6, IL-8, IFN-gamma, and RANKL mRNA levels were higher in the diabetic group when compared with the control non-periodontitis group (P < 0.05), whereas IL-10 and OPG were lower (P < 0.05). No difference was observed for TNF-alpha between diabetic and control groups (P > 0.05). Diabetes lowered IL-1beta, IL-8, IL-10, TNF-alpha, RANKL, and OPG mRNA levels in sites with comparable type of periodontitis (P < 0.001). Moreover, increased RANKL:OPG and IL-6:IL-10 ratios were found. CONCLUSION AND CLINICAL RELEVANCE: Taken together, these data suggest that decreased levels of IL-10 and OPG may play an important role in the periodontal breakdown in diabetic patients.     

IL-1β and compressive forces lead to a significant induction of RANKL-expression in primary human cementoblasts

AIM: The aim of this study was to investigate the response of primary human cementoblasts to conditions as they occur on the pressure side during orthodontic tooth movement. METHODS: In our previous study, the cementoblasts were characterized using markers for osteoblastogenic differentiation and the cementoblast-specific marker CEMP-1. Initially, primary human cementoblasts were compressed for 1?h, 4?h, and 6?h (30?g/cm(2)). In the second experiment, the cementoblasts were stimulated with interleukin (IL)-1β for 24?h and for 96?h with 1?ng/ml and 10?ng/ml and subsequently compressed for 1?h and 6?h. Changes in mRNA expression for receptor activator of NF-κB (RANK), RANK ligand (RANKL), osteoprotegerin (OPG), and cyclooxygenase-2 (COX-2) were measured by quantitative real-time polymerase chain reaction (RT-PCR). RANK and RANKL were also examined by immunocytochemical staining at the protein level. RESULTS: Compression (30?g/cm(2)) led to a significant increase in RANKL expression after 6?h. OPG expression in compressed cementoblasts was significantly reduced after 1?h. RANK remained unchanged during the course of the experiment. Stimulation with IL-1β induced RANKL and OPG expression. However, IL-1β-dependent induction of RANKL was more prominent than the induction of OPG, leading to a (significant) increase in the RANKL/OPG ratios. The expression of RANK remained unchanged after 24?h of stimulation with IL-1β and decreased significantly after 96?h. Compression of the prestimulated cells resulted in a further increase in RANKL expression significant after 6?h. OPG and RANK expression remained unchanged compared to the unstimulated sample. COX-2 increased significantly after both compression and stimulation with IL-1β. Combined stimulation and compression resulted in a significant further increase after 6?h compared to IL-1β stimulation alone. CONCLUSION: Primary human cementoblasts in vitro express increased levels of RANKL, in particular during the combination of inflammation and compression. The increase in RANKL expression is not compensated by an increase in OPG expression. The induction of RANKL expression was associated with a significant increase in COX-2 expression. Since RANKL attracts osteoclasts, its increase might be associated with the progression of root resorption. The in vitro alterations in cementoblasts we observed may be indicators of cellular mechanisms that lead to the increased root resorption during orthodontic treatment.     

Prostaglandin E(2) is a main mediator in receptor activator of nuclear factor-kappaB ligand-dependent osteoclastogenesis induced by Porphyromonas gingivalis, Treponema denticola, and Treponema socranskii

BACKGROUND: Periodontitis is an inflammatory disease that often leads to destruction of alveolar bone; a number of bacteria in subgingival plaque are associated with bone destruction in periodontitis. To understand the mechanism of how periodontopathogens induce osteoclastogenesis, we determined which mediators are involved in the osteoclastogenesis. METHODS: We investigated effects of sonicates from three periodontopathic bacteria, Porphyromonas gingivalis, Treponema denticola, and Treponema socranskii, on osteoclast formation in a co-culture system of mouse calvaria-derived osteoblasts and bone marrow cells. The osteoclast formation was determined by tartrate resistant acid phosphatase (TRAP) staining. The expression of the receptor activator of nuclear factor-kappa B ligand (RANKL), prostaglandin E(2) (PGE(2)) and osteoprotegerin (OPG) in mouse calvaria-derived osteoblasts was determined by immunoassay. RESULTS: Each bacterial sonicate induced the osteoclast formation in the co-culture system. These bacterial sonicates increased the expression of RANKL and PGE(2), and decreased the expression of OPG in osteoblasts. The addition of OPG, an inhibitor of RANKL, in the co-culture completely suppressed the osteoclastogenesis that was stimulated by each bacterial sonicate. Indomethacin, which is an inhibitor of PGE(2) synthesis, reduced more than 88% of the osteoclast formation induced by each bacterial sonicate. Indomethacin inhibited more than 80% of RANKL expression in osteoblasts induced by T. denticola and T. socranskii, and 59% by P. gingivalis. Indomethacin completely recovered the depression of OPG expression in osteoblasts by T. denticola and T. socranskii to the level of the untreated osteoblasts. Indomethacin recovered the reduction of OPG expression by P. gingivalis to 67%. CONCLUSION: These findings suggest that the osteoclastogenesis by P. gingivalis, T. denticola, and T. socranskii is mediated by a RANKL-dependent pathway and that PGE(2) is a main factor in the pathway by the enhancing of RANKL expression and the depression of osteoprotegerin, a RANKL inhibitor.