Apple- and hop-polyphenols protect periodontal ligament cells stimulated with enamel matrix derivative from Porphyromonas gingivalis

BACKGROUND: Enamel matrix derivative (EMD) is a tissue regenerative agent used clinically as an adjunct to periodontal surgery. It was previously demonstrated that Porphyromonas gingivalis, a periodontal pathogen, significantly diminished the efficacy of EMD with periodontal ligament (PDL) cells through the proteolytic actions of Arg- and Lys-gingipains (Rgp and Kgp). Thus, antiproteolytic supplements are considered clinically desirable for effective periodontal regenerative therapies. In the present study, we examined apple- (AP) and hop-polyphenols to determine their ability to protect EMD-stimulated PDL cells from P. gingivalis. METHODS: AP, apple condensed tannin (ACT), hop bract polyphenol (HBP), high and low molecular weight fractions of HBP (HMW-HBP and LMW-HBP), and epigallocatechin gallate (EGCg) were used. PDL cells were grown on EMD-coated dishes and infected with P. gingivalis, and cellular migration and proliferation were evaluated with an in vitro assay of wound healing assay in the presence or absence of the polyphenols. RESULTS: Each polyphenol significantly enhanced the viability of PDL cells infected with P. gingivalis, whereas only EGCg demonstrated cytotoxicity. Further, all polyphenols significantly inhibited Rgp activity, with AP, ACT, and HBP more effective toward Kgp. P. gingivalis markedly diminished the migration and proliferation of EMD-stimulated PDL cells, whereas the addition of AP, ACT, HBP, and HMW-HBP significantly protected the cells from bacterial cytotoxicity. In contrast, EGCg and LMW-HBP did not show protective effects. CONCLUSION: These results suggest that AP, ACT, AP, HBP, and HMW-HBP protect EMD-stimulated PDL cells from P. gingivalis and may be therapeutically useful supplements for EMD therapy.     

Mixed infection of Porphyromonas gingivalis and Bacteroides forsythus in a murine abscess model: involvement of gingipains in a synergistic effect

Several microorganisms including Porphyromonas gingivalis and Bacteroides forsythus have been implicated to be etiologically important agents of periodontal disease. In this study, we determined the ability of combinations of periodontopathogenic microorganisms to cause tissue destruction in a murine abscess model. Although all bacterial combinations used in this study produced larger abscesses than did monoinfection of each bacterium, the combination of P. gingivalis and B.forsythus showed a synergistic effect on abscess formation. Since these two bacteria have been frequently found together in lesions of periodontitis, these results suggest the significance of their co-infection in the progression of periodontitis. P. gingivalis produces extracellular and cell-associated cysteine proteinases (gingipains) which appear to be involved in its virulence. The rgpA rgpB double and kgp mutants induced significantly smaller abscesses than the wild type. Moreover, the rgpA rgpB kgp triple (gingipain-null) mutant hardly showed lesion formation at all with the experimental conditions used in this study, indicating that these genes encoding gingipains are important for virulence of P. gingivalis. Mixed infection of these P. gingivalis mutants with B. forsythus showed an additive effect on abscess formation, indicating that the gingipains of P. gingivalis may play an important role in the pathological synergism between P. gingivalis and B. forsythus.     

Antibody responses to Porphyromonas gingivalis infection in a murine abscess model--involvement of gingipains and responses to re-infection

BACKGROUND: Porphyromonas gingivalis is one of the most important periodontopathogens. It produces cysteine proteinases named gingipains. We previously examined the effect of gingipains on abscess formation in a murine model. The rgpA rgpB double and kgp mutants induced smaller abscesses than the wild type. Moreover, the rgpA rgpB kgp triple (gingipain-null) mutant hardly showed lesion formation at all under the experimental conditions used, indicating that genes encoding gingipains are important for P. gingivalis virulence. OBJECTIVES: Here, we further report the humoral immune responses induced by P. gingivalis strains. METHODS: After the lesions were apparently cured, sera were collected from the mice and immunoglobulin G (IgG) responses against the whole cell antigens of wild-type P. gingivalis were measured. RESULTS: Wild-type strain was found to induce a strong antibody reaction. On the other hand, the rgpA rgpB kgp triple and kgp mutants induced significantly lower antibody responses compared to the wild type. Western blotting analysis confirmed the differences in antibody production. Next, these mice were re-infected with wild-type strain. Mice that were first infected with wild-type strain showed significantly smaller lesion formation than control mice that were first infected with medium only. On the other hand, mice that were first infected with mutant strains devoid of gingipain activities did not show resistance to re-infection and immunoglobulins directed against gingipains may be protective. CONCLUSIONS: These results suggest that gingipains play an important role in abscess formation in mice, and humoral immune responses seem to be partly responsible for the resistance to re-infection by P. gingivalis.     

大黄素-8-O-β-D-吡喃葡萄糖苷对牙龈卟啉单胞菌唾液酸酶活性及其毒力基因表达影响研究

目的    探讨大黄素-8-O-β-D-吡喃葡萄糖苷对牙龈卟啉单胞菌（P. gingivalis）唾液酸酶活性及其毒力基因表达的影响。方法    使用不同质量浓度的大黄素-8-O-β-D-吡喃葡萄糖苷（0.2、0.5、2、5、10 mg/mL）处理P. gingivalis W83（实验组）,用未加药物的P. gingivalis W83作对照（对照组）,采用荧光法检测大黄素-8-O-β-D-吡喃葡萄糖苷对P. gingivalis唾液酸酶活性的作用。5 mg/mL大黄素-8-O-β-D-吡喃葡萄糖苷作用于P. gingivalis W83,Real-time PCR法检测毒力基因fimA、fimR、fimS、kgp、rgpA和rgpB的表达情况。结果    大黄素-8-O-β-D-吡喃葡萄糖苷对P. gingivalis唾液酸酶活性产生了抑制作用,当其质量浓度为0.2、0.5、2、5、10 mg/mL时,对唾液酸酶活性的抑制率分别为11.4%、32.23%、40.21%、73.54%、84.31%。与对照组比较,实验组（5 mg/mL大黄素-8-O-β-D-吡喃葡萄糖苷处理）的fimA、fimR、fimS、kgp、rgpA和rgpB基因表达均下降,差异均有统计学意义（均P < 0.05）。结论    大黄素-8-O-β-D-吡喃葡萄糖苷可有效抑制P. gingivalis唾液酸酶活性,其抑制作用会降低细菌毒力基因表达,有望成为预防及治疗牙周炎的新型药物。     

Susceptibility of type 2 diabetic mice to low-virulence bacterial infection: induction of abscess formation by gingipain-deficient Porphyromonas gingivalis

BACKGROUND AND OBJECTIVE: Type 2 diabetes mellitus is considered an important risk factor of adult periodontitis. However, recent studies have revealed that the subgingival microbial flora of diabetes mellitus patients does not differ from that of healthy individuals. In this study, we examined the response of type 2 diabetes mellitus hosts to low-virulence bacteria in a murine abscess model. MATERIAL AND METHODS: Porphyromonas gingivalis ATCC 33277 or KDP128 (rgpA rgpB kgp) were injected into two mouse strains - C57BL/6J and its derivative, KK/A(Y), which becomes diabetic spontaneously. RESULTS: Lesions of KK/A(Y) mice injected with either low-virulence P. gingivalis KDP128 or wild-type 33277 were significantly larger than those of C57BL/6J mice injected with the same strains. Histologically, more neutrophils and macrophages migrated to the lesions in the KK/A(Y) mice injected with P. gingivalis 33277 and KDP128 compared with those of C57BL/6J mice injected with the same respective strains. CONCLUSION: These results suggest that severe inflammation is observed in response to low-virulence bacteria in addition to the highly virulent bacteria in type 2 diabetes mellitus hosts.     

牙龈卟啉菌蛋白酶基因rgpA与rgpB蛋白酶区的克隆和表达

目的克隆牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)rgpA、rgpB蛋白酶区,构建rgpA、rgpB蛋白酶区原核表达系统。方法采用聚合酶链式反应(PCR)从PgATCC33277菌株中扩增rgpA、rgpB蛋白酶区,T-A克隆后测定核苷酸序列,构建pET42a的rgpA/rgpB蛋白酶区表达载体,在E.coli BL21DE3宿主菌中用不同浓度的IPTG诱导其表达。结果所克隆的PgATCC33277菌株rgpA、rgpB基因蛋白酶区的核苷酸序列与报道的相应核苷酸序列同源性分别为98.9%、99.0%,氨基酸序列同源性分别高达99.2%、99.1%。pET42a-rgpA/rgpB-E.coli BL21DE3系统的蛋白表达量为细菌总蛋白的60%左右。结论本研究成功地构建PgrgpA、rgpB蛋白酶区高效表达系统,为测定RgpA、RgpB免疫原性及作用提供前提。     

kgp,rgpA, and rgpB DNA Vaccines Induce Antibody Responses in Experimental Peri‐Implantitis

Background: Peri‐implantitis is the key factor for implant failure. This study aims to evaluate kgp, rgpA, and rgpB DNA vaccines to induce an immune response and prevent peri‐implantitis. Methods: The kgp, rgpA, and rgpB genes were amplified by polymerase chain reaction (PCR) from Porphyromonas gingivalis (Pg) ATCC 33277 and cloned into the pVAX1 vector. Titanium implants were placed into the mandibular bone of dogs. Three months later, the animals were divided into four groups, immunized with pVAX1‐kgp, pVAX1‐rgpA, pVAX1‐rgpB, or pVAX1. Cotton ligatures infiltrated with Pg were tied around the neck of the implants. Immunoglobulin (Ig)G and IgA antibodies were detected by enzyme‐linked immunosorbent assay before and after immunization. Results: The kgp, rgpA, and rgpB genes were successfully cloned into the pVAX1 plasmid. Animals immunized with pVAX1‐kgp and pVAX1‐rgpA showed higher titers of IgG and IgA antibodies compared to those before immunization (P <0.05) and compared to those that were immunized with pVAX1 and pVAX1‐rgpB, whereas there were no significant differences in the animals treated with pVAX1 and pVAX1‐rgpB. Furthermore, among these, the kgp DNA vaccine was more effective. The bone losses of the groups with pVAX1‐kgp and pVAX1‐rgpA were significantly attenuated. Conclusion: pVAX1‐kgp and pVAX1‐rgpA DNA vaccines enhanced immunity responses and significantly retarded bone loss in experimental peri‐implantitis animal models, whereas pVAX1‐rgpB was ineffective.     

大鼠实验性牙周炎中牙龈卟啉单胞菌rgpB和kgp变化的研究

目的:通过建立大鼠实验性牙周炎模型,检测不同时段大鼠上颌第一磨牙龈下人工定植的牙龈卟啉单胞菌、牙龈素rgpB和kgp相对含量的改变,动态观察牙周炎发展不同阶段牙龈卟啉单胞菌致病基因的变化。方法:选择6周的成年雄性大鼠13只,应用钢丝结扎法和细菌种植法建立大鼠实验性牙周炎模型。分别在4周和8周取上颌第一磨牙龈下菌斑,提取细菌DNA,应用PCR方法进行牙龈卟啉单胞菌、牙龈素基因rgpB和kgp特异引物扩增,应用SPSS13.0统计软件,分析大鼠牙周炎不同时段rgpB和kgp的相对变化。结果:大鼠实验性牙周炎模型,龈下菌斑中牙龈卟啉单胞菌的相对含量实验组明显高于对照组,8周组高于4周组;实验组牙龈卟啉单胞菌牙龈素rgpB和kgp明显高于对照组,4周组和8周组rgpB和kgp相对含量无显著性差异。结论:rgpB基因和kgp基因与牙周炎的致病性有关而与牙周炎的严重程度可能无直接相关。     

Sequence variations in rgpA and rgpB of Porphyromonas gingivalis in periodontitis

Objective:  The aim of the present study was to determine sequence variations in the active centre of the Arg-X-specific protease encoding genes rgpA and rgpB of clinical Porphyromonas gingivalis isolates and to analyse their prevalence in periodontitis patients before and 3 months after mechanical periodontal therapy.
Background:  Genetic diversity at nucleotides 281, 283, 286 and 331 has been shown to result in amino acid substitutions in the catalytic domain of RgpA and RgpB that affect the substrate specificity and thus may influence the efficacy of Arg-X-protease specific inhibitors.
Methods:  Sequence analysis of rgpA and rgpB genes in clinical P. gingivalis strains isolated from subgingival plaque samples of 82 periodontitis patients before and 3 months after mechanical supra- and subgingival debridement was performed.
Results:  No specific variation within the rgpA sequence was observed. However, the rgpB sequence in the region of the active centre showed five different rgpB genotypes, which were named NYPN, NSSN, NSSK, NYPK and DYPN according to the derived amino acid substitution. Porphyromonas gingivalis genotype NYPN was detected in 27 patients (32.9%) before and in 8 patients (9.8%) after therapy, NSSN in 26 (31.7%) and 10 (12.2%), NSSK in 22 (26.8%) and 2 (2.4%), NYPK in 5 (6.2%) and 1 (1.2%), and DYPN in 1 patient (1.2%) and 0 patients (0%), respectively. Only one patient (1.2%) harboured two P. gingivalis rgpB genotypes (NSSK/NYPN) before treatment; these were no longer detected after therapy.
Conclusion:  The results indicate that five rgpB genotypes are maintained in natural populations of P. gingivalis. These data may be of importance with regard to the development of specific rgpB inhibitors.     

The role of gingipains in the pathogenesis of periodontal disease

Gingipains are trypsin-like cysteine proteinases produced by Porphyromonas gingivalis, a major causative bacterium of adult periodontitis. HRgpA (95 kDa) and RgpB (50 kDa), products of 2 distinct but related genes, rgpA and rgpB, respectively, are specific for Arg-Xaa peptide bonds. Kgp, a product of the kgp gene, is specific for Lys-Xaa bonds. HRgpA and Kgp are non-covalent complexes containing separate catalytic and adhesion/ hemagglutinin domains, while RgpB has only a catalytic domain with a primary structure essentially identical to that of the catalytic subunit of HRgp. HRgpA and RgpB induce vascular permeability enhancement through activation of the kallikrein/kinin pathway and activate the blood coagulation system, which, respectively, are potentially associated with gingival crevicular fluid production and progression of inflammation leading to alveolar bone loss in the periodontitis site. Kgp is the most potent fibrinogen/fibrin degrading enzyme of the 3 gingipains in human plasma and is involved in the bleeding tendency at the diseased gingiva. HRgpA activates coagulation factors and degrades fibrinogen/fibrin more efficiently than RgpB due to the adhesion/hemagglutinin domains, which have affinity for phospholipids and fibrinogen. Gingipains degrade macrophage CD14, thus inhibiting activation of the leukocytes through the lipopolysaccharide (LPS) receptor, and thereby facilitating sustained colonization of P. gingivalis. Gingipains play a role in bacterial housekeeping and infection, including amino acid uptake from host proteins and fimbriae maturation. Based on the important activities of gingipains in the bacterial infection and the pathogenesis of periodontitis, the bacterial proteinases can be targets for periodontal disease therapy. Immunization with RgpB, HRgpA, or a portion of HRgpA catalytic domain attenuated P. gingivalis induced disorders in mice. In addition, a trypsin-like proteinase inhibitor retarded P. gingivalis growth specifically. Gingipains are potent virulence factors of P. gingivalis, and are likely to be associated with the development of periodontitis. It is, therefore, suggested that gingipain inhibition by vaccination and gingipain-specific inhibitors is a useful therapy for adult periodontitis caused by P. gingivalis infection.     

Effects of enamel matrix derivative and transforming growth factor-beta1 on human periodontal ligament fibroblasts

AIM: The objective of this study was to evaluate the effects of enamel matrix derivative (EMD), transforming growth factor-beta1 (TGF-beta1), and a combination of both factors (EMD+TGF-beta1) on periodontal ligament (PDL) fibroblasts. MATERIAL AND METHODS: Human PDL fibroblasts were obtained from three adult patients with a clinically healthy periodontium, using the explant technique. The effects of EMD, TGF-beta1, or a combination of both were analysed on PDL cell proliferation, adhesion, wound healing, and total protein synthesis, and on alkaline phosphatase (ALP) activity and bone-like nodule formation. RESULTS: Treatment with EMD for 4, 7, and 10 days increased cell proliferation significantly compared with the negative control (p<0.05). At day 10, EMD and EMD+TGF-beta1 showed a higher cell proliferation compared with TGF-beta1 (p<0.01). Cell adhesion was significantly up-regulated by TGF-beta1 compared with EMD and EMD+TGF-beta1 (p<0.01). EMD enhanced in vitro wound healing of PDL cells compared with the other treatments. Total protein synthesis was significantly increased in PDL cells cultured with EMD compared with PDL cells treated with TGF-beta1 or EMD+TGF-beta1 (p<0.05). EMD induced ALP activity in PDL fibroblasts, which was associated with an increase of bone-like nodules. CONCLUSION: These findings support the hypothesis that EMD and TGF-beta1 may play an important role in periodontal regeneration. EMD induced PDL fibroblast proliferation and migration, total protein synthesis, ALP activity, and mineralization, while TGF-beta1 increased cellular adhesion. However, the combination of both factors did not positively alter PDL fibroblast behaviour.     

Participation of endogenous IGF-I and TGF-beta 1 with enamel matrix derivative-stimulated cell growth in human periodontal ligament cells

Previous studies have provided the biological basis for the therapeutic use of enamel matrix derivative (EMD) at sites of periodontal regeneration. A purpose of this study is to determine effects of EMD on cell growth, osteoblastic differentiation and insulin-like growth factor-I (IGF-I) and transforming growth factor-beta 1 (TGF-beta 1) production in human periodontal ligament cells (HPLC). We also examined participation of endogenous IGF-I and TGF-beta 1 with EMD-stimulated cell growth in these cells. HPLCs used in this study were treated with EMD alone or in combination with antihuman IGF-I antibody (anti-hIGF-I) or anti-hTGF-beta 1, recombinant human bone morphogenetic protein-2 (rhBMP-2), 1,25-dihydroxyvitamin D3[1,25(OH)2D3], rhTGF-beta 1 or rhIGF-I. After each treatment, cell growth, the production of IGF-I and TGF-beta 1 and the expression of osteoblastic phenotypes were evaluated. EMD stimulated cell growth in dose-dependent and time-dependent manners. EMD was also stimulated to express IGF-I and TGF-beta 1 at protein and mRNA levels. The EMD-stimulated cell growth was partially suppressed by cotreatment with anti-hIGF-I or anti-hTGF-beta 1, and cell growth was also stimulated by treatment with rhIGF-I or rhTGF-beta 1. rhBMP-2 stimulated alkaline phosphatase (ALPase) activity and ALPase mRNA expression, and 1,25(OH)2D3 stimulated ALPase and osteocalcin mRNA expression. However, EMD showed no effect on the osteoblastic phenotypes expression. These results demonstrated that EMD has no appreciable effect on osteoblastic differentiation, however it stimulates cell growth and IGF-I and TGF-beta 1 production in HPLC, and that these endogenous growth factors partially relate to the EMD-stimulated cell growth in HPLC.     

Cytokine production induced by a 67-kDa fimbrial protein from Porphyromonas gingivalis

Fimbriae have been reported to play an important role in the adherence of Porphyromonas gingivalis to oral surfaces and possibly in triggering host responses. P. gingivalis ATCC 33277 has two distinctly different fimbriae expressed on the cell surface. The 67-kDa fimbriae differ in size and antigenicity from the earlier reported FimA, a major 41-kDa fimbrial component of P. gingivalis. Expression of the 67-kDa fimbriae on the cell surface of a fimA mutant was investigated by electron microscopy. The 67-kDa fimbrial protein was purified from the fimA mutant by sonication, precipitation, and chromatography on a DEAE Sepharose CL-6B column. The N-terminal amino acid sequence of the 67-kDa fimbrillin was distinct from that of the 41-kDa fimbrillin. Moreover, we have found that the 67-kDa fimbrial protein from P. gingivalis ATCC 33277 induced IL-1alpha, IL-beta, IL-6 and TNFalpha cytokine expression in mouse peritoneal macrophages. These results suggest that P. gingivalis 67-kDa fimbriae may play a part in the inflammatory response during the development of periodontal diseases.     

Unaltered expression of the major protease genes in a non-virulent recA-defective mutant of Porphyromonas gingivalis W83

Porphyromonas gingivalis FLL32, a recA mutant, was isolated during construction of a recA defective mutant of P. gingivalis W83 by allelic exchange mutagenesis. In contrast to W83 and FLL33, the typical recA- mutant previously reported, FLL32 was non-pigmented, lacked beta-hemolytic activity on blood agar and produced significantly less proteolytic activity. The proteolytic activity in FLL32 was mostly soluble. Expression of the rgpA, rgpB and kgp protease genes was unaltered in FLL32 when compared to FLL33 and the wild-type strain. FLL32 exhibited reduced virulence in a murine model and partially protected the animals immunized with that strain against a subsequent lethal challenge by the wild-type strain. These results indicate that the reduced level of proteolytic activity in FLL32 may be due to a defect in the processing of the proteases. Further, immunization with a non-virulent recA defective mutant of P. gingivalis can partially protect against a lethal wild-type challenge. The results from this study suggest that the recA locus may be involved in expression and regulation of proteolytic activity in P. gingivalis.     

Characterization of biologically active cell surface components of a periodontal pathogen. The roles of major and minor fimbriae of Porphyromonas gingivalis

BACKGROUND: We previously reported that the presence of 2 different types of fimbriae expressed on the cell surface of Porphyromonas gingivalis ATCC 33277. The initial event in most infectious diseases involves adhesion of pathogens to host tissues and subsequent invasion by the pathogens. To define the role of fimbriae in Porphyromonas gingivalis adherence to and invasion of epithelial cells, we have constructed fimbrial mutants. The involvement of P. gingivalis fimbriae in the invasion process and alveolar bone resorption in rats was examined. METHODS: Inactivated mutants of 41-K fimbrillin gene (fimA) and/or the 67-K fimbrillin gene (mfa1) were constructed by a homologous recombination technique and compared among fimA mutant (MPG1), mfa1 mutant (MPG67), and double knockout mutant (MPG4167). Adherence and invasion of P. gingivalis was assessed in human oral epithelial KB cells. We used a rat model to examine the role of each type of fimbriae in alveolar bone loss by oral infection. RESULTS: The adherence and invasion levels of the mutants were lower than the wild-type strain. The bone loss of rats infected with the MPG1 was higher than that of those infected with MPG67. Moreover, the bone loss of rats infected with the double knockout mutant was significantly decreased compared to that of rats infected with the wild-type strain. CONCLUSIONS: Data from this study suggest that not only the 41-K fimbrial protein, but also the 67-K fimbrial protein, play important roles in the pathogenesis of periodontal disease.     

Ⅰ、ⅣfimA型牙龈卟啉单胞菌对内皮细胞与平滑肌细胞共培养体系产生内皮素-1及一氧化氮的影响

［摘要］ 目的 研究不同fimA基因型牙龈卟啉单胞菌（Porphyromonas gingivalis,P.gingivalis）刺激人脐静脉血管内皮细胞（human umbilical vein endothelial cells,HUVECs）及人脐动脉血管平滑肌细胞（human umbilical artery smooth muscle cells,HUASMCs）共培养体系产生内皮素-1（endothelin-1,ET-1）和一氧化氮（NO）的水平。方法 用ⅠfimA型P.gingivalis（ATCC 33277）和ⅣfimA型P.gingivalis（W83）分别刺激HUVECs-HUASMCs共培养体系,于2、8、24、48 h时收集细胞培养上清液,酶联免疫反应检测ET-1的含量,硝酸还原酶法测定NO的含量。各组均设阴性对照组（纯培养基）及阳性对照组（1 μg/mL E.coli-LPS）。结果 HUVECs-HUASMCs共培养体系在Ⅰ、ⅣfimA型P.gingivalis刺激作用下产生ET-1和NO的量以及ET-1/NO水平,与阴性及阳性对照组比较存在差异。ⅠfimA型P.gingivalis刺激细胞共培养模型分泌ET-1和NO情况的总趋势与阴性对照组相似、而ⅣfimA型P.gingivalis的刺激作用总趋势则与阳性对照组相似,ⅣfimA型P.gingivalis较ⅠfimA型P.gingivalis刺激共培养细胞可分泌更多的ET-1、而NO量减少,ⅣfimA型P.gingivalis感染48 h的细胞共培养模型表现出明显的ET-1/NO的失衡。结论 不同fimA型P.gingivalis刺激共培养模型后产生ET-1及NO的情况及ET-1/NO的水平有明显差异,可能与其本身毒力相关,ⅣfimA型P.gingivalis比ⅠfimA型P.gingivalis更易引起内皮功能紊乱。     

Autocrine growth factors in human periodontal ligament cells cultured on enamel matrix derivative

OBJECTIVE: Enamel extracellular matrix proteins in the form of the enamel matrix derivative EMDOGAIN (EMD) have been successfully employed to mimic natural cementogenesis to restore fully functional periodontal ligament, cementum and alveolar bone in patients with severe periodontitis. When applied to denuded root surfaces EMD forms a matrix that locally facilitates regenerative responses in the adjacent periodontal tissues. The cellular mechanism(s), e.g. autocrine growth factors, extracellular matrix synthesis and cell growth, underlying PDL regeneration with EMD is however poorly investigated. MATERIAL AND METHODS: Human periodontal ligament (PDL) cells were cultured on EMD and monitored for cellular attachment rate, proliferation, DNA replication and metabolism. Furthermore, intracellular cyclic-AMP levels and autocrine production of selected growth factors were monitored by immunological assays. Controls included PDL and epithelial cells in parallel cultures. RESULTS: PDL cell attachment rate, growth and metabolism were all significantly increased when EMD was present in cultures. Also, cells exposed to EMD showed increased intracellular cAMP signalling and autocrine production of TGF-beta1, IL-6 and PDGF AB when compared to controls. Epithelial cells increased cAMP and PDGF AB secretion when EMD was present, but proliferation and growth were inhibited. CONCLUSION: Cultured PDL cells exposed to EMD increase attachment rate, growth rate and metabolism, and subsequently release several growth factors into the medium. The cellular interaction with EMD generates an intracellular cAMP signal, after which cells secrete TGF-beta1, IL-6 and PDGF AB. Epithelial cell growth however, is inhibited by the same signal. This suggest that EMD favours mesenchymal cell growth over epithelium, and that autocrine growth factors released by PDL cells exposed to EMD contribute to periodontal healing and regeneration in a process mimicking natural root development.     

Molecular genetics and nomenclature of proteases of Porphyromonas gingivalis

The strategies used by bacterial pathogens to establish and maintain themselves in the host represent one of the fundamental aspects of microbial pathogenesis. Characterization of these strategies and the underlying molecular machinery offers new opportunities both to our understanding of how organisms cause disease in susceptible individuals and to the development of novel therapeutic measures designed to undermine or interfere with these determinants of successful survival. With respect to the microbial aetiology of the periodontal diseases, a growing body of evidence suggests that the proteolytic enzymes of Porphyromonas gingivalis represent key survival and, by extrapolation, virulence determinants of this periodontal bacterium. This in turn has led to international efforts to characterize these enzymes at the gene and protein level. Approximately 20 protease genes of P. gingivalis with different names and accession numbers have been deposited in the gene databases and a correspondingly heterogeneous nomenclature system is employed for the products of these genes in the literature. However, it is evident, through comparison of these gene sequences and through gene inactivation studies, that the genetic structure of the proteases of this organism, particularly those with specificity for arginyl and lysyl peptide bonds, is less complicated than originally thought. The major extracellular and surface associated arginine specific protease activity is encoded by 2 genes which we recommend be designated rgpA and rgpB (arg-gingipains A & B). Similarly we recommend that the gene encoding the major lysine specific protease activity is designated kgp (lys-gingipain). These three genes, which account for all the extracellular/surface arginine and lysine protease activity in P. gingivalis, belong to a family of sequence-related proteases and haemagglutinins.     

牙龈卟啉单胞菌对牙龈上皮细胞焦点黏附成分paxillin和FAK的作用

目的:研究牙龈卟啉单胞菌(P.gingivalis)对牙周组织的破坏机制,探讨菌毛的致病作用.方法:应用Western blot法检测P.gingivalis ATCC 33277及其fimA缺陷突变株对Hela细胞及永生化人牙龈上皮细胞 (immortalized human gingival epithelial cells, IHGE细胞) 的焦点黏附成分——焦点黏附蛋白paxillin和焦点黏附激酶 (focal adhesion kinase,FAK) 蛋白表达的影响.结果:P.gingivalis ATCC 33277野生株和fimA缺陷突变株能够使HeLa细胞和IHGE细胞的paxillin和FAK发生降解,fimA缺陷突变株对paxillin和FAK的降解能力较野生株显著减弱.在IHGE细胞中,paxillin和FAK的降解呈时间依赖性和MOI依赖性.结论:菌毛介导的P.gingivalis的黏附和侵入可能对焦点黏附成分的降解起促进作用,IHGE细胞较Hela细胞更适用于牙周致病菌的研究.