Showing papers in "Journal of Periodontal Research in 1997"

Porphyromonas gingivalis proteinases as virulence factors in the development of periodontitis.

Abstract: Porphyromonas gingivalis contains exceedingly high concentrations of cysteine proteinases with trypsin-like activity which have been implicated as virulence factors in adult-onset periodontitis. These enzymes, referred to as gingipains, cleave protein and peptide substrates after arginine (gingipain R) and lysine residues (gingipain K), and it has been found that neither is easily inhibited by host proteinase inhibitors. Examination of the properties of each proteinase clearly indicates a role(s) for both in the dysregulation of a number of normally tightly controlled pathways. The effects of such uncontrolled proteolysis are the development of edema (kallikrein/kinin pathway activation by gingipain R), neutrophil infiltration (complement pathway activation by gingipain R), and bleeding (degradation of fibrinogen by gingipain K). Since three of the major hallmarks of periodontitis involve increased crevicular flow, neutrophil accumulation at infected sites and bleeding on probing, it seems likely that both P. gingivalis-derived proteinases are important virulence factors in the development of periodontal disease.

Gingival crevicular interleukin-1 and interleukin-1 receptor antagonist levels in periodontally healthy and diseased sites.

Abstract: Interleukin-1 (IL-1) molecules, IL-1 alpha and IL-1 beta are cytokines involved in the acute-phase response against infection and in the pathogenesis of periodontal destruction. Administration of exogenous IL-1 receptor antagonist (IL-1ra) is effective in reducing the inflammatory reactions mediated by IL-1. However, the relationship between these three naturally occurring IL-1 molecules and periodontal diseases has been poorly characterized. We investigated the correlation of gingival crevicular IL-1 molecules and the clinical status of patients with different severities of periodontitis. IL-1 alpha, IL-1 beta, IL-1ra and the total IL-1/IL-1ra ratio (IL-1 activity index; IL-1AI) were measured in 75 gingival crevicular fluid (GCF) samples from non-inflamed gingiva sites in 2 healthy subjects and diseased sites in 7 patients with several types of periodontitis. IL-1 alpha, IL-1 beta and IL-1ra were measured by specific non-cross-reactive enzyme linked immunosorbent assay. The probing depth, gingival index and alveolar bone loss of each site was recorded at the time of GCF sampling. The total amount of IL-1 alpha, IL-1 beta and the IL-1AI, but not total IL-1ra, were found to be correlated with alveolar bone loss score. Three IL-1 molecules were also measured in the gingival tissue of patients with periodontitis. A similar progressive decrease of the IL-1AI was detected in gingival tissue with periodontitis. These results suggest that the amounts of both crevicular IL-1 and IL-1AI are closely associated with periodontal disease severity.

Secretion of IL-1 beta, TNF-alpha, IL-8 and IL-1ra by human polymorphonuclear leukocytes in response to lipopolysaccharides from periodontopathic bacteria.

Abstract: Polymorphonuclear leukocytes (PMN) are the first cells that migrate into periodontal tissues and gingival crevices in response to invading pathogens It was recently demonstrated that PMN have the ability to synthesize and release cytokines following appropriate stimulation, while it is not clear whether these capacities are directly related to periodontal destructive processes We therefore investigated the amounts of the cytokines interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), IL-8 and IL-1 receptor antagonist (IL-1ra) secreted by PMN from healthy donors following stimulation with lipopolysaccharide (LPS) from 4 periodontopathic bacteria, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Capnocytophaga ochracea and Fusobacterium nucleatum, and the non-oral bacterium Escherichia coli A actinomycetemcomitans, F nucleatum and E coli LPS stimulated the release of significantly greater amounts of IL-1 beta, TNF-alpha and IL-8 than the control unstimulated PMN (p < 001) The levels of IL-1 beta, TNF-alpha and IL-8 released from cells stimulated with P gingivalis or C ochracea LPS were significantly lower than those of cells stimulated with A actinomycetemcomitans or E coli LPS (p < 005) On the other hand, substantially greater amounts of IL-1ra were released from PMN stimulated with each LPS and from control unstimulated PMN during the first 6 h, and then significantly greater amounts of IL-1ra were secreted by PMN stimulated with A actinomycetemcomitans and Ecoli LPS during the following 12 h (p < 001) The inhibitory effects of IL-1ra on the biological activity of IL-1 in the supernatants of PMN were examined by the thymocyte comitogen proliferation assay The supernatants of PMN stimulated with each LPS showed less biological IL-1 activity as compared with the same doses of recombinant human IL-1 beta detected by enzyme-linked immunosorbent assay Furthermore, no activity was detected in the supernatants of PMN stimulated with P gingivalis or C ochracea LPS These findings demonstrated that LPS from periodontopathic bacteria were capable of stimulating PMN to release not only pro-inflammatory cytokines but also their inhibitors such as IL-1ra Different secretion levels of these cytokines and their biological activities induced by the various LPS might be important in the onset and progression of periodontal diseases

Effects of basic fibroblast growth factor on human periodontal ligament cells.

Abstract: In order to clarify the regulatory mechanisms of periodontal regeneration by basic fibroblast growth factor (bFGF), effects of bFGF on proliferation, alkaline phosphatase activity, calcified nodule formation and extracellular matrix synthesis of human periodontal ligament (PDL) cells were examined in this study. bFGF enhanced the proliferative responses of PDL cells in a dose-dependent manner. The maximum mitogenic effect of bFGF on PDL cells was observed at the concentration of 10 ng/ml. In contrast, bFGF inhibited the induction of alkaline phosphatase activity and the mineralized nodule formation by PDL cells. Moreover, employing the reverse transcription-polymerase chain reaction (RT-PCR) technique, we observed that the levels of laminin mRNA of human PDL cells was specifically upregulated by bFGF stimulation, but that of type I collagen mRNA was downregulated. On the other hand, the expression of type III collagen and fibronectin mRNA were not altered even when the cells were activated by bFGF. These results suggest that suppressing cytodifferentiation of PDL cells into mineralized tissue forming cells, bFGF may play a role in wound healing by inducing growth of immature PDL cells and that in turn accelerates periodontal regeneration.

Fibroblast heterogeneity in the periodontium and other tissues

Abstract: Fibroblasts are the major resident cells which inhabit the periodontal tissues. As such, they are crucial for maintaining the connective tissues which support and anchor the tooth. Little is known of their origins, synthesis of regulatory cytokines and growth factors in health and disease, and importance in soft tissue regeneration. An emerging concept is that fibroblasts are not homogeneous, but instead consist of subsets of cells which can regulate bone marrow-derived cells such as T lymphocytes. Fibroblasts can be separated into subsets on the basis of morphology, size and expression of intermediate filaments as well as collagen subtypes. Differential surface marker expression has also been a key feature to distinguish fibroblast subsets from many tissues. Antigens such as Thy-1, class II MHC, and C1q are among those surface proteins which have been employed successfully to separate fibroblasts. Importantly, these fibroblast subsets are not only antigenically diverse, but also possess distinct functions. Thy 1+ pulmonary fibroblasts can display class II MHC antigens, synthesize IL-1 and can activate T lymphocytes, whereas the Thy 1+ subset is devoid of these functions. Recently, fibroblasts from the human orbit have also been shown to be separable on the basis of Thy 1 surface marker expression. Fibroblasts derived from human gingiva and periodontal ligament also appear to be composed of subsets with a heritable pattern of surface markers which will permit their separation into functional subpopulations. This paper will review findings of fibroblast heterogeneity in periodontal and other tissues. Evidence will be presented for the use of surface markers to delineate functional subsets. The ability to discriminate subsets of fibroblasts will aid in studies of periodontal disease pathogenesis and wound healing.

Underlying mechanisms at the bone‐surface interface during regeneration

Abstract: The goal of regenerative therapy around teeth and implants is to create a suitable environment in which the natural biological potential for functional regeneration of periodontal ligament and/or bone can be maximized. In order for the regenerative process to be successful, the following factors must be addressed: prevention of acute inflammation from bacteria, mechanical stability of the wound, creation and maintenance of blood clot-filled space, isolation of the regenerative space from undesirable competing tissue types, and the creation of a desirable surface chemistry, energy, roughness and microtopography that can directly influence cellular response, ultimately affecting the rate and quality of new tissue formation and, therefore, the regeneration process. This paper will review how surface characteristics (chemistry and roughness) can affect cell response and local factor production. To evaluate the effect of surface chemistry on cell proliferation and differentiation costochondral chondrocytes were grown on standard tissue culture plastic dishes sputter-coated with different materials. The results indicate that surface materials can elicit differential responses in cell metabolism and phenotypic expression in vitro. In a second study, the effect of varying titanium surface roughnesses on osteoblast-like cell behavior was examined. Surface roughness was found to alter osteoblast proliferation, differentiation and matrix production in vitro. In addition, production of PGE2 and TGF beta by these cells was also shown to increase with increasing surface roughness, indicating that substrate surface roughness also affects cytokine and growth factor production. The role of surface roughness in determining cellular response was further explored by comparing the response of osteoblasts grown on new and previously used surfaces. The results of these latter studies showed that cell proliferation, expression of differentiation markers and overall matrix production are not altered when cells are grown on used vs. virgin surfaces. This suggests the possibility that implants may be re-used, especially in the same patient, if they are appropriately treated. In this context, it should also be noted that rougher titanium surfaces may require more extensive cleaning procedures. From a global perspective, these studies provide some insight into how bone regeneration can be optimized in the presence of an implant or tooth root residing at the site of a bony defect. Since the new bone being produced, during regeneration, grows from a distal area toward the implant or tooth root surface, it is hypothesized that the osteoblasts growing on the surface of the implant may produce local factors that can affect the bone healing process distally. In short, it appears that the surface characteristics of an implant, particularly roughness, may direct tissue healing and, therefore, subsequent implant success in sites of regeneration by modulating osteoblast phenotypic expression.

Molecular and cellular mechanisms for periodontal diseases: role of Th1 and Th2 type cytokines in induction of mucosal inflammation

Abstract: An accumulation of elevated numbers of macrophages (M phi) and Ig producing cells is associated with localized and chronically inflamed gingiva of patients with adult periodontitis. When gingival lymphocytes were isolated from inflamed tissues and examined by flow cytometry, approximately 20-30% of lymphocytes were CD4+ T cells. For the analysis of Th1 and Th2 cytokine expression by these CD4+ T cells, RNA was extracted and reverse transcriptase polymerase chain reaction (RT-PCR) was performed by using specific 5' and 3' primers for IFN-gamma and IL-2 (Th1), IL-4, IL-5, IL-6, IL-10 and IL-13, (Th2) and beta-actin (housekeeping gene). Two distinct cytokine profiles were noted based on the expression of selected Th1 and Th2 cytokines. Thus, one pattern was represented by the expression of mRNA for IFN-gamma, IL-6, IL-10 and IL-13, while the other case consisted of mRNA for IFN-gamma, IL-6, and IL-13. Except for a few cases, messages for IL-2, IL-4 and IL-5 were not detected by cytokine-specific RT-PCR. The predominant expression of Th2 cytokines (e.g. IL-6, IL-10 and IL-13) may contribute to the induction of high B cell responses in local disease sites. On the other hand, lack of IL-4 may be responsible for the accumulation of M phi in diseased periodontium. We also investigated whether a relationship exists between IL-4 receptor (IL-4R) expression and M phi persistence in the absence of exogenous IL-4. Gingival M phi, when compared with monocytes (MN)/M phi from peripheral blood mononuclear cells (PBMC), expressed high levels of IL-4R mRNA. When gingival M phi were incubated with recombinant IL-4 (rIL-4), the cell viability was dramatically reduced by apoptosis. These findings clearly show that the lack of IL-4 may contribute to the persistent occurrence of M phi at the disease site and addition of exogenous rIL-4 to gingival M phi cultures leads to cell death by apoptosis.

Interleukin-6 production in human fibroblasts derived from periodontal tissues is differentially regulated by cytokines and a glucocorticoid

Abstract: Interleukin-6 (IL-6) is thought to be a major mediator of the host's defense against infection, and it regulates immune responses in inflamed tissue. In this study, we investigated the regulation of IL-6 production in human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPLF). Pro-inflammatory cytokines including interleukin (IL)-1 alpha, IL-1 beta and tumor necrosis factor (TNF)-alpha stimulated IL-6 production in HGF and HPLF in a time- and dose-dependent manner. This IL-1 alpha, IL-1 beta, or TNF-alpha-induced IL-6 production was enhanced, but the cAMP accumulation they induced was inhibited by the addition of indomethacin. This result suggests that endogenous prostaglandin E2 (PGE2) partially inhibits IL-1 or TNF-alpha-induced IL-6 production and that the enhancement of IL-6 production by IL-1 or TNF-alpha may not be caused through endogenous PGE2-induced cAMP-dependent pathway. Dexamethasone (DEX), a glucocorticoid which is a inhibitor of nuclear factor kappa B (NF-kappa B activation, markedly inhibited IL-1 (alpha or beta) or TNF-alpha-induced IL-6 production; so this production may be partially mediated through NF-kappa B. IL-1 (alpha or beta) and TNF-alpha enhanced IL-6 production synergistically. IL-6 production in HGF or HPLF stimulated with IL-1 beta was augmented by the addition of interferon (IFN)-gamma, but was slightly suppressed by the addition of IL-4. Endogenous IL-6 enhanced IL-1 (alpha or beta)-induced IL-6 production in the presence of IL-6 soluble receptor (IL-6sR). Accordingly, in inflamed periodontal tissues, gingival fibroblasts and periodontal ligament fibroblasts stimulated with pro-inflammatory cytokines such as IL-1 or TNF-alpha, may produce IL-6, and this production can be differentially modulated by endogenous PGE2, IL-6sR, T cell-derived cytokines such as IFN-gamma or IL-4, and glucocorticoids.

Molecular factors associated with compartmentalization of gingival immune responses and transepithelial neutrophil migration

Abstract: PMN migration into the gingival sulcus is a tightly regulated process aimed at selectively increasing leukocyte availability at the site of bacterial plaque aggression, i.e. the superficial portion of the junctional epithelium. The evidence reviewed in this paper indicates that, besides the action of complement fragments, arachidonic acid metabolites, formyl peptides and other bacterial products, the establishment of a gradient of ICAM-1 expression across the junctional epithelium and the expression of IL-8 in its superficial layers probably represent important regulatory mechanisms leading to PMN migration into the gingival sulcus. Such mechanisms can be regulated by the autocrine and paracrine action of some pro-inflammatory cytokines and could, possibly be initiated by specific bacteria-keratinocyte interactions. The advantage of such a redundant regulatory mechanism leading to PMN transepithelial migration is probably related to the key role of the neutrophil in the maintenance of a local host-parasite equilibrium on one side, and on the tissue injury associated with PMN persistence or random migration within periodontal tissues on the other. Several investigations are in progress aimed at identifying the initial environmental stimuli leading to PMN recruitment into the gingival sulcus and at further exploring the important regulatory events.

Effects of metronidazole on Porphyromonas gingivalis biofilms

Abstract: Subgingival bacteria exist within a biofilm consisting of cells and extracellular matrix which may afford organisms protection from both antibiotics and components of the host immune system. MIC values for planktonic Porphyromonas gingivalis treated with metronidazole were compared with those obtained for the same strain in biofilms associated with hydroxyapatite (HA) surfaces. The treated biofilms were examined for growth and studied by scanning electron microscopy. A broth assay resulted in an MIC of 0.125 microgram/ml for metronidazole against P. gingivalis, P. gingivalis biofilms exhibited growth after treatment with 20 micrograms/ml metronidazole, which was 160 times the MIC for planktonic organisms. The results of this study indicate that biofilm-associated P. gingivalis may be resistant to metronidazole at concentrations which are usually attained by systemic administration.

Attachment loss with postmenopausal age and smoking

Abstract: To determine whether postmenopausal bone loss and factors associated with osteoporosis affect tooth retention, we examined vertebral and proximal femoral (postcranial) bone mineral density in relation to tooth loss and attachment loss in a cross-sectional study of 135 postmenopausal women (age range 41-70 yr). Women had at least 10 teeth and no evidence of moderate or severe periodontal disease. Full-mouth attachment loss measurements were made using a pressure-sensitive probe, and bone density was determined by dual-energy X-ray absorptiometry. Attachment loss was correlated with tooth loss (number of remaining teeth, radiologically determined), but not with vertebral or proximal femur bone density. Multivariate analysis showed current smoking (p = 0.01), years since menopause (p = 0.02) and the interaction of age and current smoking (p < 0.01), to be statistically significant predictors of attachment loss in our study population.

Coating of titanium alloy with soluble laminin-5 promotes cell attachment and hemidesmosome assembly in gingival epithelial cells: potential application to dental implants

Abstract: The formation of a biological seal around the transmucosal portion of dental implants may be crucial for the long-term success of these therapies. Data to date suggest that the gingival epithelium attaches to dental implants through the formation of hemidesmosomes. Biochemical and genetic data indicate that the laminin isoform, laminin-5, a component of basement membranes, plays a crucial role in the assembly and maintenance of hemidesmosomes. We report the use of soluble laminin-5 as a biological coating of titanium-alloy to promote cell attachment of the gingival epithelial cell line, IHGK. Monoclonal antibodies reactive with laminin-5 depleted the coating solution of all cell attachment activity and blocked cell attachment to laminin-5-coated disks. Immunodepletion with antibodies to fibronectin had no effect. Finally, we demonstrate that IHGK cells assembled hemidesmosomes within 24 h of attachment to laminin-5-coated titanium alloy but not to the titanium alloy alone. These results suggest that soluble laminin-5 may have clinical applications as a dental implant coating to promote the formation of a biological seal.

Are bacterial proteases important virulence factors

Abstract: The contribution of bacterial proteases to virulence has been relatively understudied. It is a simple matter to argue that bacterial proteases have the potential to destroy the structural and functional proteins that constitute host tissues as well as to destroy proteins important in host defense. Systematically demonstrating that such interactions occur during disease pathogenesis is more difficult, although a few studies have suggested that the ability of a pathogen to use proteases to cross proteinaceous barriers within the host contributes to bacterial virulence. This manuscript reviews concepts of bacterial virulence. Next, it describes how the host regulates the activities of its own proteases to maintain a state of health, and examines evidence suggesting that dysregulation of host proteases results in disease. Finally, evidence supporting a role for endogenous microbial proteases or acquisition of host proteases by microbes as virulence determinants is discussed as are suggestions for future directions for research in this area.

The effect of race, smoking and immunoglobulin allotypes on IgG subclass concentrations

Abstract: In previous studies we have demonstrated that serum IgG subclass concentrations are influenced by both race and periodontal disease diagnosis. Furthermore, we have shown that smoking habits modify the concentrations of some IgG subclasses in specific racial and diagnostic groups. In view of a large amount of data showing strong associations between immunoglobulin allotypes and IgG subclass concentrations we have investigated the effects of race, smoking and IgG allotype on IgG subclass concentration in a population of subjects with or without various forms of periodontitis. The results indicated that there are complex relationships between these factors in their effects on individual IgG subclass levels, and that effects unique to black or white subject groups, or to specific periodontal diagnostic groups and racial subgroups, were evident. In blacks with chronic adult periodontitis IgG1 was lower in smokers, while in generalized early-onset periodontitis patients IgG2 was lower in smokers. IgG4 was independently affected by gender (males higher), smoking (smokers lower) and GM23 (GM23 positive subjects higher), in black subjects only. In white subjects, complex relationships between smoking and allotypic markers were noted but no influence of periodontal diagnosis was found. White GM23 negative subjects who smoked had lower levels of IgG1 than GM23 positive subjects. White GM2 negative subjects who smoked had lower levels of IgG2, than did those who did not smoke. In contrast, smoking had no effect on IgG2 levels in GM2 positive subjects. Thus, in addition to immunoglobulin allotype, smoking is associated with IgG subclass concentrations; furthermore, in black subjects, periodontal diagnosis, gender and smoking all influence IgG subclass concentrations. These results demonstrate that genetic and environmental factors can interact to influence levels of individual subclasses.

A 5-year study of attachment loss and tooth loss in community-dwelling older adults.

Abstract: Tooth loss is a widely recognized endpoint measure for the effects of periodontal diseases and the impact of periodontal therapy. In fact, traditional clinical measures of periodontal status often are considered to be surrogate endpoints in that they are assumed to be related to tooth loss. However, the strength of the relationship between attachment loss and tooth loss in a representative population of untreated subjects has not been studied extensively. The purpose of this paper is to present the trends in attachment loss over a 5-yr period in a population of community-dwelling elderly blacks and whites. Specifically, this paper presents attachment loss trends both at the person and tooth level to address the following issues; 1) whether teeth that experience attachment loss during 1 time period are more likely to be lost at the next time period; and 2) given similar levels of attachment loss, why are some people more likely to lose teeth? In 1988, the University of North Carolina School of Dentistry initiated the Piedmont 65 + Dental Study, which was designed to elicit 800 dentate respondents in the 5-county area who were examined again at 18, 36 and 60 months. Our findings indicated that teeth with poorer attachment level at baseline had a higher probability of being lost during the next 5 yr and teeth that experienced attachment loss during a time period were more likely to be lost during the next time period than teeth without additional attachment loss. In addition, it appears that there are person-level characteristics associated with increasing tendency towards tooth loss in people with similar periodontal status, a finding that may clarify the relationship between attachment loss and tooth loss.

A 5-year study of attachment loss in community-dwelling older adults : incidence density

Abstract: This is the second of three papers that present trends in attachment loss and tooth loss over a 5-yr period in a population of community-dwelling elderly black and whites. The first paper in this series showed that in addition to subject attrition during the 5 yr of the study, teeth also were lost. This loss of subjects and teeth resulted in trends that were not always consistent over time, because people were lost from the study and teeth with more active and advanced periodontal disease were more likely to be lost. In these instances, the incidence density (time-to-event) analytic strategy is useful. Incidence density is the average rate of occurrence for a fixed follow-up period. In 1988, the University of North Carolina School of Dentistry initiated the Piedmont 65+ Dental Study, which was designed to elicit 800 dentate respondents in the 5-county area who were examined again at 18, 36 and 60 months. Our findings indicated that for every 1000 sites followed for 1 yr in this population, 20.6 sites will experience attachment loss of 3+mm. Incidence densities varied greatly by subgroup, indicating that certain characteristics predispose sites for attachment loss. A multivariate logistic regression model indicated that people who are smokers, Porphyromonas gingivalis positive, have 5 or more missing teeth, are not high school graduates, and have not had a dental visit in the last 5 yr are at higher risk of attachment loss. Posterior teeth and mesiobuccal sites are at higher risk. We conclude that incidence density analyses are useful for longitudinal periodontal data and we illustrate the use of incidence density rates to plan clinical trials.

Histomorphological and biochemical differentiation capacity in organotypic co-cultures of primary gingival cells.

Abstract: To establish a three-dimensional in vitro test system mimicking the physiological situation of the oral cavity, organotypic co-cultures consisting of primary gingival cells on a collagen matrix with fibroblasts were generated. The histomorphological development after 7 and 14 d revealed close similarity with the non-keratinized gingiva epithelium. Furthermore, as epithelial specific markers synthesis and localization of keratins as well as the deposition of basement membrane components were assessed on frozen sections by immunofluorescence and keratin expression by in situ hybridization. Primary keratinocytes in conventional culture stained positive for keratin K14 and the mucosal differentiation-specific keratins K4 and K13, while primary fibroblasts, isolated from the same tissue source, and also some keratinocytes, were positive for vimentin. In organotypic co-cultures the keratinocytes formed a multilayered epithelium within 14 d containing basal cells and flattened cells in the uppermost layers. Comparable to native non-keratinized gingiva keratin 14 gene expression was clearly detectable in the basal cell compartment but showed extending immunolocalization. In addition, particularly at the early stage (7 d), basally located keratinocytes were also vimentin positive. According to morphological differentiation K4 and K13 were detectable in suprabasal position at the RNA and protein level. The major basement membrane constituents collagen type IV and laminin increased with time revealing first an interrupted and later a fully extended staining underneath the basal cells. Maintenance of basal cell function was further demonstrated by cell proliferation (BrdU incorporation) which was initially high (7 d) but declined towards the later stages (14-21 d). The results demonstrate i) that this co-culture system leads to a stratified surface epithelium with morphological and biochemical characteristics of the non-keratinized gingiva epithelium and ii) that a state of physiological tissue balance was reached, thus rendering a suitable model for tissue compatibility studies.

Interactions between periodontopathogenic bacteria and cytokines

Abstract: Cytokines produced in response to plaque bacteria clearly play a key role in the periodontal diseases. However, we know very little about the interactions between cytokines and periodontopathogenic bacteria. The aims of this study were to determine whether the key pro-inflammatory cytokines interleukin-1 beta (IL-1 beta) and IL-6 could affect the growth of Actinobacillus actinomycetemcomitans or Porphyromonas gingivalis and to determine whether these organisms could hydrolyse IL-1 beta, IL-6 or the anti-inflammatory IL-1 receptor antagonist (IL-1ra). Culture medium containing up to 100 ng/ml of IL-1 beta or IL-6 was inoculated with A. actinomycetemcomitans (serotypes a, b and c) or P. gingivalis and growth was monitored by measuring changes in electrical conductivity every 3 min for up to 48 h. IL-1 beta, IL-6 or IL-1ra were added to culture supernatants and incubated for up to 24 h. Samples were taken at various times, analysed by SDS-PAGE and the separated proteins transferred by Western blotting to PVDF membranes and probed with anticytokine antibodies. None of the cytokines tested had any effect on the rate of growth or yield of A. actinomycetemcomitans or P. gingivalis. Supernatants front P. gingivalis cultures, but not those from A. actinomycetemcomitans, hydrolysed IL-1 beta, IL-6 and IL-1ra. The hydrolysate from the P. gingivalis supernatant-treated IL-1 beta was unable to stimulate the release of IL-6 from human gingival fibroblasts showing that it had lost biological activity. These results suggest that P. gingivalis can perturb the cytokine network, not only by stimulating the release of cytokines from host cells, but also by removing them from its local environment.

An immunohistochemical study on the localization of Porphyromonas gingivalis, Campylobacter rectus and Actinomyces viscosus in human periodontal pockets

Abstract: The localization and distribution of Porphyromonas gingivalis, Campylobacter rectus and Actinomyces viscosus were studied in human periodontal pockets. After obtaining voluntary consent from 9 patients, 12 teeth and their surrounding periodontal tissue with advanced adult periodontitis were extracted carefully so as not to change the structure of the periodontal pockets. The specimens were processed into serial sections. One of the sections was stained with Brown & Brenn-modified Gram stain to observe the distribution of bacteria. The others were stained immunohistochemically by the Labelled Streptavidin Biotin method (LSAB method) using specific rabbit antibodies against selected bacteria. Some bacteria could be found within epithelial cells. P. gingivalis was found in 9/12 of the samples examined. Small aggregates of P. gingivalis were scattered in all parts of the periodontal pockets, and some of these aggregates could be seen in close contact with the epithelium. Conversely, C. rectus was observed in 5/12 of the samples examined and was predominantly located in the middle and deep pocket zones. C. rectus tended to form large clumps in both the tooth-attached and epithelium-associated plaque area. A. viscosus was observed in 7/12 of the samples examined and was localized predominantly in the tooth-attached plaque area, especially in the shallow and middle pocket zones. Although unexpected spills of unattached plaque from periodontal pockets was possible, immunohistochemical staining with species-specific antibodies was extremely sensitive and revealed the localization and the distribution of periodontal disease-associated bacteria in human periodontal pockets.

Immunolocalization of platelet‐derived growth factor A and B chains and PDGF‐α and β receptors in human gingival wounds

Abstract: Platelet-derived growth factor (PDGF) is a polypeptide growth factor which has been implicated as a major mitogen involved in wound healing. The PDGF appears to promote periodontal regeneration; however, its distribution in gingival tissues is not known and how it participates in gingival wound healing is unclear. Using highly specific antibodies we have studied the distribution of PDGF A and B chains and alpha- and beta-PDGF receptors in healing human gingival wounds. Wounds were created by making a 0.75 mm deep incision in the papilla and healthy gingiva and biopsies were obtained from the same site after 8 h and 1, 3, 7, 14 and 21 d. Frozen sections were immunostained with affinity purified antibodies. The results showed that both epithelium and fibrin clot manifested positive immunostaining for anti-PDGF-A and B-chain antibodies. Staining was present in unwounded and wounded epithelia, and in the fibrin clot it appeared to be more intense for the PDGF-A chain. Blood vessels in connective tissue were also positive while other areas were largely negative. No significant staining was detectable in healthy tissues for anti-PDGF-alpha or -beta receptor antibodies. However, the wound site began to manifest positive immunostaining fro anti-beta-receptor antibody after 3 d of healing, became maximal at 7 d, and then decreased. Our data indicate, but do not prove, that gingival epithelium may be a source of PDGF A and B chains and that the A chain may have a more prominent role to play during early stages of healing. Expression of PDGF beta-receptor appears later at the wound site, indicating that the PDGF B isomer may regulate later wound healing events.

Effective group behavioral intervention for older periodontal patients.

Abstract: A randomized clinical trial assessed the effect of a group-based behavior modification intervention on oral hygiene skills, adherence and clinical outcomes for older periodontal patients. Subjects (n = 107) were aged 50-70 yr with moderate periodontal disease. They were randomly assigned to usual care or intervention. Intervention consisted of 5 weekly, 90-min sessions that included skill training, self-monitoring, weekly feedback about bleeding points and group support focused on long-term habit change. Four-month follow-up indicated significant improvements in the intervention versus the usual periodontal maintenance group for oral hygiene skills and self-reported flossing (p < 0.001), plaque, gingival bleeding, bleeding upon probing throughout the mouth, and pocket depth for sulcus depths that measured between 3 and 6 mm at baseline (p < 0.009). Group oral health intervention provides an effective and relatively inexpensive means of helping patients improve their self-care skills and achieve high levels of adherence to an effective self-care regimen.

Prostaglandin E2 and interleukin-1 concentrations in nicotine-exposed oral keratinocyte cultures

Abstract: Oral keratinocytes are the first cells in contact with tobacco components and are capable of producing various inflammatory mediators, including PGE2 and IL-1. The purpose of this study was to examine PGE2 and IL-1 concentrations in nicotine-exposed oral keratinocyte cultures. Gingival keratinocyte cultures were established from healthy gingival tissues obtained from 7 subjects. Cultures were divided into 4 groups exposed to serum free medium (control), 0.1 microM, 10 microM or 1 mM nicotine for 4, 24 or 48 h. Using enzyme-linked immunosorbent assays, PGE2 and IL-1 alpha were quantified in culture supernatants; IL-1 alpha and beta were also measured in lysed cells. A repeated measures analysis of variance was used to identify significant differences over time and treatment. Nicotine exposure did not significantly alter PGE2 levels at any given time period; however, PGE2 quantities declined significantly (p = 0.0001) over time. At both 24 and 48 h, IL-1 alpha concentrations in lysates from 1 mM nicotine-exposed cells were significantly (p < 0.01) greater than those for all other treatments. Interleukin-1 alpha quantities also declined significantly (p = 0.037) over time in the cultures. Interleukin-1 beta concentrations were elevated, albeit not significantly, in the 1 mM treated cells at 24 and 48 h. Cell viability, mass and counts were not affected by nicotine treatment; these parameters increased significantly (p < 0.005) over time. In summary, nicotine treatment significantly increased IL-1 alpha concentrations in cultured keratinocytes; however, PGE2 synthesis was not altered. Elevated IL-1 production by keratinocytes may have implications in tobacco-induced lesions, given the central role IL-1 plays in tissue response to injury.

Biological effects of drug-loaded biodegradable membranes for guided bone regeneration.

Abstract: The biological effects of drug-loaded biodegradable novel membrane for guided bone regeneration (GBR) was evaluated. The membranes were polyglycolic acid mesh coated with poly-L-lactic acid containing flurbiprofen, tetracycline or PDGF-BB. Porous structure was generated in the membranes by using a phase inversion method. The membrane was less toxic, nicely biodegradable and biocompatible for 8 wk after implantation in the dorsal skin of the rat. The drugs released from the membranes were shown to be effective for new bone formation. Tetracycline, flurbiprofen or PDGF-BB loaded membrane was markedly effective for osteoid tissue and new bone formation in the bony defect prepared in rat calvaria to compare with that by unloaded membrane. These results suggested that drug-loaded biodegradable barrier membrane might be a potential tool for GBR in periodontal therapy.

Immunoregulatory roles of adhesive interactions between lymphocytes and gingival fibroblasts

Abstract: Chronic adult periodontitis is usually characterized by inflammatory cell accumulation in the extravascular periodontal connective tissue. In order to reveal how the lymphocyte migration and retention in periodontal lesions is regulated, we have focused on the molecular basis for the adhesive interactions between lymphocytes and human gingival fibroblasts (HGF). In this study, we investigated the involvement of cell adhesion molecules in adhesive interactions between lymphocytes and HGF. We found that activated lymphocytes bound strongly to HGF and VLA integrins, extracellular matrix receptors, play crucial roles in the binding. Interestingly, we first revealed that CD44 molecules (hyaluronate receptor) on lymphocytes also participated in lymphocyte-HGF interactions and that hyaluronate anchored on the surface of HGF functioned as the ligand for CD44. In addition, when HGF were stimulated with inflammatory cytokines such as IL-1, TNF alpha and IFN gamma, the binding avidity between lymphocytes and HGF was significantly increased and the adhesion was mainly mediated by LFA-1/ICAM-1 pathway. We then examined the possibility whether lymphocyte-HGF interaction may cause activation of HGF. When HGF directly interacted with lymphocytes for 3 h, IL-1 beta mRNA expression was clearly increased in HGF. These findings suggested that the adhesive interactions between lymphocytes and HGF was mediated at least by VLA integrins, LFA-1/ICAM-1 and CD44/hyaluronate and that the heterotypic cell-cell interactions could mutually cause intracellular signal transduction.

Furcation involvement: comparison of dental radiographs and HR‐CT‐slices in human specimens

Abstract: In this in vitro study we compared dental radiographs and high resolution computed tomography (HR-CT) regarding identification and classification of the degree of horizontal and vertical furcation involvement. After removal of the soft tissue and metallic restorations of 18 dentate upper and lower jaws in the interradicular furcation region of 28 molars, bony defects of different dimensions were experimentally produced. The specimens were examined radiographically with standardized dental radiographs and 1.0 mm thick contiguous axial CT-scans. After identification of molars with artificial furcation involvement in the dental radiographs and axial CT-scans, the horizontal and vertical grades of furcation involvement were classified. Radiological identification and classification were compared with the macroscopic findings in the specimens. For quantitative histological-radiological comparison, corresponding microsections were prepared in the same plane as the axial CT-scans. In the dental radiographs the artificial furcation involvement in 6 of 28 (21%) molars was identified. In contrast, all 28 molars with involved furcations (100%) were identified in the axial CT-scans. The horizontal and vertical grades of furcation involvement were classified in the same way as the macroscopic findings, permitting comparison of histological sections and axial CT-scans. The HR-CT technique offers 3-dimensional assessment of the interradicular bone morphology in furcation involvement without overlying structures. The periradicular alveolar bone can be assessed on all sides of the roots. HR-CT scanning thus permits a high identification rate and classification of molars with involved furcations.

Characterization of the Porphyromonas gingivalis antigen recognized by a monoclonal antibody which prevents colonization by the organism.

Abstract: Monoclonal antibody (MAb) 61BG1.3 prevented recolonization of deep pockets by Porphyromonas gingivalis in patients with periodontitis. The aim of this work was to identify the antigen recognized by the MAb. This was carried out by dose-dependent inhibition with materials extracted from P. gingivalis and assessed by a radioimmunoassay. A protease preparation and a capsular extract inhibited about 95% of the binding activity, whereas LPS or fimbriae had no effect. However, about 125 times greater concentration of the capsular than the protease material was needed to inhibit 50% of the antibody activity, suggesting that the MAb recognizes the protease preparation and that the capsular extract contained some protease. Western blotting of MAb 61BG1.3 with recombinant prpR1 protein expressed in Escherichia coli confirmed that MAb 61BG1.3 recognizes the haemagglutinating protease and mapped its epitope to residues 748-1130 of the beta component of the polyprotein. Three major bands of M(r) 45,000, 38,300 and 31,400 were detected in native whole cells of the virulent P. gingivalis strain W50 by Western blotting with MAb 61BG1.3. The MAb inhibited haemagglutination of human red blood cells by P. gingivalis or by a native protease extract. Blocking adhesion of P. gingivalis to the receptors on erythrocytes might be a mechanism by which the MAb inhibits recolonization by the microorganism.

Intra-familial transmission and distribution of Prevotella intermedia and Prevotella nigrescens.

Abstract: The periodontal bacteria Prevotella intermedia and Prevotella nigrescens have been recently separated from each other. The purpose of this study was to investigate the distribution and routes of transmission of these bacteria among family members. Seven patients with moderate to severe periodontitis were selected. These probands, their spouses and 14 of their children were investigated. The presence of Pr. intermedia and Pr. nigrescens was determined by culture techniques in pooled subgingival plaque samples, in the saliva, on the tongue, tonsils and buccal mucosa. Differentiation of Pr. intermedia and Pr. nigrescens was performed by enzyme electrophoretic mobility. From all 7 patients, as well as 4 spouses and 3 of the children, Pr. intermedia could be isolated. Pr. nigrescens was found in 2 of the 7 patients, in 5 of the spouses and in 5 of the 6 children aged 5-10 yr. In the 8 children aged 0-4 yr both species were seldom isolated. These data are in accordance with earlier findings that Pr. intermedia is associated with periodontitis and Pr. nigrescens with a relatively healthy periodontal condition. Ribotyping of bacteria was performed by hybridization of HindIII restriction endonuclease digests of chromosomal DNA with ribosomal DNA. Isolates from unrelated individuals always had distinct ribotypes. Indistinguishable ribotypes of Pr. intermedia and Pr. nigrescens were found both among married couples and among parents and children. This indicates that intrafamilial transmission of Pr. intermedia and Pr. nigrescens is possible both between adults and between parents and children.

Evaluation of a dental subtraction radiography system

Abstract: The aim of this in vitro study was to evaluate the ability of a dental subtraction radiography system to quantitatively detect differences in density between radiographic image pairs. Four periapical radiographs were taken of the upper first permanent molars on five human skulls using the Digora radiographic imaging system. The 4 images were a "baseline" image and 3 containing test objects consisting of either 0.5, 1 or 2 mm thick aluminium cylinders, 2.5 mm in diameter. Semi-automated image processing software was used to "warp" the 3 images with test objects into the same geometric/density registration as the corresponding baseline image using a process called patch minimization. "Difference" images were than produced and their contrast stretched. For regions of interest, with and without test objects present, the difference in density between the baseline and "test object" images was calculated using a reference aluminium step wedge. The test objects were clearly visible in all the "difference" images. The mean difference between the actual and estimated volume of the test object was 0.31 (95% CI [-0.55, 1.17]) mm3 Al. There was a strong association (r = 0.83) between the actual and estimated aluminium volumes. It is concluded that this system provides adequate precision for clinical evaluation.

The potential role of α2-macroglobulin in the control of cysteine proteinases (gingipains) from Porphyromonas gingivalis

Abstract: Porphyromonas gingivalis is closely associated with the development of some forms of periodontitis. The major cysteine proteinases released by this bacterium hydrolyze peptide bonds only after arginyl (gingipain R) or lysyl residues (gingipain K). No target protein inhibitors have been identified for either enzyme, leading us to investigate their inhibition by human plasma alpha 2-macroglobulin (alpha 2M). Both 50- and 95 kDa gingipain R were efficiently inhibited by alpha 2M, whereas the catalytic activity of gingipain K could not be eliminated. All 3 enzymes were, however, inhibited by a homologous macroglobulin from rat plasma, alpha 1-inhibitor-3 (alpha 1I3). alpha-Macroglobulins must be cleaved in the so-called "bait region" in order to inhibit proteinases by a mechanism involving physical entrapment of the enzyme. A comparison of the amino acid sequences of the 2 macroglobulins indicates that the lack of lysyl residues within the bait region of alpha 2M protects Lys-specific proteinases from being trapped. On this basis, other highly specific proteinases might also not be inhibited by alpha 2M, possibly explaining the inability of the inhibitor to control proteolytic activity in some bacterially induced inflammatory states, despite its abundance (2-5 mg/ml) in vascular fluids.