bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of receptors for basic fibroblast growth factor on human periodontal ligament cells
Authors: Takayama S. , Murakami S. , Nozaki T. , Ikezawa K. , Miki Y. , Asano T. , Terashima A. , Okada H.
Published: July 2006

Basic fibroblast growth factor (FGF‐2; bFGF) is a major mitogen for connective tissue cells, and participates in the healing process. It has already been reported that FGF‐2 could be applicable to enhance periodontal regeneration. In the present study, we examined FGF receptor (FGFR) expression on human periodontal ligament (PDL) cells. The binding of [125I]‐labeled FGF‐2 to human PDL cells was studied by radioreceptor assay. The binding of [125I]‐FGF‐2 to PDL cells reached a plateau after 2.5 h incubation at 4°C and was inhibited by the addition of unlabeled FGF‐2 and acidic FGF (FGF‐1; aFGF), but not insulin‐like growth factor‐I, platelet‐derived growth factor and transforming growth factor‐β1. Scatchard analysis revealed the presence of approximately 1.0 × 105 FGF‐2 binding sites per cell with an apparent Kd of 1.2 × 10−10 M. Interestingly, the binding of [125I]‐FGF‐2 on PDL cells reached its maximum at d 6 of the culture and then gradually decreased. Scatchard analysis also demonstrated that the number of FGFRs on a PDL cell was altered during the course of the culture, while the affinity between FGF‐2 and its receptor was not. The responsiveness of PDL cells to FGF‐2, which was monitored by the inhibitory effect on alkaline phosphatase activity, was reduced in proportion to the decrease in the number of FGFRs on the PDL cells. The present study suggests that PDL cells alter the responsiveness to FGF‐2 during the course of the culture by changing the density of its receptor, and that the density of FGFR expression might be a marker of the cytodifferentiation of PDL cells into mineralized tissue forming cells.
Fluid dynamics of gingival tissues
Authors: Del Fabbro Massimo , Galardi Elena , Weinstein Roberto , Bulfamante Gaetano , Miserocchi Giuseppe
Published: July 2006

Gingival hydraulic interstitial pressure was measured with glass micropipettes in 18 anesthetized rabbits at the level of the free gingiva, attached gingiva and oral mucosa facing the incisors and molar teeth. Samples of gingival interstitial tissue fluid were also collected by inserting nylon wicks in the subepithelial layer of the oral mucosa. Colloid osmotic pressure of interstitial fluid samples was measured with an osmometer whose membrane had a molecular cutoff of 30 kD. Hydraulic interstitial pressure from the free gingiva, at an average distance of 300 μm from sulcular space, was ‐ 1.3 ± 0.9 (SD) cmH2O. Mean colloid osmotic pressure of gingival tissues interstitial fluid was 13.1 cmH2O, corresponding to a protein concentration of 2.8 g/dl. The thickness of the sulcular epithelium and of the oral gingival epithelium (data from 2 rabbits) were ∼ 100 μm the minimal distance of microvessels from the surface of the sulcular epithelium was ∼ 150 μm. Based on hydraulic and colloid osmotic data, the Starling balance of pressures causes fluid filtration from gingival capillaries to gingival interstitium; however, across the sulcular epithelium, the pressure gradient sustains fluid absorption from the sulcus into the gingival interstitium. Plasma proteins may leak from microvessels into gingival interstitium, down convective bulk flow and via a concentration dependent diffusive component. At sulcular level, proteins may leak into the sulcus down a concentration gradient. Thus, at sulcular level a peculiar condition occurs in that there is an absorption gradient for water but a filtration gradient for plasma proteins.
The effect of transforming growth factor beta one (TGF‐β1) on wound healing, with or without barrier membranes, in a Class II furcation defect in sheep
Authors: Mohammed S. , Pack A. R. C. , Kardos T. B.
Published: July 2006

The purpose of this study was to analyse the effect of TGF‐β1 on wound healing in standardized Class II furcation defects of 48 mandibular second premolar teeth in 24 sheep. The experimental design included a control group (carrier only, 25% pluronic F‐127), and 2 experimental groups: group A (80 μg/ml TGF‐β1+ carrier) and group B (80 μg/ml TOF‐β1+ carrier covered with a barrier membrane). Sheep were killed either 2 wk or 6 wk after surgery. Mesiodistal sections of the decalcified specimens were quantified histologically using stereology. Percentage volumes of regenerated bone, fibrous connective tissue and cementum were calculated for each furcation defect. Mean values were analysed using multiple ANOVA; p values were calculated using paired and unpaired Student's t tests. After 2 wk there was more bone in group B than either of the other 2 groups, but this was not statistically significant. By 6 wk more bone was present in group A than in the control group (p<0.02) and also in group B when compared with both group A and the control group (p<0.02 and p<0.44), respectively. In the 4 wk between sampling significantly more bone had formed (group A <0.05 and group B p<0.003, respectively). A negative correlation existed between volumes of bone and fibrous connective tissue and no significant differences between the volumes of cementum were evident between any of the groups. This study demonstrated that TGF‐β1 encouraged bone regeneration in Class II furcation defects in sheep, an effect enhanced by the presence of a barrier membrane. This is the first report on the use of TGF‐β1 in conjunction with GTR in periodontal defects.
Distribution of macrophage lineage cells in rat gingival tissue after topical application of lipopolysaccharide: an immunohistochemical study using monoclonal antibodies: OX6, ED1 and ED2
Authors: Miyauchi Mutsumi , Takata Takashi , Ito Hiroshi , Ogawa Ikuko , Kudo Yasusel , Takekoshi Toshitsugu , Nikai Hiromasa
Published: July 2006

To discuss the role of macrophage lineage cells on the periodontal tissue destruction, we immunohistochemically examined the phenotype and the dynamics of macrophage lineage cells 1 or 3 h or 1, 2, 3 or 7 d after topical application of LPS (5 mg/ml in physiological saline) from the rat gingival sulcus using 3 monoclonal antibodies: OX6 (antigen‐presenting cells), ED1 (monocytes, macrophages and dendritic cells) and ED2 (resident macrophages). We could detect at least 3 different types of macrophage lineage cells, namely OX6+/ED1+/ED2− dendritic cells and exudate macrophages and ED2+ resident macrophages. After LPS application the majority of macrophage lineage cells accumulated in the subjunctional epithelial area were newly extravasated OX6+/ED1+/ED2− dendritic cells or macrophages. The number Department of Oral Pathology, Hiroshima of these cells increased progressively with time and reached a maximum level at University School of Dentistry, d 2. On the other hand, number and tissue distribution of ED2+ resident macrophages did not change. These results indicate that several types of macrophage lineage cells exist in rat gingival tissue and suggest that dendritic cells and exudate macrophages transiently accumulated after LPS application are responsible for various host immune response and tissue destruction caused by LPS.
Detection of Prevotella intermedia in subgingival plaque of adult periodontitis patients by polymerase chain reaction
Authors: Riggio M. P. , Lennon A. , Roy K. M.
Published: July 2006

A PCR assay was developed that could specifically amplify DNA from the periodontal pathogen Prevotella intermedia. A pair of primers was selected from regions of the 16S rRNA gene of P. intermedia that were both divergent in sequence at their 3′ ends with respect to the corresponding regions of the 16S rRNA gene of P. nigreseens, its most closely related species, and used in the PCR assay. Positivity was indicated by amplification of an 855 bp product. Using purified genomic DNA from these 2 species, assay conditions were determined under which only P. intermedia DNA and not P. nigrescens DNA was amplifiable. Absolute specificity of the assay was confirmed by the fact that no amplification products were obtained when using DNA from several other important periodontal organisms. The optimized PCR assay was used to identify P. intermedia in subgingival plaque samples of patients with adult periodontitis. Confirmation of amplification of P. intermedia DNA was achieved by digestion of PCR products with the restriction endonuclease Rsal, which gives different restriction patterns for P. intermedia and P. nigrescens. Of the 97 samples analysed, 38 (39%) were positive for P. intermedia. The results obtained confirm P. intermedia as a possible aetiological agent of adult periodontitis. Additionally, PCR primers targeting the corresponding region of the 16S rRNA gene of P. nigrescens were shown to be specific for the organism when used in a PCR assay, although P. nigrescens was not detectable in any of the subgingival plaques analysed.
Evidence that laminin‐5 is a component of the tooth surface internal basal lamina, supporting epithelial cell adhesion
Authors: Mullen L. M. , Richards D. W. , Quaranta V.
Published: July 2006

Laminin‐5 (Ln‐5) is an extracellular matrix (ECM) glycoprotein found in epithelial basal laminae. We studied its expression on the surface of rat molars, in relationship to the location of the internal basal lamina (IBL) of the junctional epithelium (JE). In order to avoid disruption of the JE‐tooth interface as much as possible, the surface of molars was prepared by mechanical removal of tissue debris and detergent/osmotic lysis of epithelial cell layers, and directly stained by immunohistochemistry, without sectioning. Antibodies to Ln‐5 specifically stained a narrow band in the region of the cemento‐enamel junction (CEJ), consistent with the expected location of the IBL. Western blotting of ECM material detergent—solubilized from the prepared tooth surfaces confirmed the molecular nature of Ln‐5 identified by immunohistochemistry. By the use of a high‐definition 3‐D microscope, it appeared that Ln‐5 coated the most apical part of the enamel and the most coronal portion of the cementum, on either side of the CEJ. In adhesion assays performed directly on tooth surfaces, epithelial cells adhered preferentially to the Ln‐5 coated area of the tooth compared to the root surface, which is coated by other ECM components. Adhesion to the Ln‐5 coated surface was specifically inhibited by a function‐blocking monoclonal antibody to Ln‐5. These results suggest that Ln‐5 is a component of the IBL, and that it may be important in promoting adhesion of JE cells onto the tooth surface.
Clinical and microbiological characteristics of smokers with early onset periodontitis.
Authors: Kamma J. J. , Nakou M. , Baehni P. C.
Published: July 2006

Cigarette smoking is a potential risk factor which has recently been associated with periodontal disease progression. The objective of this study was to compare the microbial profile of smokers and non‐smokers in a group of patients with early onset periodontitis. The study population consisted of 60 healthy individuals, 40 males and 20 females aged 22 to 35 yr, exhibiting early onset periodontitis. Thirty patients were smokers (30.9 cigarettes/d) and 30 non‐smokers. Smokers had a higher proportion of deep pockets (PD>5 mm), especially in the maxilla anterior and premolar regions (p <0.001) and presented a significantly greater mean probing depth and attachment loss (p<0.05) in diseased sites and a significantly greater alveolar bone loss (p<0.01) compared to non‐smokers. Two pooled bacterial samples were obtained from each patient. Samples were collected from the deepest periodontal pockets of each quadrant. The samples were cultured anaerobically and in 10% CO2 plus air for bacterial isolation using selective and non‐selective media. Isolates were characterized to species level by conventional biochemical tests and various identification kits. Smokers harboured a greater number of bacteria in total. Analysis of bacterial counts using the ANOVA (Mann‐Whitney U‐test) showed that Staphylococcus aureus, Peptostreptococcus micros, Campylobacter concisus, Escherichia coli, Bacteroides forsythus, C. gracilis, C. rectus, Porphyromonas gingivalis, Selenomonas sputigena, Candida albicans and Aspergillus fumigatus were found in significantly higher numbers and more frequently in smokers while Joanna Streptococcus intermedius, A. naeslundii, A. israelii and Eubacterium lentum were detected more frequently and in significantly higher proportions in non‐smokers. The isolation of bacteria belonging to the exogenous flora such as E. coli, C. albicans, A. fumigatus and S. aureus in smokers' microbiota underscores the importance of the host that is adversely affected by cigarette smoking.
Effect of microbial siderophores on matrix metalloproteinase‐2 activity
Authors: Gendron R. , Grenier D. , Sorsa T. , Uitto V.‐J. , Mayrand D.
Published: July 2006

Matrix metalloproteinases (MMPs) are capable of cleaving almost all macromolecules of the extracellular connective tissue matrix and are thought to play a major role in tissue destructive inflammatory diseases such as periodontitis. The aim of this study was to determine the effects of siderophores, which are iron‐chelating molecules produced by a variety of microorganisms, on the activity of MMP‐2. Heat‐denatured type I collagen (gelatin) was incubated with p‐aminophenylmercuric acetate‐activated MMP‐2 and siderophores. Degradation of gelatin was monitored by SDS‐PAGE and Coomassie blue staining. Ferrichrome, rhodotorulic acid, desferoxamine mesylate and 2,3‐dihydroxybenzoic acid were found to inhibit the MMP‐2 activity whereas β‐phenylpyruvic acid had no effect. The inhibition could be reversed by adding an excess calcium chloride or ferric chloride to the assay mixtures. Our study suggests that microbial siderophores may represent new‐potential therapeutic molecules for the treatment of destructive inflammatory diseases involving excess MMP‐2 activity, such as periodontitis.
Phagocytosis of new cementum‐like islets formed inside the gingival connective tissue in cyclosporin‐A treated rats
Authors: Ayanoglou Charles M.
Published: July 2006

Three control and 3 experimental rats were administered vehicle or cydosporin‐A solutions. Animals were anaesthetized, tissues fixed and jaws processed for Epon inclusion. Histological examination of serially cut areas revealed the presence inside the gingival connective tissue of new cementum‐like islets (NCLIs) associated to or engulfed by voluminous multinucleated cells (MCs). These complexes were located adjacent to blood vessels, at 250–350 μm from the root surface. Histomorphometric study indicated that the volume of the NCLIs varied from 3900 to 72,900 μm3 and that of the MCs from 822 to 56,190 μm3. The latter bore up to 14 nuclear profiles. Comparative evaluation of the NCLI–MC associations to other complexes “multinucleated cell(s)–resorbed material” seems to indicate that the phagocytosis of the NCLIs is dictated by their ectopic location, rather than their nature (new cementum‐like structures). Therefore the MCs should be considered as foreign body giant multinucleated cells rather than cementoclasts.
In vitro studies of lymphocyte apoptosis induced by the periodontal pathogen Porphyromonas gingivalis
Authors: Geatch D. R. , Harris J. I. , Heasman P. A. , Taylor J. J.
Published: July 2006

Apoptosis has a physiological role in lymphocyte development and function serving to remove self‐reactive T‐cells in the thymus as well as activated peripheral T‐cells when they are no longer required in the immune response. Evidence from the study of several pathogenic bacteria indicate that induction of premature cell death by apoptosis may be an important pathogenic mechanism promoting infection, inflammation and concomitant disease. In this paper we demonstrate that cultures of the periodontal pathogen Porphyromonas gingivalis (P. gingivalis) promote lymphocyte apoptosis in peripheral blood mononuclear cells (PBMC). We have used assays designed to investigate the different molecular and cellular changes associated with apoptosis. Thus flow cytometry revealed that whole cultures of P. gingivalis promoted cell shrinkage in the lymphocyte fraction of PBMC and analysis of hypodiploidy confirmed that the cellular changes were associated with nuclear changes characteristic of apoptosis. We also found that apoptosis was promoted in PBMC exposed to both whole P. gingivalis cultures and culture supernatant but not washed bacterial cells; this indicates that molecule(s) secreted into the medium were responsible for this activity and not a factor intrinsic to the bacterial cell. Furthermore heat treatment has no effect on the ability of P. gingivalis cultures to induce lymphocyte apoptosis. In summary, a soluble heat stable component of the supernatant from P. gingivalis cultures promotes lymphocyte apoptosis. These data establish the principle that bacteria‐induced apoptosis may be an important feature of the pathogenesis of periodontal disease.
The proteoglycans of human cementum: immunohistochemical localization in healthy, periodontally involved and ageing teeth
Authors: Ababneh K.T. , Hall R.C. , Embery G.
Published: July 2006

Cementum is believed to play a regulatory rote in periodontal regeneration through a variety of macromolecules present in its extracellular matrix (ECM), among which are the proteoglycans (PG). The PG of human cementum have not been fully characterized. This study has used a standard indirect immunoperoxidase technique to investigate the presence and distribution of PG species within the ECM of human cementum. Freshly extracted human permanent teeth were separated into 8 age groups; each group was subdivided to include healthy and periodontally involved teeth, which were then fixed, demineralized and wax‐embedded. Sections were incubated with polyclonal antibodies recognizing protein core epitopes in the large chondroitin sulphate PG versican and the small interstitial PG decorin, biglycan, fibromodulin and lumican. Immunoreactivity to versican, decorin, biglycan and himican was evident at the borders and lumina of a proportion of lacunae and canaliculi surrounding cementocytes in cellular cementum, as well as on inserted periodontal ligament (PDL) fibres. Biglycan was also present along incremental lines in cellular cementum, whereas staining for fibromodulin was negative. In acellular cementum, no immunoreactivity was evident with any of the antibodies used except on inserted PDL fibres. These results indicate that versican, decorin, biglycan and lumican are components of the ECM of cellular, but not of acellular cementum. Neither age nor periodontal diseases appear to qualitatively influence the PG population of cementum. The distribution of PG epitopes around a proportion of cementocytes suggests the existence of different cementocyte subpopulations, or a differential response of these cells to yet undefined stimuli.
Prostaglandin E2 and I2 regulate intercellular adhesion molecule‐1 expression in interleukin‐1β‐stimulated human gingival fibroblasts
Authors: Iwasaki Kengo , Noguchi Kazuyuki , Ishikawa Isao
Published: July 2006

The present study investigated the effect of prostaglandin (PG) E2 and PGI2 on intercellular adhesion molecule‐1 (ICAM‐1) expression in interleukin‐1β (IL‐1β)‐stimulated human gingival fibroblasts (HGF). IL‐1β potently induced ICAM‐1 expression in HGF and indomethacin, a cyclooxygenase inhibitor, enhanced ICAM‐1 expression in the cells. These data showed that endogenous PGs generated by HGF stimulated with IL‐1β downregulated ICAM‐1 expression. IL‐1β significantly increased the levels of PGE2 and, to a lesser extent, those of 6‐keto‐PGF1α (a stable metabolite of PGI2) in the culture media of HGF. Indomethacin completely inhibited the production of PGE2 and 6‐keto‐PGF1α in IL‐1β‐stimulated HGF. Exogenous PGE2 and carbacyclin (a stable derivative of PGI2) in the presence of indomethacin dose‐dependently suppressed ICAM‐1 expression in IL‐1β‐challenged HGF. Since PGE2 and PGI2 are known to elevate intracellular cyclic AMP (cAMP) levels, we examined the effect of dibutyryl cAMP, a cAMP analogue, and isobutylmethylxanthine, a phosphodiesterase inhibitor, on ICAM‐1 expression. Both agents downregulated ICAM‐1 expression in IL‐1β‐stimulated HGF. These results suggest that PGE2 and PGI2 downregulate ICAM‐1 expression in IL‐1β‐stimulated HGF through a cAMP‐dependent mechanism and that intracellular cAMP elevation in HGF may control inflammatory and immune responses in periodontal disease.
Expression of transforming growth factor‐beta receptors types II and III within various cells in the rat periodontium
Authors: Gao Jin , Symons Anne L. , Bartold P. Mark
Published: July 2006

This study reports the immunohistochemical localization of TGF‐β receptor type II (TβR‐II) and type III (TβR‐III) in cells of the forming periodontal ligament (PDL) in rat first molar roots. Mandibular periodontium was obtained from 3, 6 and 12‐wk‐old rats. This represented tissue from the initial, premature and post‐mature stages of root and periodontal development, respectively. Mandibular bone chips and molar roots were used to isolate osteoblasts, fibroblasts and cementoblasts. Cells were obtained using a 2‐step trypsinization and explant technique, and cultured in Dulbecco's modification of Eagle's medium (DMEM) under routine cell culture conditions. Cells were cultured on coverslips for the purpose of detecting TGF‐β receptors, and compared with whole tissue sections using the same detection method. Cells which stained positively for TβR‐II and TβR‐III on both paraffin sections and cultured cell slides were counted. Both receptors were expressed in the various periodontal tissue compartments. PDL fibroblasts, cementoblasts and osteoblasts were stained positively for TβR‐II and TβR‐III. Endothelial cells were noted to be positive for TβR‐II only. TβR‐II was more widely distributed in cells than TβR‐III, but TβR‐III was extensively localized in the extracellular matrix. Both receptors were expressed on the cell membrane and also localized in the cytoplasm. The findings for paraffin sections were consistent with the immunohistochemical staining of cultured cells. The percentage of cells which stained positively for TβR‐II was greater (∼85%) than that for TβR‐III (∼60%) in all major types of the PDL cells on both paraffin sections and cultured cell slides. Extensive location of TGF‐β receptors in both cells and extracellular matrix suggests that several binding sites are available for TGF‐βs to interact with target cells during development and following maturation of the periodontium.
Alkaline phosphatase activity is up regulated in regenerating human periodontal cells
Authors: Kuru Leyla , Griffiths Gareth Steven , Petrie Aviva , Olsen Irwin
Published: July 2006

The activity of alkaline phosphatase (ALP) is considered to indicate the presence of osteoblast cells and the formation of new bone. In the present study this enzyme was investigated in cells obtained from retrieved polytetrafluoroethylene membranes (M cells) of periodontal disease patients treated by guided tissue regeneration (GTR) and from the regenerated tissue underlying the membrane (RT cells). Normal periodontal ligament (PL) and gingival cells were also grown from the corresponding healthy tissues of human subjects. ALP activity was measured colourimetrically, using paranitrophenyl phosphate as the substrate, after 4 and 7 d of culture in the absence and presence of dexamethasone (DEX), a synthetic glucocorticoid which induces osteoblast differentiation. The results showed that basal levels of ALP activity were expressed by all the cells and that DEX upregulated ALP levels in the M, RT and PL cells but not in the gingival cells. Moreover, both the basal and DEX‐induced ALP activities were statistically significantly higher in the RT cells than in any of the other cells. Our results suggest that both the GTR‐associated and normal PL cells express osteoblast‐like characteristics and, furthermore, that the RT cultures in particular contain a high proportion of osteoprogenitor cells.
Flow cytometry to monitor phagocytosis and oxidative burst of anaerobic periodontopathogenic bacteria by human polymorphonuclear leukocytes
Authors: Conrads G. , Herrler A. , Moonen I. , Lampert F. , Schnitzler N.
Published: July 2006

The reduced susceptibility to phagocytosis found among some periodontopathogenic anaerobes may account for the differences between invasive and non‐invasive strains. We applied flow cytometry as a powerful tool to analyze and quantify phagocytosis using standardized cultures of oral anaerobes (Porphyromonas gingivalis, Prevotella intermedia, P. nigrescens, Capnocytophaga gingivalis, C. ochracea, C. sputigena, Fusobacterium nucleatum and Peptostreptococcus micros) and heparinized whole blood. Bacteria were labeled by a fluorescein‐methylester and their esterase activity, resulting in green fluorescence. Ingested bacteria could be detected easily and quantified by a shift towards green fluorescence in the PMNL population involved and a concomitant decrease in the bacterial population. Furthermore, the oxidative burst of PMNLs was detected in parallel assays using the dye DHR123 which becomes fluorescent upon oxidation during the oxidative burst process. We found a great diversity in phagocytosis susceptibility determined by estimating the portion of phagocytosing PMNLs, ranging from 10.6% (strain W83) to >99.4% (e.g. ATCC 33277T) in P. gingivalis and from 15.9% (strain MH5) to >95% (ATCC 33563T) in P. nigrescens. In contrast, almost all P. intermedia strains as well as the representatives of the other anaerobic, putative periodontopathic species tested showed no or only moderate resistance in the phagocytosis assay. Comparison of clinical data of patients and the extent of phagocytosis resistance of the corresponding P. gingivalis strains suggests that this virulence factor may contribute to the clinical outcome.
Phenytoin metabolism to 5‐(4‐hydroxyphenyl)‐5‐phenylhydantoin (HPPH) in man, cat and rat in vitro and in vivo, and susceptibility to phenytoin‐induced gingival overgrowth
Authors: Kamali F. , Ball D. E. , McLaughlin W. S. , Seymour R. A.
Published: July 2006

Interspecies differences in phenytoin (PHT) metabolism to 5‐(4‐hydroxyphenyl)‐5‐phenylhydantoin (HPPH) were examined in human, cat and rat hepatic microsomes in vitro. Rat liver microsomes were 25 and 650 times more efficient at the conversion of PHT to HPPH than human and cat liver microsomes, respectively. Sulphaphenazole (83%) and tolbutamide (TOL) (64%) were the most potent inhibitors of HPPH formation in human liver microsomes, while ciprofloxacin (27%), enoxacin (27%) and TOL (26%) produced the greatest inhibition in cat liver microsomes. TOL was tested for its effect on HPPH formation and gingival overgrowth in cats in vivo. Eight cats received PHT sodium (4 mg/kg/d) and another 8 cats received PHT sodium together with TOL (20 mg/kg/d) for 10 wk. Six cats (75%) in the PHT group and 4 cats (50%) in the PHT & TOL group developed significant gingival overgrowth by the end of the study. However, the extent and incidence of the overgrowth were similar in the 2 groups. There were no significant differences in mean AUC0‐10weeks for plasma PHT (552.90 ± 29.36 μg·d/mL [PHT alone] vs. 582.41 ± 24.49 μg·d/mL [PHT & TOL]) and unconjugated HPPH (1016.4 ± 295.5 ng·d/mL [PHT alone] vs. 1174.5 ± 397.2 ng·d/mL [PHT & TOL]) concentrations between the 2 groups of cats. Neither PHT nor HPPH were detectable in the plasma of 8 rats which received PHT (4 mg/kg/d) over a 10‐wk period. The rats showed no sign of gingival inflammation (mean gingival index = 0) or gingival overgrowth (mean gingival overgrowth index=0). Thirty‐six adult epileptic patients on chronic PHT therapy were examined; 17 (47%) of the patients demonstrated clinically significant overgrowth. The mean steady‐state plasma PHT concentration was comparable to, and the mean plasma unconjugated HPPH concentration 5‐fold greater than, that observed in the cats. The results suggest that the rapid metabolism and elimination of PHT and HPPH in the rat may enable it to become more resistant towards developing gingival overgrowth, compared to the cat and man.
Periodontal‐derived cells attach to cementum attachment protein via α5β1 integrin
Authors: Ivanovski S. , Komaki M. , Bartold P. M. , Narayanan A. S.
Published: July 2006

A specific collagenous cementum attachment protein (CAP) has been identified in human cementum which promotes selective cell migration towards and attachment of various periodontal derived cell populations to root surfaces in vitro. The CAP is known to support attachment of periodontal‐derived cell via an RGD motif, which suggests an integrin‐mediated mode of attachment. The purpose of the present study was to ascertain which integrin(s) are involved in the attachment of periodontal‐derived cells to CAP. The integrins examined comprised subunits of the major receptors for fibronectin (α5) and collagen (α2, α3), as well as the common β1 subunit which is present in many extracellular matrix receptors. The wells of 48‐well non‐tissue culture treated plates were coated with CAP (2 μg/ml). For negative and positive controls the wells were coated with bovine serum albumin and fibronectin (5 μg/ml), respectively. Human gingival fibroblasts and periodontal ligament fibroblasts were labeled with [3H]‐proline, incubated with anti‐integrin antibodies and added to the pre‐coated wells. Attachment was assessed after incubating the cells for 1 h at 37°C in the presence of the antibodies. Antibodies to α5 and β1 inhibited the attachment of both human gingival fibroblasts and human periodontal ligament fibroblasts to CAP, while anti α2 and α3 antibodies did not affect the attachment. The binding of the fibroblasts to fibronectin was also inhibited by anti‐α2 and α3 antibodies, both of which are components of the “classical” fibronectin receptor and remained unaffected by the addition of anti‐α2 and α3 antibodies. Proteins migrating in SDS‐polyacrylamide gels in positions similar to the α5 and β1 integrin subunits were present in fractions bound to a column of CAP coupled to Sepharose CL‐4B. These results indicate that the attachment to CAP of the periodontal‐derived cells, human gingival fibroblasts and human periodontal ligament fibroblasts, is mediated primarily via the integrin α5β1.
Calcitonin gene‐related peptide acts as a mitogen for human Gin‐1 gingival fibroblasts by activating the MAP kinase signalling pathway
Authors: Kawase Tomoyuki , Okuda Kazuhiro , Wu Chung‐Hsien , Yoshie Hiromasa , Hara Kohji , Burns Douglas M.
Published: July 2006

In many peripheral tissues, calcitonin gene‐related peptide (CGRP) is released from peptidergic sensory nerve fibres and acts like a growth factor during tissue development and regeneration. However, the ability of CGRP to influence gingival tissue has not been studied. To address this question, we have now examined the effects of CGRP on the proliferation of human gingival fibroblasts (Gin‐1) in vitro. Gin‐1 cells have approximately 3100 specific CGRP‐binding sites with a Kd of 38.6 pM on their surface. Treatment with CGRP (0.1‐100 nM) significantly stimulated cell proliferation in a dose‐dependent manner, with maximal effects at 1‐10 nM CGRP after 2 d. As one early cellular response to CGRP, p44‐MAPK protein (also known as the extracellular signal response kinase [ERK]) was tyrosine‐ and threonine‐phosphorylated within 2 min, and this phosphorylation was sustained for at least 1 h. The dose‐response curve of MAPK activation was very similar to that observed for CGRP's stimulation of ceil proliferation. In addition, CGRP's activation of MAPK stimulated its ability to phosphorylate the Elk‐1 transcription factor. When cells were pretreated with PD98059, a selective inhibitor of MAPK kinase (also known as MEK), CGRP not only failed to induce phosphorylation of MAPK but also failed to stimulate Gin‐1 cell proliferation. Our present data indicate that CGRP rapidly activates the MAPK signalling pathway, an effect which consequently stimulates the proliferation of gingival fibroblasts. Our data demonstrate specific cellular responses to CGRP by gingival fibroblasts and support the possibility that CGRP acts as a targeted local factor in the regulation of development, generation and or regeneration of gingival tissues.
Clonal diversity of Actinobacillus actinomycetemcomitans isolates from young adults with minimal periodontal disease
Authors: Macheleidt A. , Müller H.‐P. , Eger T. , Putzker M. , Zölier L.
Published: July 2006

Actinobacillus actinomycetemcomitans is a major periodontal pathogen which is associated with both early‐onset periodontitis and adult cases refractory to conventional periodontal therapy, although the organism has also been shown to be widely distributed among dentate healthy individuals. The observed disease status may be associated with a variation in virulence of different strains or clones. The aim of the present study was to analyse genotype distribution as assessed by an arbitrarily primed polymerase chain reaction (AP‐PCR) among 51 isolates of A. actinomycetemcomitans recovered from more than 200 young adult recruits with no or minor periodontal disease. In addition, isolates from 25 periodontitis patients as well as reference strains were genotyped. Primers amplifying (i) a specific sequence in the 1txA region, (ii) a specific 16S rRNA sequence and (iii) sequences in the leukotoxin promoter region were used to verify species identity of the strains. Three random oligonucleotide primers were employed to analyse genomic polymorphisms of the organism by means of PCR. A total of 19 genotypes could be distinguished, which were grouped by cluster analysis into 5 major clusters based on genetic similarity and a complete linkage sort. Whereas 3 clusters assembled A. actinomycetemcomitans genotypes isolated from both healthy subjects and periodontitis patients, one cluster containing 4 different genotypes exclusively comprised isolates from healthy or gingivitis subjects. Another cluster with 2 genotypes consisted of strains originating from periodontitis patients (p<0.05). One strain characterized by a specific 530 bp deletion in the promoter region of the leukotoxin region was identified in a Ghanese patient with localized juvenile periodontitis. It was concluded that there is considerable clonal diversity of A. actinomycetemcomitans strains isolated from healthy or periodontally diseased subjects, and that genetically closely related groups might be associated with health or disease
Signal recognition particle receptor (SRPR) is downregulated in a rat model of cyclosporin A‐induced gingival overgrowth
Authors: Marley J. J. , Phenix K. V. , Irwin C. R. , Thompson J. , Robinson P. A. , Linden G. J.
Published: July 2006

Differential display is a powerful technique which can be used to identify those genes whose expression is altered between two or more tissues under investigation. We have applied differential display to a rat model of cyclosporin A‐induced gingival overgrowth (CIGO) to identify genes which are differentially expressed as a result of drug treatment. Ten weanling Wistar rats were fed with a pelleted diet containing cyclosporin A (CsA) at 120 mg/kg for 10 d and then 200 mg/kg for a further 30 d prior to culling. Experimental rats were compared with 10 age/sex‐matched rats on a control diet. Significant evidence of overgrowth was observed in the interdental papilla between the mandibular first and second molar teeth in the CsA group. Differential display was performed on total cellular RNA extracted from the mandibular buccal gingiva. A cDNA product was isolated which was underexpressed in the overgrowth tissue and demonstrated a 95% sequence homology to the human signal recognition particle receptor (Human Docking Protein). Preliminary studies indicate that this gene is also underexpressed in human CIGO tissue. The method of approach and the potential implications of our findings are discussed.
Antigen activation of THP‐1 human monocytic cells after stimulation with lipopolysaccharide from oral microorganisms and granulocyte‐macrophage colony‐stimulating factor
Authors: Baqui A. A. M. A. , Meiller Timothy F. , Kelley Jacqueline I. , Turng Been‐Foo , Falkler William A.
Published: July 2006

A human THP‐1 monocyte cell line culture system has been utilized to evaluate the morphological changes in THP‐1 cells and to measure expression of activation antigens (CD‐11b, CD‐11c, CD‐14, CD‐35, CD‐68, CD‐71 and HLA‐DR) as evidence of maturation of THP‐1 cells in response to stimulation by lipopolysaccharide (LPS) from the oral microorganisms, Fusobacterium nucleatum and Porphyromonas gingivalis, and granulocyte‐macrophage colony‐stimulating factor. THP‐1 cells were stimulated with LPS (1 μg/ml) of P. gingivalis or F. nucleatum for different time periods (1, 2, 4 and 7 d). Detection of different activation antigens on THP‐1 cells was performed by indirect immunohistochemical staining followed by light microscopy. Confirmational studies were performed in parallel using indirect immunofluorescence and immunogold electron microscopy for detection of the corresponding activation antigens. Expression of different activation antigens by resting THP‐1 cells revealed HLA‐DR to be on 3% of the cells; CD‐11b, 9%; CD‐11c, 8%; CD‐14, 22%; CD‐35, 9% and CD‐68, 7%. The CD‐71 activation antigen was not expressed in untreated THP‐1 cells. LPS stimulation increased expression of all activation antigens. A significant (p < 0.05) increase in expression of CD‐11b, CD‐11c, CD‐14, CD‐35, CD‐68 and CD‐71 was observed when GM‐CSF (50 IU/ml) was supplemented during the treatment of THP‐1 cells with LPS of F. nucleatum or P. gingivalis. Activation and differentiation of THP‐1 cells by LPS from oral microorganisms in the presence of GM‐CSF supports a role for human macrophages in acute and chronic periodontal diseases and may explain the clinically observable periodontal exacerbations in some patients after GM‐CSF therapy.
Transepithelial electrical resistance and tight junctions of human gingival keratinocvtes
Authors: Meyle J. , Güttig K. , Rascher G. , Wolburg H.
Published: July 2006

Human gingival keratinocytes (HGKs) were studied by means of freeze‐fracture technique, conventional electron microscopy and the transepithelial electrical resistance for the investigation of intercellular contacts. For the purpose of comparison, MDCK cells and HaCat cells were also included. An unexpected finding was the presence of tight junctions in the HGKs. In vivo the tight junctions, which were of low complexity and P‐face‐associated, were co‐distributed with desmosomes; in one case, the strands ran directly through desmosomal plaques. Where tight junctions and desmosomes occurred together, no gap junctions were seen. In contrast, where no tight junctions were present, gap junctions and desmosomes were co‐localized. However, the unfavourable fracture planes through the tissue did not allow a clearcut allocation of gap junction/tight junction occurrence to certain strata. In vitro, HGKs also expressed tight junctions which formed networks of low complexity and high P‐face association. Whereas desmosomes were highly expressed, gap junctions were not observed in cultured keratinocytes. Transepithelial electrical resistances (TEER) of cultured HGKs were higher than the values in low resistance‐MDCK cells and HaCat cells but considerably lower than the values in high resistance MDCK cells, supporting the fundamental correlation between tight junction morphology and TEER. The results with this cell culture model of the human gingiva provide some valuable information about in vitro differentation and concommittent changes in cellular contacts of human gingival keratinocytes.
Cytokine secretion of periodontal ligament fibroblasts derived from human deciduous teeth: effect of mechanical stress on the secretion of transforming growth factor‐β1 and macrophage colony stimulating factor
Authors: Kimoto S. , Matsuzawa M. , Matsubara S. , Komatsu T. , Uchimura N. , Kawase T. , Saito S.
Published: July 2006

The periodontal ligament may play an important role in tooth eruption, root development and resorption. The tissue physiologically receives mechanical force during mastication. We focused on the effects of intermittent mechanical strain on the cytokine synthesis of periodontal ligament (PDL) fibroblasts in vitro. The cells were derived from human periodontal ligament of deciduous teeth (HPLF‐Y) and permanent teeth (HPLF). The two kinds of PDL cells and human gingival fibroblasts (HGF) were cultured in flexible bottomed culture plates. The cells were mechanically stretched at 5% elongation. 3‐cycles/min for 24 h on d 7 in culture using a Flexercell® strain unit. After the stretching, we measured DNA content and alkaline phosphatase activity in the cell layer, transforming growth factor beta 1 (TGF‐β1) and macrophage colony stimulating factor (M‐CSF) contents in the conditioned medium. The TGF‐β1 level in the conditioned medium of HPLF was significantly higher than that of HPLF‐Y and HGF. It was stimulated by mechanical stretching only on HPLF, whereas no significant effect was observed on HPLF‐Y and HGF. M‐CSF secretion was inhibited by the stretching on all of HPLF, HPLF‐Y and HGF. Lα,25 dihydroxy vitamin D3 (D3) stimulated M‐CSF secretion into the culture medium of both HPLF and HPLF‐Y, but the stretching inhibited M‐CSF secretion and completely blocked the enhancement by D3. These data suggest that periodontal ligament cells synthesize and secrete the molecules as autocrine or paracrine factors that affect bone remodelling and root resorption and the level of those factors change in response to mechanical stress.
Effect of recombinant human platelet‐derived growth factor‐BB and bone morphogenetic protein‐2 application to demineralized dentin on early periodontal ligament cell response
Authors: Zaman K. U. , Sugaya T. , Kato H.
Published: July 2006

The purpose of this study is to investigate the early responses of human periodontal ligament cells attached to recombinant human platelet‐derived growth factor‐BB and bone morphogenetic protein‐2 applied EDTA‐demineralized dentin. One hundred and seventy‐four root‐planed flat dentin blocks were prepared from the mid‐third of periodontally diseased human tooth roots. After demineralization with 24% EDTA (pH 7.02) 120 dentin blocks were treated with 0.5 and 1 μg/ml rhPDGF‐BB, 1 and 3 μg/ml rhBMP‐2 and only MEM as control (24/group). Human periodontal ligament cells (HPLC) were seeded on these dentin surfaces and incubated. The alkaline phosphatase (ALP) activity and protein concentration of the attached cell were assessed at d 2,4 and 7. Fifty‐four dentin blocks were seeded with HPLC after application of 1 μg/ml rhPDGF‐BB, 3 μ/ml rhBMP‐2 and MEM (18/group) and then incubated. At d 2, 4 and 7, the attached cells were stained and counted under light microscope. The results showed a significant increase of protein concentration and cell number in PDGF‐BB treated groups than control (p < 0.05, p < 0.01) but not the ALP activity, and a significant increase of ALP activity was observed in BMP‐2 treated groups than control (p <0.05) but protein concentration and cell number remained almost the same over time. Thus, rhPDGF‐BB and rhBMP‐2 application to EDTA demineralized dentin surfaces promote the early human periodontal ligament cell responses by increasing cell proliferation and differentiation, respectively, which would ultimately enhance periodontal regeneration.
Prostaglandin F2α upregulates intercellular adhesion molecule‐1 expression in human gingival fibroblasts
Authors: Noguchi Kazuyuki , Iwasaki Kengo , Ishikawa Isao
Published: July 2006

Prostaglandin F2α (PGF2α) is a bioactive lipid mediator, which has been suggested to be involved in the pathogenesis of periodontal disease. However, the roles of PGF2α in the disease are not well understood. In the present study, we investigated the effect of PGF2α on intercellular adhesion molecule‐1 (ICAM‐1) expression in human gingival fibroblasts (HGF) and the effect of PGF2α on ICAM‐1 expression elicited by proinflammatory cytokines, interferon‐γ (IFN‐γ) and tumour necrosis factor α (TNFα) in the cells. PGF2α‐stimulated HGF expressed ICAM‐1 expression in a time‐ and dose‐dependent manner. IFN‐γ‐elicited ICAM‐1 expression was synergistically increased by PGF2α whereas TNFα‐induced ICAM‐1 expression was slightly inhibited by PGF2α. Fluprostenol, a selective FP receptor agonist, could mimic PGF2α‐induced effect on ICAM‐1 expression. Furthermore, signal transduction for the regulation of ICAM‐1 by PGF2α was investigated using N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulphonamide (W‐7), a calcium calmodulin antagonist, and l‐(5‐isoquinolinylsulphonyl)‐2‐methylpiperazine (H‐7), an inhibitor of protein kinase C (PKC). W‐7 and H‐7, remarkably, suppressed PGF2α‐induced ICAM‐1 expression and synergistic increase of ICAM‐1 expression by combination of PGF2α and IFN‐γ, while IFN‐γ‐elicited ICAM‐1 expression was only partially inhibited by W‐7 and H‐7. From these data, we suggest that PGF2α upregulates ICAM‐1 expression in HGF and synergistically enhances IFN‐γ‐induced ICAM‐1 expression through FP receptor by calcium calmodulin‐dependent and PKC‐dependent pathways. PGF2α may be involved in the pathology of periodontal disease by upregulating ICAM‐1 expression in HGF.
Protein recovery from several paper types used to collect gingival crevicular fluid
Authors: Johnson R. B. , Streckfus C. F. , Dai X. , Tucci M. A.
Published: July 2006

The purpose of this study was to compare the efficiency of protein elution from several types of gingival crevicular fluid (GCF) collection papers when the volume of the inoculated protein and the elution methods were constant. Various concentrations of bovine serum albumin (BSA) and 14C‐BSA were placed onto strips of Whatman #1 [W1], Whatman 3 MM chromatographic [W3]. Periopaper™ (ProFlow) [P] and Periopaper™ (Harco) [H], and recovered proteins measured following a non‐optimized centrifugal elution technique. There were significant differences in % recovery of BSA and 14C‐BSA from the papers, which was dependent on both the type of paper and the concentration of the inoculated protein; that is, proteins at the lowest concentrations were less efficiently eluted from GCF collection papers than those at higher concentrations. Equations for regression lines of elution efficiency were quadratic. Thus, our data suggest significant differences in the efficiency for elution of BSA from absorbent papers when the volume of the inoculated fluid and the elution technique were constant. Previous variable or conflicting experimental data between research groups may have resulted from incomplete elution of proteins from GCF collection papers, possibly due to entrapment within, or binding of GCF proteins to the paper.
Role of soluble interleukin‐6 receptor in inflamed gingiva for binding of interleukin‐6 to gingival fibroblasts
Authors: Naruishi Koji , Takashiba Shogo , Chou Hsin‐Hua , Arai Hideo , Nishimura Fusanori , Murayama Yoji
Published: July 2006

Interleukin‐6 (IL‐6), frequently detected in periodontitis, is known to mediate important signals in the inflammatory cylokine network. Gingival fibroblasts (GF) secrete cytokines upon stimulation with inflammatory mediators. However, it is not clear if GF respond to IL‐6. We examined the IL‐6 receptor gene expression in GF. Furthermore, we tested whether GF are target cells for IL‐6 by examination of binding of IL‐6. GF were found to contain trace amounts of mRNA for IL‐6 receptor (IL‐6R), but had high levels of mRNA for 130‐kDa glycoprotein (gp 130), which is a signal transducer for IL‐6/IL‐6R complex. Based on this observation, we hypothesized that IL‐6 could bind GF if exogenous soluble forms of IL‐6R (sIL‐6R) existed in the gingiva or culture condition. Thus, we investigated the existence of sIL‐6R in gingiva using enzyme‐linked immunosorbent assay and whether sIL‐6R influenced the binding of IL‐6 to GF in vitro. In inflamed gingival, sIL‐6R was detected and its concentration ranged from 150 to 700 pg/μg protein. The sIL‐6R enhanced the binding of IL‐6 to GF in a dose‐dependent manner. This enhancement was inhibited by an antibody against gp130, suggesting that the IL‐6/sIL‐6R complex bound to the fibroblasts via gp130. These data demonstrated that gingival fibroblasts can be target cells for IL‐6 in the presence of appropriate amounts of sIL‐6R. This situation may exist during inflammation in periodontal tissue.