bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of volatile thiol compounds on protein metabolism by human gingival fibroblasts
Authors: Johnson P. W. , Yaegaki K. , Tonzetich J.
Published: July 2006

The levels of volatile sulphur compounds (VSC) in periodontal pockets and mouth air have been found to correlate with severity of the disease process. The purpose of this study was to examine the influence of hydrogen sulphide and methyl mercaptan on protein metabolism of human gingival fibroblasts. The incorporation of labelled amino acids into protein was used to evaluate effects on total protein content. Changes in collagenous protein concentration were monitored by release of radioactivity following collagenase digestion as well as direct analysis of hydroxyproline. Both thiols were found to reduce total protein synthesis, with mercaptan exerting a greater adverse effect. In cultures exposed to mercaptan, total protein was reduced by 35%. The changes in total protein were accompanied by a corresponding decrease in collagenase‐digestible protein. Hydroxyproline analysis of CH3SH‐exposed cultures confirmed the changes associated with collagenous proteins. It indicated that in comparison to the controls the CH3SH‐exposed cultures had a 70% reduction in collagen which resulted from a combined effect of suppressed synthesis and increased rate of collagen degradation. The possibility of thiol reaction with collagen was determined using in vitro systems in which type I collagen was reacted with varying concentrations of [35S]‐H2S. The carboxymethyl (CM) cellulose assays of resulting reaction mixtures indicate that [35S]‐radioactivity was incorporated directly into αl, α2, β11, β12 peptide chains. Furthermore, upon exposure of collagen to elevated H2S concentrations, the H2S converted some of the acid‐soluble collagen to a more soluble product which could be extracted in neutral salt and analyzed by CM‐cellulose chromatography. This effect on collagen solubility may make it more susceptible to enzymatic degradation and contribute to the increased destruction of collagen observed in the thiol‐treated fibroblast cultures.
Fine structure of adrenergic nerve fibers in human periodontal ligament
Authors: Nakamura Takako K. , Nakahara Hiroshi , Nakamura Motooki , Kiyomura Hiroshi , Tokioka Takao
Published: July 2006

The presence of adrenergic nerve fibers was demonstrated ultrastructurally in the human periodontal ligament obtained from extracted premolar teeth from 8 young patients. The nerve endings were located close to arterioles. The results suggest that they seem to control blood flow in the human periodontal ligament.
Influence of temperature and foil hardness on interocclusal tactile threshold
Authors: Jacobs R. , Schotte A. , van Steenberghe D.
Published: July 2006

Determinations of interocclusal tactile threshold levels so far have involved neither appropriate psychophysical approaches nor an assessment of the mechanical and thermal properties of the foils used. Twenty subjects (12 females) aged 18 to 50 (mean age 35) were tested for their absolute threshold level (RL). Both the method of limits and the staircase method were applied to determine the active or passive RL. For the active RL assessment, foils of different thicknesses were placed between edge‐to‐edge opposed incisors during gentle biting. Inner ear receptors of the blindfolded subjects were blocked by broad band noise applied through earphones, because vibrations induced by occlusal contact and conducted through bone might be perceived by these receptors. The foils presented were aluminum (Al), tin (Sn), polyester (PE) and calibrated steel (St) (thickness ranging from 8 to 50 μm) which offer different physical and thermal properties. The range of RL of the group varied between 8 μm for aluminum to 46 μm for polyester for 50% correct assessments. Increasing the foil temperature from room temperature (20°C) to body temperature (35°C) significantly increased the RL for conducting materials (one‐way blocked ANOVA). These results indicate that temperature exchange takes place while presenting conducting foils at 20°C (cold stimulus) interocclusally, which influences the RL by activating thermosensitive receptors. The passive RL determination with classical von Frey‐hairs resulted in a mean axial RL of 3.0 g. Both psychophysical RL assessments (method of limits, staircase method) gave reproducible and similar results as ascertained by ANOVA. Furthermore, a positive correlation was established between active and passive RL (Pearson correlation test). It is concluded that the physical and thermal properties of the foils need careful consideration in future experiments on threshold determination. Although active RL determination may involve the activation of non‐periodontal receptors, it remains a realistic parameter to monitor tactile function of teeth.
The mechanisms of Eikenella corrodens aggregation by salivary glycoprotein and the effect of the glycoprotein on oral bacterial aggregation
Authors: Ebisu Shigeyuki , Nakae Hideaki , Fukuhara Hironobu , Okada Hiroshi
Published: July 2006

The mechanism of aggregation of Eikenella corrodens 1073 with E. corrodens aggregating factor (EcAF) which was purified from submandibular‐sublingual (SM‐SL) saliva was investigated. Heating (100°C, 10 minutes) or protease treatment of E. corrodens cells abolished the aggregating activity. The aggregation was inhibited by adding N‐acetyl‐D‐galactosamìne (GalNAc) and saccharides which contain a galactose residue at the non‐reducing end. The aggregating activity was sensitive to EDTA and was restored by Ca2+ but not by Mn2+ or Mg2+. Neuraminidase treatment of EcAF increased their ability to aggregate. E. corrodens, suggesting that the sialic acids on EcAF interfere with aggregation. These data suggest that the aggregation of E. corrodens 1073 with EcAF is mediated by specific interactions between a bacterial cell surface lectin‐like substance and a complementary GalNAc‐like receptor. EcAF also aggregated 16 strains of oral bacteria including periodontopathic bacteria such as Porphyromonas (Bacteroides) gingivalis 381 and Actinobacillus actinomycetemcomitans ATCC29522; however, those aggregations were not inhibited by GalNAc. Therefore, EcAF appears to have more than two types of bacterial binding site and plays important roles in accumulation of dental plaque by forming a complex network of plaque bacteria including periodontopathic strains.
A sensitive enzymatic method (SK‐013) for detection and quantification of specific periodontopathogens
Authors: Ishihara Kazuyuki , Naito Yuko , Kato Tetsuo , Takazoe Ichiro , Okuda Katsuji , Eguchi Toru , Nakashima Koichi , Matsuda Naoki , Yamasaki Kyoko , Hasegawa Kenji , Suido Hirohisa , Sugihara Kunio
Published: July 2006

Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola have been found to predominate in periodontal pockets of patients with adult periodontitis. These microorganisms hydrolyze the synthetic peptide N‐benzoyl‐DL‐arginine‐2‐naphthylamide (BANA). In this study, we developed an enzymatic method, designated SK‐013, to detect the existence of these microorganisms in subgingival plaque bacteria. This enzymatic method was based on the observation of the hydrolysis of N‐carbobenzoxy‐glycyl‐glycyl‐arginyl‐3,5‐dibromo‐4‐hyd‐roxyaniline (N‐CBz‐Gly‐Gly‐Arg‐DBHA) and made more sensitive by adding an enhancing system. The SK‐013 was specifically positive for P. gingivalis, B. forsythus, T. denticola, and some strains of Capnocytophaga species, but was not specific for any of the other bacterial strains tested. This SK‐013 system may be valuable for detection and quantification of periodontal disease‐associated bacteria in subgingival plaque and thus for diagnosis of periodontal infections.
Primate dentine extracellular matrix induces bone differentiation in heterotopic sites of the baboon (Papio ursinus)
Authors: Moehl Torsten , Ripamonti Ugo
Published: July 2006

In rodents, demineralized dentine matrix induces local differentiation of endochondral bone. This study investigated the osteoinductive potential of primate dentine matrices when implanted extraskeletally in allogeneic recipients. Demineralized dentine cylinders prepared from adult baboon incisors and demineralized dentine matrix pulverized to a particle size of 74–420 μm were implanted into the rectus abdominis of 4 subadult male baboons (Papio ursinus). Specimens were harvested 30 and 90 d after implantation. Histological analysis on serial sections showed bone differentiation in demineralized dentine cylinders after partial resorption of the external demineralized layer, and in resorption lacunae and excavation chambers within the matrix. Implants of demineralized dentine matrix of 74–420 μm particle size showed no osteoinductive activity as determined biochemically (alkaline phosphatase activity) and histologically. The demonstration of bone induction by primate dentine prepared from fully erupted tooth matrix suggests that putative osteogenic proteins may be conserved after dentinogenesis and embryonic tooth development, and may play a role during healing after surface demineralization of exposed root surfaces during regenerative procedures in humans.
Cystatin activity in gingival crevicular fluid from periodontal disease patients measured by a new quantitative analysis method
Authors: Ichimaru Eiji , Imura Kenji , Hara Yoshitaka , Kato Yuzo , Kato Ihachi
Published: July 2006

Cystatins are protein inhibitors of cysteine proteinases, which are believed to play an important role in the pathogenesis of periodontal disease. In this study, we report a new sensitive method for the quantitative analysis of cystatin activity in a small amount of crude sample such as gingival crevicular fluid. Cystatin activity in the crude sample was determined by using active site‐titrated papain, which is a cysteine proteinase from the plant Carica papaya. Crude samples usually contain endogenous cysteine proteinases. These competed with the added papain for the active sites of the cystatins. The cystatin‐cysteine proteinase complex was able to be dissociated by the addition of papain. This competition and dissociation could interfere with the determination of cystatin activity, since some of the cysteine proteinases, such as cathepsin B, hydrolyzed the specific substrate for papain during titration with the papain. In order to exclude this interference and measure total cystatin activity, the crude sample must be alkalinized (pH 11.0) for 5 min at 4°C followed by 10 min at 40°C before titration with papain. The minimum detectable amount of cystatins was 20 fmol/ assay when it was calculated per mole of papain inhibitory sites. Using this method, significant levels of cystatin activity were detected in all the samples of gingival crevicular fluid taken from periodontal disease patients. These results suggest that cystatins could regulate the cysteine proteinases in gingival crevicular fluid and that this new method could be useful to clarify the role of cystatins in the pathogenesis of periodontal disease.
Effects of destructive periodontitis, induced by diet, on the mechanical properties of the periodontal ligament of the mandibular first molar in golden hamsters
Authors: Yamazaki Y.
Published: July 2006

To examine progressive changes in the mechanical properties of the periodontal ligament in the hamster mandibular first molar with experimental periodontitis – induced by feeding a high‐carbohydrate diet – load‐deformation curves obtained by extracting the tooth from its socket in the dissected jaw were analyzed. The maximum shear load, elastic stiffness and failure energy in shear in the experimental groups decreased significantly during the experimental period. On the other hand, significant differences in the maximum deformation were not found between the experimental and the relevant control groups. Radiographic and histological observations showed that the alveolar bone loss and destruction of the periodontal tissues occurred at the cervical region of the tooth in the experimental animals at 8 and 12 weeks after the start of the experiment. It is suggested that the reduction of the mechanical strength of the periodontal ligament in the experimental animals may be due to the alveolar bone loss, destruction of the periodontal tissues at the cervical region – particularly on the lingual aspect – followed by a decrease in the surface area of the ligament. It is also suggested that degenerative changes in the remaining periodontal ligament occurred in hamsters with periodontal disease, causing a reduction in the mechanical strength of the ligament.
Five parameters of gingival crevicular fluid from eight surfaces in periodontal health and disease
Authors: Smith Quenton T. , Au Gladys S. , Freese Pamela L. , Osborn Joy B. , Stoltenberg Jill L.
Published: July 2006

Volume and amounts of myeloperoxidase (MPO), lactoferrin (LF), aryl sulfatase (AS) and lactate dehydrogenase (LDH) were measured in gingival crevicular fluid (GCF) collected from the mesial and distal proximal surfaces of the premolars and first and second molars of 3 subject groups. Group assignment was based on subject mean gingival index (GI) and probing depth (PD) of sampled sites as follows: healthy, GI <0.5, PD >3.0; disease 1, GI > 1.0. PD >3.0 mm; disease 2, PD>4.0 mm. Attachment loss (ATL) of most sites in the 3 groups was: healthy, 0–1 mm; disease 1, 1–2 mm; and disease 2, 4–9 mm. GCF volume differed among surfaces and teeth in each of the 3 groups. The greater amount of GCF collected from posterior locations was not related to the GI and PD. Differences with sampling location in amounts of GCF constituents were restricted to MPO and LF. Most of these differences (greater amounts at posterior sites) were associated with more severe disease. Variability in amount and composition of GCF collected from different sites, therefore, should be considered in experiments which include quantitation of GCF parameters. The ratio of MPO in disease group 2 to disease group 1 was greater than similar ratios for GCF volume and LF, AS and LDH. The quantity of MPO was the only measure which differed between the 2 disease groups at all surfaces. MPO thus appears to have the greatest potential, among the measured parameters, to serve as a marker for advanced periodontal disease.
Modulation of human gingival fibroblast cell metabolism by methyl mercaptan
Authors: Johnson P. W. , Ng W. , Tonzetich J.
Published: July 2006

Methyl mercaptan (CH3SH) is a malodorous compound whose levels are elevated in mouth and crevicular air of individuals with active periodontal disease. Since it may play a role in the disease process, its effects were evaluated using human gingival fibroblast cultures and viable porcine unkeratinized oral mucosal tissue sections. Results showed that the protein content of CH3SH‐exposed cell cultures pulsed with [14C]‐labelled glycine and proline was decreased by approximately 25%. Furthermore, this deleterious effect was irreversible in test cultures subsequently incubated for 24 h in a control 95% air/5% CO2mercaptan‐free environment. The supporting slab‐gel electrophoresis profiles yielded evidence that exposure to CH3SH caused an alteration in collagen metabolism and a pooling of Type I procollagens. In addition, DNA synthesis was suppressed in CH3SH‐exposed cultures by 44.1% at the 24 to 26 h peak of DNA synthesis. This is a true inhibition and not a shift in peak of maximum DNA synthesis as the shape and location of time‐course curves of control and test systems is very similar. Proline transport study using [14C]‐proline indicated a reduction in proline transport in the range of 40 to 50% in cultures exposed for 24 to 30 h to CH3SH. Significantly even 15 min exposure to 6.7 ng CH3SH/ml of incubating atmosphere suppressed proline transport by approximately 24%). This indicates that even brief exposure to low concentrations of CH3SH has a significant adverse effect on proline transport. Fluorescent staining of tissue sections exposed to mercaptan indicated that the agent elevated the number of cells stained with vital dye. The results demonstrate that CH3SH at concentrations employed in this study significantly decreased total protein content, collagen content, DNA synthesis and proline transport in fibroblast cell cultures, as well as exerting an adverse effect on the cellular integrity of intact oral mucosal tissues.
Analysis of chondroitin sulfate isomers in the periodontium of the monkey using high‐performance liquid chromatography
Authors: Okazaki Joji , Kamada Aiko , Gonda Yoshimichi , Sakaki Tetsuya
Published: July 2006

Glycosaminoglycan (GAG) was extracted from monkey periodontium, consisting of gingiva, periodontal ligament, alveolar bone and cementum, and from dental pulp and dentin by digestion with Pronase E. Unsaturated disaccharide isomers formed by chondroitinase AC digestion from chondroitin sulfate were labeled with dansylhydrazine and analyzed by high‐performance liquid chromatography. These tissues showed different molar ratios of the unsaturated chondroitin sulfate disaccharides. The ratio of ΔDi‐4S to ΔDi‐6S was lowest in the dental pulp, followed by the gingiva, periodontal ligament, dentin, alveolar bone, and cementum, in that order. It was greater in the calcified than in the uncalcified tissues.
Detection of, and anti‐collagen antibody produced by, CD5‐positive B cells in inflamed gingival tissues
Authors: Sugawara Masako , Yamashita Kaoru , Yoshie Hiromasa , Hara Kohji
Published: July 2006

This study was performed to investigate the frequency and distribution of CD5‐positive (CD5+) B cells in inflamed gingival tissues using flow cytometric and immunohistochemical analyses. The ability of CD5+ B cells to produce antitype I collagen antibody was also examined. CD5+ B cells expressed “low” fluorescence intensity in the peripheral blood of both healthy subjects and patients with adult periodontitis. However, in inflamed gingival tissues the intensity of this surface marker was high. The percentage of B cells bearing CD5 surface marker was statistically higher in gingiva than in peripheral blood obtained from both the patients and healthy subjects. These CD5+ B cells were observed in gingival subepithelial connective tissues from the bottom to the middle of the periodontal pocket. This area showed destruction of collagen fibers and dense cell infiltrations. Anti‐collagen IgG antibody level in patients’ gingival crevicular fluids (GCF) was higher than that in sera from healthy subjects, and slightly higher than in autologous sera. IgM anti‐collagen antibody in GCF was lower than in autologous sera and in sera from healthy subjects. EBV‐transformed CD5+ B cells produced considerably more IgM and IgG antibody to collagen than CD5− B cells. Therefore CD5+ B cells may contribute to the pathogenesis of inflamed gingival tissues.
Longitudinal monitoring of humoral antibody in subjects with destructive periodontal diseases
Authors: Taubman M. A. , Haffajee A. D. , Socransky S. S. , Smith D. J. , Ebersole J. L.
Published: July 2006

Fifty‐one subjects (16–61 years old) with evidence of prior destructive periodontal disease were monitored clinically and immunologically at bi‐monthly intervals for up to 5 yr. Periodontal disease activity, determined as new attachment loss, was detected in 33 of these subjects. Only 4 of 51 subjects failed to show an elevated serum antibody level to any of the 18 subgingival species tested. The antibody level threshold established for periodontally healthy subjects was exceeded most often in diseased subjects with serum antibody to Actinobacillus sp., P. gingivalis, E. corrodens, C. concisus, F. nucleatum and P. intermedia in that order. In general, most serum antibody levels to subgingival species remained relatively consistent for periods as long as 5 yr. However, major increases and decreases in antibody could be detected to at most one or two species in individual subjects. In addition, prolonged, steady increases and decreases in antibody to specific species could be detected in certain subjects. These findings suggest that major changes occurring in serum antibody may reflect fluctuations in the nature of the infection. Differences were observed in the antibody level to specific species when subjects were divided into subsets on the basis of clinical criteria. These included high levels of antibody to A. actinomycetemcomitans Y4 in LJP and RPP subjects and to A. actinomycetemcomitans 29523 in LJP and GJP subjects.
A preliminary evaluation of the use of a redox agent in the treatment of chronic periodontitis
Authors: Wilson M. , Gibson M. , Strahan D. , Harvey W.
Published: July 2006

A small‐scale clinical trial was carried out to evaluate the effect of a redox agent, methylene blue, on microbiological and clinical indices of chronic periodontitis. Methylene blue was applied subgingivally on a daily basis for 7 d to 25 test sites in 7 patients and the sites evaluated clinically and microbiologically for up to 14 d: 25 control sites in the same patients received sterile water. The test sites showed statistically significant differences from the control sites in terms of changes in a number of clinical and microbiological indices. In the test sites the proportions of anaerobes. Gram‐negative anaerobes, spirochetes and motile bacteria decreased, as did the crevicular fluid flow, while the proportions of facultative anaerobes and cocci increased. These changes are indicative of a shift towards periodontal health. No significant differences were observed between the test and control sites in terms of bleeding on probing or pocket depth. The encouraging results of this preliminary study suggest that the use of redox agents in the treatment of chronic periodontitis warrants further investigation.
Fibrinolytic activity of human gingiva in the presence or absence of plaque bacteria
Authors: Cortellini P. , Prato G. Pini , Clauser C.
Published: July 2006

Activators of fibrinolysis are found in the gingival connective tissue and in the sulcular epithelium. The influence of plaque bacteria and the related inflammatory reaction on the fibrinolytic activity has been evaluated in the human gingiva. An autohistographic technique was applied to sections taken from three groups of 6 specimens each: group A from clinically healthy sites with plaque index = 1 and no bleeding on probing; group B from areas with unambiguous visual signs of gingivitis, with plaque index = 2–3 and bleeding on probing; group C from sites previously treated with professional toothcleaning twice a week for 3 months and with chlorhexidine mouthrinses twice a day for the final 3 wk, in order to obtain a virtually complete elimination of the plaque bacteria. In group C the plaque index was 0 and there was no bleeding on probing. Connective tissue fibrinolytic activity was present in all the sections from the three groups. The sulcular fibrinolytic activity was observed in all the sections taken from the specimens of groups A and B. In contrast, no fibrinolytic activity was observed over the sulcular area in any section taken from the specimens of group C. Therefore, this study does not support previous claims that healthy sulcular epithelium is capable of releasing activators of fibrinolysis. It can be concluded that the presence of any amount of plaque bacteria is associated with sulcular fibrinolytic activity. Contrarily, the elimination of plaque bacteria is associated with the absence of any detectable sign of fibrinolytic activity in the gingival sulcus.
Cathepsin B/L‐, elastase‐, tryptase‐, trypsin‐ and dipeptidyl peptidase IV‐like activities in gingival crevicular fluid: Correlation with clinical parameters in untreated chronic periodontitis patients
Authors: Eley B. M. , Cox S. W.
Published: July 2006

20 untreated chronic periodontitis patients were given a full periodontal examination, including measurements of probing depth (PD), clinical attachment loss (CAL), gingival index (GI), bleeding index (BI) and plaque index (Pl.I.). At a second visit, gingival crevicular fluid (GCF) was collected on filter paper strips from the deepest accessible probing site of each tooth. GCF volumes were determined and the samples eluted into buffer. Protease activities in the resulting eluates were assayed with peptidyl derivatives of 7‐amino‐4‐trifluoromethyl coumarin (AFC). Cathepsin B/L‐like activity was determined with Bz‐Val‐Lys‐Lys‐Arg‐AFC, elastase‐like activity with MeOSuc‐Ala‐Ala‐Pro‐Val‐AFC, tryptase‐like activity with Z‐Ala‐Ala‐Lys‐AFC, trypsin‐like activity with Z‐Gly‐Gly‐Arg‐AFC and dipeptidyl peptidase (DPP) IV‐like activity with Ala‐Pro‐AFC. Total enzyme activities and enzyme concentrations both correlated positively with all clinical parameters in linear regression analysis. This was true on both a patient level, using mean patient values, and a site level, using either individual patient or pooled patient data. Most of these correlations were statistically significant, although the proportion was greater for total enzyme activity than concentration. With total activities, correlations with different enzymes and parameters generally followed the order: cathepsin B/L‐>elastase‐.> DPP IV‐> trypsin‐> tryptase‐like activity and PD>CAL>GI>BI> Pl.I respectively. Total enzyme activities had good diagnostic specificity and sensitivity as predictors of clinical parameters in this cross‐sectional study, suggesting that GCF proteases might provide useful information on the periodontal condition.
Neutrophil‐mediated damage to human gingival epithelial cells
Authors: AItman Leonard C. , Baker Coralie , Fleckman Philip , Luchtel Daniel , Oda Dolphine
Published: July 2006

Polymorphonuclear leukocytes (PMNs) have been implicated in the pathogenesis of inflammatory gingivitis and periodontitis. To further study the role of PMNs in mediating gingival injury, we cocultured these cells in vitro with monolayers of human gingival epithelial cells. Scanning electron microscopy revealed that the epithelial cells were homogeneous and SDS‐PAGE/immunoblot analysis identified the presence of keratins K3, K13 and the K.6/16 pair which authenticated the oral origin of the cells. Injury to the gingival cells was determined by scanning electron microscopy and measurement of cell detachment and cytolysis. Unstimulated PMNs produced minimal lysis or detachment, but PMNs stimulated by phorbol myristate acetate produced marked epithelial cell detachment without lysis, which was time‐ and PMN‐dose‐dependent. Supernatants of activated PMNs were similarly effective, indicating that the mediator was a stable soluble substance. Elastase and cathepsin G, two neutral proteases of PMN origin, produced time‐ and concentration‐dependent detachment of gingival epithelial cells, suggesting that these enzymes may mediate this form of injury. In other studies, gingival epithelial cells were exposed to PMN myeloper‐oxidase (MPO), chloride and glucose plus glucose oxidase (GO) as a hydrogen peroxide (H2O2) generating system. The toxic oxygen species produced by this system caused lysis of the epithelial targets which was dependent on the duration of incubation and the concentrations of MPO and GO. Azide, an inhibitor of MPO, and catalase, a scavenger of H2O2, inhibited the lytic activity of this system. Scanning electron micrographs of gingival epithelial cells cocultured with activated PMNs showed lifting of the cells from the plating surface, while target cells attacked by the MPO system revealed extensive damage of cell membranes. These studies indicate that activated PMNs cause nonlytic detachment injury to gingival epithelial cells which may be mediated by digestion of their extracellular matrix by granule neutral proteases. Furthermore, PMN MPO is capable of generating toxic oxygen species which can lyse these epithelial cells. Collectively, these actions could have profound adverse effects on the function and integrity of the gingival epithelium.
Partial characterization of proteoglycans synthesized by human gingival epithelial cells in Culture
Authors: Potter‐Perigo Susan , Prather Phillip , Baker Coralie , Altman Leonard C. , Wight Thomas N.
Published: July 2006

Proteoglycans (PGs) were extracted from the [35S]‐sulfate labelled medium and cell layer of proliferating human ginigival epithelial cells and anlyzed by ion exchanged and molecular sieve chromatorgraphy, and by SDS‐PAGE. The majority of the incorporated radioactivity secreted into the medium eluted from a DEAE Sephacel ion exchange column as a single peak at 0.44 M NaCl with a small shoulder at 0.52 M NaCl. This material, when chromatographed on Sephartose CL‐6B contained two spieces – a quantitatively major peak at Kav= 0.30 (Mt≃ 235 000 on SDS‐PAGE) and a qantitatively major peak at Kav= 0.39. The major peak was sensitive to alkaline borohydride, shifting to Kav= 0.45, and nitrous acid degradtion, indicating the presence of heparan sulfate PG with glyscosaminoglycan chins with Mt≃ 26 000. The minor peak is chondroitin/dermatan sulfateP with glycpsminoglycan chains of Mt= 22 200 as indicted by sensitivity to alkaline borohydride (shifting to Kav= 0.48) and chondroitin ABC lyase digestion. The [35S]‐sulfate labelled material from the cell layer eluted in a broad peak between 0–0.50 M NaCL from DEAE Sephacel. Chromatography of this material on Sepharose CL‐6B revealed the presence of three peaks at Kav=0.20, 0.31, and 0.75. The largest peak (Kav=0.20 and Mr≃ 245 000 on SDS‐PAGE) shifted elution position to nitrous acid degradation. These results indicate that this peak contains heparan sulfate PG with glycosaminoglycan chains of Mt≃ 20000. Two peaks containing [35S]‐sulfate labelled glycosaminoglycan chains were detected by chromatography of the cell layer extract over Sepharose CL‐6B with Kavs=0.42 (Mr≃ 30 500) and 0.75 (Mr≃ 5300). The larger peak was predominately chondroitin/dermatan glycosaminoglycan as indicated by susceptibility to chondroitin ABC lyase susceptibility to nitrous acid. These results indicate that cultured human gingival epithelial cells synthesize and secrete principally heparan sulfate PGs with small amounts of chondroitin/dermatan sulfate PGs. This work will serve as a basis for future studies designed to examine those factors involved in regulation of PG synthesis by these cells.
Epithelial cell‐fibroblast interactions: modulation of extracellular matrix proteins in cultured oral cells
Authors: Hou Lein‐Tuan , Kollar Edward J. , Yaeger James A.
Published: July 2006

A model system involving co‐cultures of human gingival or periodontal ligament fibroblasts with mouse epithelial root sheath cells or human gingival eipthelial cells was used to study epithelial cell‐fibroblast interactions. Double‐labeled immunofluorescence and microfluorometry were used to investigate the expression of extracellular martix molecules of collagen type I (collagen I), type III (collagen III) and fibronectin in fibroblasts. When fibroblasts from either source were cultured alone, the fluorescence for collagen I and fibronectin ranged from strongly positive to almost negative. Collagen III staining was relatively weak compared with that of collagen I. After 2‐3 days of co‐culture, gingival fibroblasts and ligament fibroblasts adjacent to the mouse sheath cells exhibitied enhanced intracellular fluorescence for collagen I and fibronectin. Very little change was observed for collagen III. Gingival fibroblasts cultured with gingival epithelial cells showed increased fluorescence for collagen I but decreased fluorescence for fibronectin. In contrast, the fluorescence intensity for both collagen I and fibronectin in ligament fibroblasts were reduced after 3 days of co‐culture with gingival epithelial cells. Ultrastructural changes in fibroblasts co‐cultured with mouse root sheath cells included increased Golgi cisternae and vesicles and an increased abundance of rough endoplasmic reticulum, polyribosomes, secretory vesicles and pinocytotic vesicles. Thus, the expression of extracellular martix proteins and the metabolic activity of fibroblasts can be modulated by oral epithelial cells.
Change detection on longitudinal data in periodontal research
Authors: Yang Mark C. K. , Namgung Yearnok Y. , Marks Ronald G. , Magnusson Ingvar , Clark William B.
Published: July 2006

Longitudinal data of attachment level (AL) or the alveolar bone level are often used to assess the progression of periodontal disease. This paper tries to identify the most efficient method to detect the changes of AL in a general periodontal research environment; that is, a sequential decision based on multiple sites. Several existing methods suggested in the periodontal research literature such as the tolerance, running median, cusum, and regression methods as well as change‐point detection methods in the statistical literature are examined. It is found that the regression method is most convenient among the several methods that are equally effetive in change detection. Formulae, tables and their usage are discussed in detail.
Fluoridation effects on periodontal disease among adults
Authors: Grembowski David , Fiset Louis , Spadafora Agnes , Milgrom Peter
Published: July 2006

Numerous studies report that water fluoridation reduces caries in children, but little current evidence exists about fluoridation's effects on the periodontal health of adults. To address this issue, we estimated fluoridation effects on periodontal disease among 1066 Washington state employees and their spouses, aged 20 to 34, with current residences divided evenly between fluoridated and nonfluoridated communities. Subjects were interviewed by telephone to collect residence histories, personal characteristics and other data. Each subject's lifetime years of fluoridation exposure (YFE) was calculated from the person's residence history and the U.S. Centers for Disease Control's Fluoridation Census. Oral assessments were conducted to measure the extent of periodontal disease. Relative to adults with no exposure, continuous lifetime exposure reduced the probability of attachment loss from 0.87 to 0.72. Similar benefits were obtained for bleeding gingiva and calculus. The estimates of fluoridation's benefits were not influenced by selection bias due to subjects' nonparticipation in the oral assessments.
Increased oxidative product formation by peripheral blood polymorphonuclear leukocytes in human periodontal diseases
Authors: Kimura S. , Yonemura T. , Kaya H.
Published: July 2006

We examined the oxidative burst (hydrogen peroxide‐dependent oxidative product formation) of polymorphonuclear leukocytes (PMNL) in the peripheral blood from the patients with various types of periodontal diseases including localized juvenile (LJP), generalized juvenile (GJP) and adult periodontitis (AP). Heparinized peripheral blood was obtained from 15 LJP. 13 GJP and 52 AP patients and from 30 healthy control subjects. The oxidative product (2′, 7′‐dichlorofluorescein: DCF) formation of PMNL by stimulation with phorbol myristate acetate was evalauted by a rapid quantitative assay using flow cytometry. The results indicated that all patient groups contained variable populations with normal or increased DCF formation, while the control subjects exhibited DCF formation as a single population. No significant differences in average DCF formation were found among the three patient groups. Although individual patients gave various values, the average DCF formation of the three patient groups was much higher than that of the control group. Statistical analysis revealed a significant positive correlation between DCF formation and the clinical periodontal parameters on an individual basis. Futhermore, after intial periodontal treatment, DCF formation decreased to normal levels. These results suggest that the capacity of peripheral blood PMNL to mount oxidative burst reactions might reflect the inflammotory status of periodontal disease.
Transmission of Actinobacillus actinomycetemcomitans in families of adult periodontitis patients
Authors: Petit M. D. A. , Van Steenbergen T. J. M. , De Graaff J. , Van der Velden U.
Published: July 2006

At present Actinobacillus actinomycetemcomitans is regarded as an important microorganism in the etiology of some forms of periodontitis. The purpose of the present investigation was to study the number of Restriction Endonuclease Analysis (REA)‐types present in the oral cavity of A. actinomycetem‐comitans positive subjects and to study the possibility of transmission of A. actinomycetemcomitans within families of adult periodontitis patients. DNA of A. actinomycetemcomitans isolates was digested with a combination of the restriction endonucleases Pst I and Bam HI, after which the DNA fragments were separated by agarose gelelectrophoresis. To study the number of REA‐types, multiple A. actinomycetemcomitans isolates obtained from 8 different sites in the oral cavity of five subjects were typed. The results showed that in most cases only one REA‐type is present. In the 13 families investigated in 4 of the 26 children (15%) and in 1 of the 13 spouses (8%) of the adult periodontitis patients an indistinguishable REA‐type was found within the families. This suggests that also in the case of adult periodontitis transmission of A. actinomycetemcomitans is possible, but does not seem to occur easily.
Effect of anaerobiosis and sulfide on killing of bacteria by polymorphonuclear leukocytes
Authors: Granlund‐Edstedt Margareta , Johansson Eva , Claesson Rolf , Carlsson Jan
Published: July 2006

Anaerobic microorganisms in periodontal pockets produce toxic amounts of hydrogen sulfide. The capacity of polymorphonuclear leukocytes to kill a capsulated and a non‐capsulated variant of a group B streptococcal strain was studied in presence and absence of sulfide. The killing was equally efficient under aerobic and anaerobic conditions. However, in presence of sulfide the killing of the capsulated variant of the strain was significantly inhibited. Since this strain required higher serum concentrations to be killed by the polymorphonuclear leukocytes, it suggested that sulfide interfered with the opsonization of the bacteria. The capacity of sulfide to split the disulfide bonds of complement factor 3 and immunoglobulin G, deposited on the bacterial surface, was evaluated by sodium dodecyl sulfate‐polyacrylamide gel electrophorests. There was no detectable effect of 2mM sulfide on immunoglobulin G. However, sulfide released from opsonized bacteria the β‐chain of C3b C3bi, and the C‐terminal part of the α‐chain of C3bi. This region of the α‐chain of C3bi has been suggested to bind to the complement receptor 3 of polymorphonuclear leukocytes. The β‐chain of C3b/C3bi may augment the binding of opsonized bacteria to the complement receptors of polymorphonuclear leukocytes. The formation of sulfide by the microflora of the periodontal pockets may provide conditions for the bacteria to escape important parts of the host immune system.
Expression of TIMP‐1, TIMP‐2 and collagenase mRNA in periodontitis‐affected human gingival tissue
Authors: Nomura Takashi , Takahashi Tokuya , Hara Kohji
Published: July 2006

Collagenolysis in periodontitis is thought to be modulated by the expression of three genes, collagenase, tissue inhibitors of metalloproteinases‐1 and ‐2 (TIMP‐1 and ‐2). We assessed the possible difference in TIMP‐1, TIMP‐2 and collagenase mRNA levels between gingival samples from patients with periodontitis and those from healthy subjects by reverse transcription‐polymerase chain reaction (RT‐PCR). This technique allows detection of transcripts from a very small sample quantity. The experiments showed that levels of TIMP‐1 and collagenase transcripts relative to β‐action are significantly higher in the diseased group than in healthy controls (8.11±0.83 versus 1.38±0.28% for TIMP‐1 and 0.50±0.10 versus 0.0075±0.0024% for collagenase, respectively). The difference in TIMP‐2 between the two groups (2.91±0.46 versus 1.84±0.87%) did not differ. Therefore, the host would have responded to the increase in collagenase level by preferentially producing TIMP‐1 against tissue destruction. The differential gene expression of TIMP‐1 and TIMP‐2 in our study may account for a distinct genetic regulation of TIMP‐1 and ‐2 in vivo.
Inhibition of epithelial cell matrix metalloproteinases by tetracyclines
Authors: Nip L. H. , Uitto V.‐J. , Golub L. M.
Published: July 2006

The effects of tetracyclines on periodontal epithelial cells were investigated by culturing cells from porcine rests of Malassez in the presence of oxytetracycline, doxycycline or one of two analogues of tetracycline bearing no antimicrobial activity. Matrix metalloproteinase activity produced by the epithelial cells was assayed by quantitation of radioactive gelatin degradation and by gelatin enzymography. The results show that all tested tetracyclines exerted a direct dose‐dependent inhibitory effect on epithelial cell gelatinases. Furthermore, epithelial cells cultured with doxycycline, oxytetracycline and de‐dimethylaminot‐etracycline in concentrations ranging from 1 to 50 μg/ml showed a marked reduction in secreted gelatinase activity when grown in alpha minimum essential medium in the absence of fetal calf serum. Viability of cells following this treatment, measured as lactate dehydrogenase activity released to the cell media, was not affected by the presence of any of these drugs at the concentrations used. Scanning electron microscopy revealed striking morphologic changes of the cells following treatment with tetracyclines in the absence of serum which include rounding, decreased intercellular contacts and increased intercellular spaces. No such effects were seen in cells cultured in the presence of serum. These results provide evidence that periodontal epithelial cells produce matrix metalloproteinases whose activities are inhibited by tetracyclines and their non‐antimicrobial analogues at concentrations present in gingival crevicular fluid following tetracycline therapy. When used as adjuncts in periodontal therapy, tetracyclines may therefore inhibit epithelial cell mediated degradation of basement membrane and subepithelial connective tissue.
Changes in crevicular fluid levels of interleukin‐1β, leukotriene B4, prostaglandin E2, thromboxane B2 and tumour necrosis factor α in experimental gingivitis in humans
Authors: Heasman P. A. , Collins J. G. , Offenbacher S.
Published: July 2006

We conducted a study to determine the sequence of cytokine and lipid mediator activation within the periodontium during the development of experimental gingivitis in humans. Crevicular fluid samples were collected from 7 previously cleaned, healthy patients at baseline, 1, 2, 3 and 4 weeks following the cessation of all oral hygiene measures. Gingival and plaque scores were taken at each visit to follow the development of gingivitis and plaque retention. Crevicular fluid samples were assayed in quadruplicate for prostaglandin E2 (PGE2), thromboxane B2 (TxB2), leukotriene B4, interleukin‐1β (IL‐1β) and tumour necrosis factor alpha (TNFα) by RIAs or ELISAs, maintaining site pairing to examine temporal changes. Redness scores (expressed as mean percentage of sites with redness present) increased almost linearly from a baseline value of zero to approximately 40% at 1 week, 59% at 2 weeks, 83% at 3 weeks and 100% at 4 weeks. The mean % of sites with bleeding on probing increased gradually from zero at baseline to 5% at 3 weeks and then rose abruptly to 25% at 4 weeks. CF‐PGE2 and CF‐TxB2 levels remained fairly stable at baseline levels for the first 3 weeks then rose sharply by 2.5‐ and 2‐fold, respectively, at 4 weeks. CF‐LTB4 levels increased 2‐fold by 1 week and 4‐fold by 4 weeks. The CF levels of IL‐1β increased abruptly from mean baseline values of 16.5±9.3 ng/ml to 131±76.0 ng/ml at 1 week and remained at this level for the duration of the experiment. CF‐TNFα levels were non‐detectable throughout the experiment (i.e. < 2 ng/ml). These data suggest that LTB4 and IL‐1β are involved in the earliest stages of gingivitis and that increases in the cyclooxygenase products PGE2 and TxB2 occur later and are associated with an increase in bleeding scores.
Analytical electron microscopy of chlorhexidine‐induced tooth stain in humans: direct evidence for metal‐induced stain
Authors: Warner R. R. , Myers M. C. , Burns J. , Mitra S.
Published: July 2006

Using analytical electron microscopy, we directly tested the hypothesis that transition metals and sulfur are the cause of chlorhexidine‐induced tooth stain. Plaque scraped from the teeth of water‐treated individuals or chlorhexidinetreated “non‐stainers” was non‐or lightly‐stained and contained low concentrations of sulfur and transition metals. Tooth scrapings from heavily‐staining chlorhexidine‐treated individuals consisted of distinct unstained and stained regions. The stained regions were organic but were in close proximity to mineralized areas. Enhanced levels of sulfur and transition metals, particularly iron, were found in stained regions, whereas unstained regions contained low sulfur and metal levels similar to the water‐treated or non‐staining individuals. Excluding decreased mineralization, the major elemental change in heavily‐stained plaque was an increase in sulfur, and to a lesser extent, iron and other transition metal. Sulphur and iron levels were directly correlated. Following chlorhexidine treatment with iron supplementation, staining was enhanced, the Fe/S ratio increased, and sulfur and iron remained correlated. These data support the hypothesis that transition metals and sulfur are the cause of chlorhexidine‐induced tooth stain. The data are consistent with chlorhexidine treatment altering the incorporation into plaque of a natural sulfur‐containing organic component of saliva or bacteria. This natural component appears to readily interact with transition metals, particularly iron, producing stained material.
A new ultrasensitive chemiluminescent assay for the site‐specific quantification of alkaline phosphatase in gingival crevicular fluid
Authors: Chapple I. L. C. , Matthews J. B. , Thorpe G. H. G. , Glenwright H. D. , Smith J. M. , Saxby M. S.
Published: July 2006

The search for markers of periodontal disease activity and progression has accelerated over the last decade, in an effort to replace existing subjective clinical measures of periodontal health status. Research is being aimed at establishing more objective and quantitative methodology, capable of rapid diagnosis prior to the appearance of clinical signs of destructive disease. Such tests need to be sensitive enough to evaluate individual periodontal sites in health as well as disease states. We report the development of a new chemiluminescent assay for the enzyme alkaline phosphatase, that is capable of quantifying the enzyme in sub‐microliter volumes of gingival crevicular fluid and serum. The technique will measure alkaline phosphatase (ALP) whilst immobilised on paper strips, without the need for an elution stage. It is simple, versatile and amenable to chair‐side use. We discuss in detail the assay procedure and have examined levels of ALP in 11 adult volunteers with clinically healthy periodontal tissues. The mean ALP concentration was 2135 IU/L for GCF and 183 IU/L for serum, a 12‐fold difference. There also appeared to be an “oral pattern” of enzyme distribution in healthy periodontal sites, with levels being higher in the anterior region of the mouth and highest in the lower anterior region.