bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of the structure of three corals on their resorption kinetics
Authors: Fricain J. Ch. , Roudier M , Rouais F. , Basse‐Cathalinat B. , Dupuy B.
Published: July 2006

The aim of this study was to investigate the influence of the structure of corals on their resorption kinetics after implantation in subcutaneous areas. Three types of coral (Porites astreoides, Montastrea annularis and Dichocenia stokesi) identical in composition but different in structure were implanted, for periods of 1 and 2 months in subcutaneous sites in OF l mice. The resorption of the implants was studied by means of qualitative (histology, scanning electron microscopy, fluorochrome labelling method) and quantitative approaches (gravimetric method). The results of the qualitative study revealed a process of irregular deterioration of the coral, linked to the detachment of crystals at the surface of the implant. The results of the quantitative study showed that the speed of resorption increases with the implantation time and the open porosity of the coral. These reactions are explained by the increase of the surface exchange area in contact with factors responsible for resorption: biological medium and cells. When considering the choice of coral as a bone substitute, these factors must be taken into account to allow the in situ maintenance of the implant over a sufficiently long period of time according to the clinical situation.
Age‐related changes of the cementogenesis in the senescence‐accelerated mouse (SAM)
Authors: Sashima Mieko , Satoh Masanobu
Published: July 2006

Cementogenesis of the molars with aging was studied using senescence accelerated mouse (SAM) which included SAMP2/Iw and SAMP8//Iw as prone strains, and SAMRl/Iw as a resistant strain. Morphometric analysis was done for the cementum thickness at 2, 6, 12 and 16 months of age on 4 parts of the mice, i.e. at the mesial (M), the distal (D), the apical (A) and the furcational (F) cementum of the maxillary first molar. SAMRl/Iw was also studied at 20 months of age. Mean cementum thickness was statistically analyzed for age and strain differences. Mean thicknesses of the M and the D cementum in SAMP2/Iw and in SAMP8//Iw were usually higher than those in SAMRl/Iw. Furthermore, there were significant differences between SAMP2/Iw and SAMRl/Iw, and between SAMP8//Iw and SAMRl/Iw, both at 12 and 16 months of age. There was no difference in mean thickness in the F cementum in either strain and at any age. In the A cementum, SAMP2/Iw displayed significantly thicker cementum than SAMRl/Iw at 6, 12 and 16 months of age. The degree of molar eruption was thought to be more accelerated in SAMP2/Iw than in SAMRl/Iw. However, the thickness of the A cementum was not different for SAMP8//Iw and SAMRl/Iw in any age group. In this study, 2 conclusions were drawn as follows: first, that SAMP2/Iw and SAMP8//Iw exhibited more accelerated cementogenesis than SAMRl/Iw in the M and the D cementum. Secondly, several factors such as intrinsic factors, occlusal forces and degree of attrition affected the cementogenesis of the M and the D, the F and the A cementum, respectively.
Interleukin‐1 alpha, interleukin‐8 and interferon‐alpha levels in gingival crevicular fluid
Authors: Mathur Ambika , Michalowicz Bryan , Castillo Monica , Aeppll Dorothy
Published: July 2006

Cytokines play an important role in the pathology associated with chronic inflammatory diseases. We measured the total amounts [picograms (pg)] and concentrations (pg/μl) of interleukin‐1 alpha (IL‐lα), interleukin‐8 (IL‐8) and interferonalpha (IFN‐α) in 20 s gingival crevicular fluid (GCF) samples obtained from 2 diseased and 2 healthy sites in 20 subjects with periodontitis, and from 2 healthy sites in 20 subjects without disease. Both the mean amount and concentration of IL‐lα were significantly higher (p < 0.001) in diseased sites compared to healthy sites in subjects with disease. The results for IL‐8 and IFN‐α differed depending on the method of reporting. Whereas the amount of IL‐8 was significantly higher (p < 0.01) in diseased sites, the mean concentration of IL‐8 was lower compared to healthy sites. The mean amount of IFN‐α was similar in health and disease; however, the concentration of IFN‐alpha was significantly lower in diseased sites (p < 0.001) corresponding to the significant increase in crevicular fluid volume (p < 0.001). There were no significant differences in the amount or concentrations of the 3 cytokines between healthy sites from subjects with disease and healthy sites from healthy controls. The total amounts of both IFN‐α and IL‐8 were correlated between healthy and diseased sites in subjects. These data suggest that, while the disease status of a site is the major determinant of the levels of these cytokines locally, subjects with high levels of IL‐8 and IFN‐α in healthy sites also tend to have high levels of these cytokines in diseased sites. Finally, both the concentrations and total amounts of IL‐8 and IFN‐α were significantly correlated in diseased sites, suggesting that levels of these two cytokines rise or fall in tandem. The combination of decreased IL‐8 and decreased IFN‐α concentrations at diseased sites may reflect the reduced anti‐bacterial host defense activity at that site.
Advanced glycation endproducts (AGEs) induce oxidant stress in the gingiva: a potential mechanism underlying accelerated periodontal disease associated with diabetes
Authors: Schmidt Ann Marie , Weidman Elliott , Lalla Evanthia , Du Yan Shi , Hori Osamu , Cao Rong , Brett Jerold G. , Lamster Ira B.
Published: July 2006

We hypothesized that one mechanism underlying advanced periodontal disease in diabetes may involve oxidant stress in the gingiva, induced by the effects of Advanced Glycation Endproducts (AGEs), the irreversible products of non‐enzymatic glycation and oxidation of proteins and lipids which accumulate in diabetic plasma and tissue. Infusion of AGE albumin, a prototypic ligand, into mice resulted in increased generation of thiobarbituric acid reactive substances (TBARS) compared with infusion of non‐glycated albumin in the gingiva, as well as in the lung, kidney and brain. Pretreatment of the animals with the antioxidants probucol or N‐acetylcysteine (NAC) prevented the generation of TBARS in the gingiva. Affinity‐purified antibody to AGEs demonstrated increased immunoreactivity for AGEs in the vasculature and connective tissues of the gingiva in streptozotocin‐induced diabetic mice compared to non‐diabetic controls. Increased immunoreactivity for AGEs was also demonstrated in the gingiva of diabetic humans compared with non‐diabetic individuals via immuno‐histochemistry and ELISA. Consistent with these data, immunohistochemistry for heme oxygenase‐1, a marker of enhanced oxidant stress, was increased in the gingival vasculature of diabetic mice and humans compared with non‐diabetic controls. These data suggest that AGEs present in diabetic gingiva may be associated with a state of enhanced oxidant stress, a potential mechanism for accelerated tissue injury.
Immunolocalization of platelet‐derived growth factor A and B chains and PDGF‐α and β receptors in human gingival wounds
Authors: Green R. J. , Usui M. L. , Hart C. E. , Ammons W. F. , Narayanan A. S.
Published: July 2006

Platelet‐derived growth factor (PDGF) is a polypeptide growth factor which has been implicated as a major mitogen involved in wound healing. The PDGF appears to promote periodontal regeneration; however, its distribution in gingival tissues is not known and how it participates in gingival wound healing is unclear. Using highly specific antibodies we have studied the distribution of PDGF A and B chains and α‐ and β‐PDGF receptors in healing human gingival wounds. Wounds were created by making a 0.75 mm deep incision in the papilla of healthy human gingiva and biopsies were obtained from the same site after 8 h and 1, 3, 7, 14 and 21 d. Frozen sections were immunostained with affinity purified antibodies. The results showed that both epithelium and fibrin clot manifested positive immunostaining for anti‐PDGF‐A and B‐chain antibodies. Staining was present in unwounded and wounded epithelia, and in the fibrin clot it appeared to be more intense for the PDGF‐A chain. Blood vessels in connective tissue were also positive while other areas were largely negative. No significant staining was detectable in healthy tissues for anti‐PDGF‐α or ‐β receptor antibodies. However, the wound site began to manifest positive immunostaining for anti‐β‐receptor antibody after 3 d of healing, became maximal at 7d, and then decreased. Our data indicate, but do not prove, that gingival epithelium may be a source of PDGF A and B chains and that the A chain may have a more prominent role to play during early stages of healing. Expression of PDGF β‐receptor appears later at the wound site, indicating that the PDGF B isomer may regulate later wound healing events.
Vital microorganisms in early supragingival dental plaque and in stimulated human saliva
Authors: Weiger R. , von Ohle C. , Decker E. , Axmann‐Krcmar D. , Netuschil L.
Published: July 2006

The aim of this study was to compare the percentage of vital microorganisms (= microbial vitality) of saliva with that of supragingival plaque both collected at various times during the early phases of de novo plaque formation. Between intervals of optimal oral hygiene, 14 healthy participants refrained from all oral hygiene measures for periods of 1, 4, 8 and 72 h. Stimulated whole saliva was collected at the beginning (= baseline) and the end of each period. Vestibular plaque was removed from teeth 13–16, and 23–26. Analysis of the pooled plaque (p) and saliva (s) samples comprised the total number of bacterial counts and colony‐forming units to estimate the percentage of viable microorganisms (PEp; PES). The microbial vitality (VFp; VFS) was determined by using a fluorescence staining to differentiate vital from dead bacterial cells. The bulk of the PES values reached 5–30%. At baseline VFS ranged between 70% and 90%. The VFS values recorded at baseline or in the presence of 1 h and 4 h‐old plaque, were significantly (α= 0.05) higher than the corresponding VFp values ranging from 5% to 30%. It was concluded that there is a considerable discrepancy between the microbial vitality of a very early dental plaque and that of whole surrounding saliva sampled at the same time. Unfavourable local environmental conditions prevailing at cervical tooth surfaces are suggested to restrain the survival of the majority of the first bacteria adhering to a particular tooth area during the early phases of supragingival plaque formation.
Histochemical study of the influence of transplanted teeth with periodontal ligament on the binding of peanut agglutinin in rat dorsal skin
Authors: Abe Yoshihiro , Hara Yoshitaka , Kato Lhachi
Published: July 2006

Our previous study showed that the expression of carbohydrate residues in junctional epithelium (JE) after resection is closely related to attachment and stratification along the dental root surface. However, the influence of the periodontal ligament on carbohydrate expression has still not been clarified. In this study we examined the relationship between the presence of periodontal ligament and the expression of carbohydrate residues on epithelium regenerating along root surfaces. We transplanted extracted rat molars with or without periodontal ligament tissue, repeatedly frozen and thawed teeth with ligament and demineralized teeth without ligament into the dorsal skin of rats. After 2, 3, 5, 7, 10, 14 or 21 d, dorsal cutaneous tissues containing transplanted teeth were resected, fixed, decalcified and embedded in paraffin. Serial sections were stained histochemically with the HRP‐conjugated lectin. peanut agglutinin (PNA) to observe the expression of carbohydrate residues in regenerating epithelium. Histochemical observation revealed that lectin binding reactions were changed from the characteristics of skin to those of JE when the regenerating epithelium was attached and stratified along the tooth with unfrozen or frozen tissue. These results suggested that the structural formation and expression of PNA in regenerating epithelium around the root surface were influenced by not only the tooth but also by the periodontal ligament.
Immunohistochemical study of cathepsin G and medullasin in inflamed gingival tissues from periodontal patients
Authors: Kunimatsu K. , Ozaki Y. , Hara Y. , Aoki Y. , Yamamoto K. , Kato I.
Published: July 2006

Cathepsin G and medullasin are 2 major serine proteinases associated with the granular fraction of polymorphonuclear leukocytes (PMNs). To know their possible involvement in the pathophysiological gingival connective tissue turnover, we have determined the distribution and localization of these 2 enzymes in inflamed gingival tissues from periodontal patients by immunohistochemistry with discriminating antibodies specific for each enzyme. The gingival connective tissues were obtained from periodontitis patients with various inflammatory conditions and control healthy subjects without any clinical signs of periodontal inflammation. In all gingival specimens examined, cathepsin G and medullasin were found mainly in neutrophil‐like cells and partly in macrophage‐like cells. No positive staining for both enzymes was obtained in endothelial cells and fibroblasts in every part of the gingival tissues. Immunoreactivity for each enzyme in the gingival tissues from the periodontitis group was stronger and greater in the intensity and frequency than that from the control group and appeared to be increased with the severity of the disease. In both groups, the number of immunoreactive cells for each enzyme was greater in the vicinity of pocket epithelium (zone I) than in the area of central connective tissue (zone II) or the area subjacent to the oral epithelium (zone III). While both enzymes in zones II and III were exclusively found in coarse granules, their stainings in zone I were not only coarse but also diffuse. These results strongly suggest that both enzymes may have some association with inflamed gingival tissue degradation.
Expression of osteocalcin in cementoblasts forming acellular cementum
Authors: Kagayama M. , Li H.C. , Zhu J. , Sasano Y. , Hatakeyama Y. , Mizoguchi I.
Published: July 2006

To determine the phenotypic expression of cementoblasts responsible for acellular cementum, an immunohistochemical study was performed using a polyclonal antibody raised against the aminoterminal peptide of rat osteocalcin (OC). Maxillary first molars of Wistar male rats aged 2 and 3 wk were used for observations. Serial sections of decalcified paraffin embedded specimens were stained either with hematoxylin and eosin or with the anti‐OC antibody. In 2‐wk‐old rats, apical roots were lined with the epithelial root sheath. A thin layer of acellular cementum was seen at most of the root surface, but was not seen near to root apex. In 3‐wk‐old rats, cellular cementum began to be formed at root apex, and acellular cementum became more thick than in 2‐wk‐old rats. Acellular and cellular cementum were lined with the fibroblast‐like cells. Osteocalcin staining was detected in cells lining root surface in both 2‐ and 3‐wk‐old rats. Almost all cells lining cellular cementum were positive for OC. In contrast OC positive cells lining acellular cementum and root surface devoid of cementum appeared at a specific site of the root. The cells at the interradicular area of root surface were positive but the cells at the outer area (the opposite side of the interradicular area) were negative for OC. Osteoblasts and odontoblasts were positive with the antibody. The present results suggest that the OC expression of cementoblasts forming acellular cementum is similar to that of cells forming cellular cementum as well as osteoblasts and odontoblasts, and has a role for calcification of acellular cementum.
Coating of titanium alloy with soluble laminin‐5 promotes cell attachment and hemidesmosome assembly in gingival epithelial cells: potential application to dental implants
Authors: Tamura R. N. , Oda D. , Quaranta V. , Plopper G. , Lambert R. , Glaser S. , Jones J. C. R.
Published: July 2006

The formation of a biological seal around the transmucosal portion of dental implants may be crucial for the long‐term success of these therapies. Data to date suggest that the gingival epithelium attaches to dental implants through the formation of hemidesmosomes. Biochemical and genetic data indicate that the laminin isoform, laminin‐5, a component of basement membranes, plays a crucial role in the assembly and maintenance of hemidesmosomes. We report the use of soluble laminin‐5 as a biological coating of titanium‐alloy to promote cell attachment of the gingival epithelial cell line, IHGK. Monoclonal antibodies reactive with laminin‐5 depleted the coating solution of all cell attachment activity and blocked cell attachment to laminin‐5‐coated disks. Immunodepletion with antibodies to fibronectin had no effect. Finally, we demonstrate that IHGK cells assembled hemidesmosomes within 24 h of attachment to laminin‐5‐coated titanium alloy but not to the titanium alloy alone. These results suggest that soluble laminin‐5 may have clinical applications as a dental implant coating to promote the formation of a biological seal.
Hen egg‐white lysozyme inhibits biological activities of lipopolysaccharides from periodontopathic bacteria
Authors: Ito H.‐O. , Hirata M. , Koga T.
Published: July 2006

Lysozyme has a bactericidal activity for some strains of Gram‐positive bacteria, by enzymatic cleavage of peptidoglycans that constitute the cell wall. Hen egg‐white lysozyme (HEL) was tested in vitro for effects on biological activities of lipopolysaccharides from periodontopathic Gram‐negative bacteria, Actinobacillus actinomycetemcomitans, Prevotella intermedia and Porphyromonas gingivalis. HEL inhibited a wide range of activities of these lipopolysaccharides, including activation of Limulus amoebocyte lysate, stimulation of human leukocytes to secrete tumour necrosis factor‐alpha, polyclonal activation of mouse B cells, and promotion of osteoclastic differentiation in mouse bone marrow cultures. The anti‐endotoxic activity of HEL may be worthy of being intended for periodontal therapy.
Is periodontal breakdown a fractal process? Simulations using the Weierstrass‐Mandelbrot function
Authors: Landini G.
Published: July 2006

This paper introduces a theoretical model of periodontal disease that is multifactorial, cumulative and produces periodontal breakdown in “bursts and remissions”. The simulation is based on the generalization of the Weierstrass‐Mandelbrot function as an integration of a series of sinusoid fluctuations that facilitate or prevent periodontal breakdown with different frequencies, amplitudes and phases. The breakdown is produced when the integration of the factors reaches a certain threshold and is stopped when it is below it. The zeroset of the function (the set of points of the function in intersection with the time axis) is a self‐similar set that corresponds to the instances when the process switches between destructive and non‐destructive phases, and its fractal nature indicates that in theory, bursts of destruction do not have a characteristic duration size. If the mechanism of periodontal disease has in principle similarities to the model presented, then accurate site‐specific predictions about periodontal destruction may prove an unrealizable task.
Effect of tension‐force on plasminogen activator activity from human periodontal ligament cells
Authors: Yamaguchi M. , Shimizu N. , Ozawa Y. , Saito K. , Miura S. , Takiguchi H. , Iwasawa T. , Abiko Y.
Published: July 2006

The plasminogen activator (PA)‐plasmin proteolytic system has recently received considerable attention because of its participation in a wide variety of biological activities and in pathological conditions involving tissue destruction. Excessive mechanical stress such as occlusal trauma is associated with alveolar bone loss in severe periodontitis. Therefore, mechanical stress may involve degradation of the extracellular matrix by occlusal trauma through activation of the PA‐plasmin proteolytic system. We examined the effects of mechanical stress on PA activity, gene expressions of tissue type (t) PA, urokinase type (u) PA and PA inhibitor‐1 (PAI‐1) in human PDL cells. Human PDL cells were cultured on flexible‐bottomed culture plates and placed on a Flexercell Strain Unit. The cells were flexed at 6 cycles (5 s strain, 5 s relaxation) at 9% and 18% elongation for 5 d. Application of tension‐force induced significantly higher PA activity in stressed PDL cells than in non‐stressed controls, and did so in a time‐ and magnitude‐dependent manner (p<0.001, ANOVA). Western‐blot analysis revealed that the high level of activity was due to tPA and not uPA. Gene expression of tPA mRNA in stressed PDL cells, as examined by RT‐PCR, increased on d 5. These findings suggest that tPA may be involved in periodontal metabolism in response to mechanical stress.
Bacterial metabolites sodium butyrate and propionate inhibit epithelial cell growth in vitro
Authors: Pöllänen M. T. , Overman D. O. , Salonen J. I.
Published: July 2006

The structural and functional barrier preventing the free advancement of microbial plaque subgingivally along the tooth surface is formed by the junctional epithelial (JE) cells directly attached to the tooth (DAT cells). The mechanism leading to degeneration of the DAT cells is not known. In the present study we examined the possible role of short chain fatty acids (SCFAs) on epithelial cells by making use of 2 epithelial cell cultures (HaCaT and ERM) and an explant culture model of human JE. The SCFAs butyrate and propionate were used in concentrations found in human plaque and gingival crevicular fluid (0.25–16.0 mM). The SCFAs had no effect on primary cell adhesion nor on the epithelial attachment apparatus (EAA). By contrast, even 0.25 mM of butyrate significantly retarded epithelial cell growth. Similar effects with propionate were first observed at concentrations higher than 1.0 mM. The retardation of epithelial cell growth was found to be due to inhibition of cell division. Furthermore, after butyrate treatment dense accumulations of intermediate filaments and cytoplasmic vacuolization were characteristically seen in cells adjacent to cells of normal appearance. This suggests that some cells of the growing epithelial cell population are more sensitive to the SCFAs than others, and agrees with previous reports on the DAT cells of periodontally‐involved teeth in vivo. The results suggest that SCFAs are microbial factors that play a role in the initiation and progression of periodontal pocket formation by impairing epithelial cell function rather than having a direct effect on the EAA.
Intra‐familial transmission and distribution of Prevotella intermedia and Prevotella nigrescens
Authors: van Steenbergen T. J. M. , Bosch‐Tijhof C. J. , Petit M. D. A. , Van der Velden U.
Published: July 2006

The periodontal bacteria Prevotella intermedia and Prevotella nigrescens have been recently separated from each other. The purpose of this study was to investigate the distribution and routes of transmission of these bacteria among family members. Seven patients with moderate to severe periodontitis were selected. These probands, their spouses and 14 of their children were investigated. The presence of Pr. Intermedia and Pr. nigrescens was determined by culture techniques in pooled subgingival plaque samples, in the saliva, on the tongue, tonsils and buccal mucosa. Differentiation of Pr. intermedia and Pr. nigrescens was performed by enzyme electrophoretic mobility. From all 7 patients, as well as 4 spouses and 3 of the children, Pr. intermedia could be isolated. Pr. Nigrescens was found in 2 of the 7 patients, in 5 of the spouses and in 5 of the 6 children aged 5–10 yr. In the 8 children aged 0–4 yr both species were seldom isolated. These data are in accordance with earlier findings that Pr. Intermedia is associated with periodontitis and Pr. Nigrescens with a relatively healthy periodontal condition. Ribotyping of bacteria was performed by hybridization of Hindlll restriction endonuclease digests of chromosomal DNA with ribosomal DNA. Isolates from unrelated individuals always had distinct ribotypes. Indistinguishable ribotypes of Pr. intermedia and Pr. Nigrescens were found both among married couples and among parents and children. This indicates that intrafamilial transmission of Pr. intermedia and Pr. Nigrescens is possible both between adults and between parents and children.
Statistical methods for the estimation of sensitivity and specificity of site‐specific diagnostic tests
Authors: Ahn C.
Published: July 2006

The performance of periodontal diagnostic tests is often evaluated by estimating their sensitivity and specificity with respect to a traditionally accepted standard test regarded as a “gold standard” in making the diagnosis. Correlated samples of binary data arise in dental research. The fundamental unit for analysis is occasionally the site rather than the patient in site‐specific dental studies. Statistical methods that take into account the within‐patient correlation should be employed to estimate the sensitivity and the specificity of diagnostic tests since site‐specific results within a patient can be highly correlated. Several statistical methods are introduced for the estimation of the sensitivity and the specificity of site‐specific diagnostic tests; these techniques are applied to the data from a study involving an enzymatic diagnostic test to motivate and illustrate the estimation of the sensitivity and the specificity of periodontal diagnostic tests.
Identification of calprotectin, a calcium binding leukocyte protein, in human dental calculus matrix
Authors: Kido J. , Nishikawa S. , Ishida H. , Yamashita K. , Kitamura S. , Kohri K. , Nagata T.
Published: July 2006

Calprotectin is a calcium binding protein produced by leukocytes, macrophages and epithelial cells, and its levels in several tissues increase during infections and in many inflamed areas, suggesting that it may be an indicator of inflammatory activity. Osteopontin is a prominent phosphorylated glycoprotein in bone matrix, having calcium binding capacity. Recently, it has been reported that calprotectin and osteopontin are present in urinary stones (pathological mineralized masses in the body), and that these proteins may be involved in their formation. Dental calculus formed by mineralization of dental plaque is an inflammatory factor which may contribute to periodontal disease. It contains many organic components involved in mineralization. We recently found osteopontin molecules in human dental calculus and suggested that the components of its matrix may be similar to those of urinary stones. In this study, we investigated the presence of calprotectin in human dental calculus by immunohistochemical and immunoblotting analyses using a specific antibody for calprotectin. After fixation and demineralization of dental calculi adhered to tooth roots, sections embedded in paraffin were immunoreacted with the antibody for calprotectin and positive immunostaining for calprotectin was observed. Dental calculus proteins were then extracted with EDTA and separated by electrophoresis on 15% polyacrylamide gels. By immunoblotting analysis, 3 or 4 bands were observed at 11, 14.5, 22–25, 28 or 36.5 kDa and these patterns corresponded to those of calprotectin subunits. When non‐immune rabbit serum was used instead of calprotectin‐specific antibody as a negative control, no immunoreactivity was observed. These findings indicate that calprotectin is associated not only with antibacterial action but also with calcium binding capacity during dental calculus formation.
Histomorphological and biochemical differentiation capacity in organotypic co‐cultures of primary gingival cells
Authors: Tomakidi P. , Fusenig N. E. , Kohl A. , Komposch G.
Published: July 2006

To establish a three‐dimensional in vitro test system mimicking the physiological situation of the oral cavity, organotypic co‐cultures consisting of primary gingival cells on a collagen matrix with fibroblasts were generated. The histomorphological development after 7 and 14 d revealed close similarity with the non‐keratinized gingiva epithelium. Furthermore, as epithelial specific markers synthesis and localization of keratins as well as the deposition of basement membrane components were assessed on frozen sections by immunofluorescence and keratin expression by in situ hybridization. Primary keratinocytes in conventional culture stained positive for keratin K14 and the mucosal differentiation‐specific keratins K4 and K13, while primary fibroblasts, isolated from the same tissue source, and also some keratinocytes, were positive for vimentin. In organotypic co‐cultures the keratinocytes formed a multilayered epithelium within 14 d containing basal cells and flattened cells in the uppermost layers. Comparable to native non‐keratinized gingiva keratin 14 gene expression was clearly detectable in the basal cell compartment but showed extending immunolocalization. In addition, particularly at the early stage (7d), basally located keratinocytes were also vimentin positive. According to morphological differentiation K4 and K13 were detectable in suprabasal position at the RNA and protein level. The major basement membrane constituents collagen type IV and laminin increased with time revealing first an interrupted and later a fully extended staining underneath the basal cells. Maintenance of basal cell function was further demonstrated by cell proliferation (BrdU incorporation) which was initially high (7d) but declined towards the later stages (14–21 d). The results demonstrate i) that this co‐culture system leads to a stratified surface epithelium with morphological and biochemical characteristics of the non‐keratinized gingiva epithelium and ii) that a state of physiological tissue balance was reached, thus rendering a suitable model for tissue compatibility studies.
Furcation involvement: comparison of dental radiographs and HR‐CT‐slices in human specimens
Authors: Fuhrmann R. A. W. , Bücker A. , Diedrich P. R.
Published: July 2006

In this in vitro study we compared dental radiographs and high resolution computed tomography (HR‐CT) regarding identification and classification of the degree of horizontal and vertical furcation involvement. After removal of the soft tissue and metallic restorations of 18 dentate upper and lower jaws in the interradicular furcation region of 28 molars, bony defects of different dimensions were experimentally produced. The specimens were examined radiographically with standardized dental radiographs and 1.0 mm thick contiguous axial CT‐scans. After identification of molars with artificial furcation involvement in the dental radiographs and axial CT‐scans, the horizontal and vertical grades of furcation involvement were classified. Radiological identification and classification were compared with the macroscopic findings in the specimens. For quantitative histological‐radiological comparison, corresponding microsections were prepared in the same plane as the axial CT‐scans. In the dental radiographs the artificial furcation involvement in 6 of 28 (21%) molars was identified. In contrast, all 28 molars with involved furcations (100%) were identified in the axial CT‐scans. The horizontal and vertical grades of furcation involvement were classified in the same way as the macroscopic findings, permitting comparison of histological sections and axial CT‐scans. The HR‐CT technique offers 3‐dimensional assessment of the interradicular bone morphology in furcation involvement without overlying structures. The periradicular alveolar bone can be assessed on all sides of the roots. HR‐CT scanning thus permits a high identification rate and classification of molars with involved furcations.
Components of a randomized clinical trial
Authors: Dennison David K.
Published: July 2006

The purpose of this paper is to discuss a number of components associated with the design, development and conduct of a randomized clinical trial (RCT). In a RCT, a patient is entered into a protocol in which therapy is randomly assigned. Thus, neither the patient nor the care provider have active roles in deciding treatment. The elements of informed consent were developed to ensure that the patient's interests are protected. The informed consent document serves as the cornerstone of a clinical study by introducing patients to the research protocol, informing them of their role as participants in the study, and educating them of their rights. Questions regarding ethics arise during all phases of a study. A study designed with insufficient sample size will provide inconclusive data, thus making participation in the study of limited to no value. Conversely, having patients enrolled in a RCT after data has proved superiority of an arm of the study postpones beneficial therapy for patients and society. Other ethical conflicts arise when subjects are recruited for clinical trials. Special care must be taken to avoid coercion in protected populations such as students, employees, the mentally challenged, and the indigent. Careful planning with adequate attention to consent form development can minimize problems and optimize patient care and research integrity.

The role of the RCT in the future is unclear. The logistics of conducting a multicentered trial has escalated the costs of new agents in terms of both time and money. In many cases, the decision to conduct a RCT is made on the potential profitability of the product/agent under test. Whatever the reasons credited for conducting an RCT, the research must be adequately designed to protect both the subjects involved in the study, as well as humankind from erroneous data.
Prostaglandin E2 and interleukin‐1 concentrations in nicotine‐exposed oral keratinocyte cultures
Authors: Johnson G. K. , Organ C. C.
Published: July 2006

Oral keratinocytes are the first cells in contact with tobacco components and are capable of producing various inflammatory mediators, including PGE2 and IL‐1. The purpose of this study was to examine PGE2 and IL‐1 concentrations in nicotine‐exposed oral keratinocyte cultures. Gingival keratinocyte cultures were established from healthy gingival tissues obtained from 7 subjects. Cultures were divided into 4 groups exposed to serum free medium (control), 0.1 μm, 10 μm or 1 mM nicotine for 4, 24 or 48 h. Using enzyme‐linked immunosorbent assays, PGE2 and IL‐1α were quantified in culture supernatants; IL‐1 α and β were also measured in lysed cells. A repeated measures analysis of variance was used to identify significant differences over time and treatment. Nicotine exposure did not significantly alter PGE2 levels at any given time period; however, PGE 2 quantities declined significantly (p=0.0001) over time. At both 24 and 48 h, IL‐1 α concentrations in lysates from 1 mM nicotine‐exposed cells were significantly (p < 0.01) greater than those for all other treatments. Interleukin‐1α quantities also declined significantly (p=0.037) over time in the cultures. Interleukin‐1β concentrations were elevated, albeit not significantly, in the 1 mM treated cells at 24 and 48 h. Cell viability, mass and counts were not affected by nicotine treatment; these parameters increased significantly (p < 0.005) over time. In summary, nicotine treatment significantly increased IL‐1 a concentrations in cultured keratinocytes; however, PGE2 synthesis was not altered. Elevated IL‐1 production by keratinocytes may have implications in tobacco‐induced lesions, given the central role IL‐1 plays in tissue response to injury.
Lipoproteins of Treponema denticola: their effect on human polymorphonuclear neutrophils
Authors: Sela M. N. , Bolotin A. , Naor R. , Weinberg A. , Rosen G.
Published: July 2006

The presence of lipoproteins and lipooligosaccharides in Treponema denticola, an oral spirochaete associated with periodontal diseases, was investigated. T. denticola ATCC 35404 and the clinical isolate GM‐1 were metabolically labeled with [3H]‐cis‐9‐octadecenoic acid and extracted with the non‐ionic detergent Triton X‐114. The extract was phase separated, precipitated with acetone and delipidated to remove non‐covalently bound lipid (dLPP). In T. denticola ATCC 35404, sodium dodecyl sulfate polyacrylamide electrophoretic separation followed by autoradiography showed [3H]‐cis‐9‐octadecenoic acid incorporation in bands with apparent molecular masses of 14, 20, 26, 31, 38, 72 and 85 kDa and a broad band running from 113 kDa to the top of the gel. This last band resolved into a 53 kDa [3H]‐cis‐9‐octadecenoic acid band upon heating for 10 min, at 100oC. The structural relationship of the outer sheath major oligomeric polypeptide of strain ATCC 35404 and the 53 kDa protein was demonstrated immunologically. Antibodies against the 113 kDa component of the oligomer cross‐reacted with the 53 kDa protein. Proteinase K degraded the [3H]‐cis‐9‐octadecenoic acid bands with the exception of the 14 kDa. The 14 kDa was also the major [3H]‐fatty acid labeled compound found in the water phase following phenol‐water extraction of whole T. denticola ATCC 35404 cells. This compound was purified from the water phase by gel filtration followed by hydrophobic chromatography. Chemical analysis showed that hexadecanoic acid was the predominant fatty acid bound to T. denticola lipoproteins. In the GM‐1 strain [3H]‐cis‐9‐octadecenoic acid incorporation was observed in the 116 kDa and 14 kDa bands. dLPP from strain ATCC 35404 caused an enhanced (0.8–8 μg/ml) luminol dependent chemiluminiscence (LDCL) effect in human polymorphonuclear neutrophils (PMN) which could be related to protein concentration. The addition of dLPP to PMN together with FMLP at submaximal concentration (1 μM) resulted in a synergistic activation of LDCL. At 21 μg/ml, dLPP also induced lysozyme release by the PMN at approximately 30% of the release induced by the chemotactic peptide at 1 μM. In addition, dLPP (21 μg/ml) increased additively the release of lysozyme caused by 1 μM FMLP. The release of β‐glucuronidase was not affected. The modulation of neutrophil activity was abolished by preincubation of dLPP with proteinase K. The purified 14 kDa had no effect on either LDCL or exocytosis of lysosomal enzymes of PMN. These data strongly suggest that T. denticola possesses several lipoproteins including outer sheath major oligomeric polypeptides (113‐234 kDa) and a lipooligosaccharide of molecular mass of 14 kDa. In addition, an enriched lipoprotein fraction from this oral spirochaete modulates oxygen dependent and independent mechanisms for controlling microorganisms by human PMN.
In situ hybridization study on tissue inhibitors of metalloproteinases (TIMPs) mRNA‐expressing cells in human inflamed gingival tissue
Authors: Kubota Takehiko , Matsuki Yutaka , Nomura Takashi , Hara Kohji
Published: July 2006

This study presents the exact cell types and localization of tissue inhibitors of metalloproteinases (TIMPs) production sites in periodontal diseased gingiva by means of in situ hybridization. Gingival tissue specimens were fixed, embedded and hybridized in situ with specific digoxigenin‐labeled cRNA probes (386 and 496 bp). TIMP‐1 and ‐2 mRNAs were expressed on macrophages, mononuclear cells, capillary endothelial cells and some fibroblasts throughout the gingival tissue. In periodontitis, TIMP‐1 and ‐2 mRNA‐expressing cells showed significantly different localization. TIMP‐1 mRNA was broadly observed in the gingival connective tissue while TIMP‐2 mRNA was predominantly expressed in the connective tissue adjacent to the pocket epithelium (p < 0.01). Fewer TIMPs mRNA were observed in minimal gingivitis than in periodontitis, especially in the middle zone of gingival tissue. Thus, TIMP‐1 and TIMP‐2 mRNA was detected differentially and site‐specifically in periodontal diseased gingival tissue.
Gingival crevicular interleukin‐1 and interleukin‐1 receptor antagonist levels in periodontally healthy and diseased sites
Authors: Ishihara Y. , Nishihara T. , Kuroyanagi T. , Shirozu N. , Yamagishi E. , Ohguchi M. , Koide M. , Ueda N. , Amano K. , Noguchi T.
Published: July 2006

Interleukin‐l (IL‐1) molecules, IL‐lα and IL‐lβ are cytokines involved in the acute‐phase response against infection and in the pathogenesis of periodontal destruction. Administration of exogenous IL‐1 receptor antagonist (IL‐1ra) is effective in reducing the inflammatory reactions mediated by IL‐1. However, the relationship between these three naturally occurring IL‐1 molecules and periodontal diseases has been poorly characterized. We investigated the correlation of gingival crevicular IL‐1 molecules and the clinical status of patients with different severities of periodontitis. IL‐lα, IL‐1β, IL‐1ra and the total IL‐1/IL‐1ra ratio (IL‐1 activity index; IL‐1AI) were measured in 75 gingival crevicular fluid (GCF) samples from non‐inflamed gingiva sites in 2 healthy subjects and diseased sites in 7 patients with several types of periodontitis. IL‐lα, IL‐1bT and IL‐1ra were measured by specific non‐cross‐reactive enzyme linked immunosorbent assay. The probing depth, gingival index and alveolar bone loss of each site was recorded at the time of GCF sampling. The total amount of IL‐lα, IL‐1β and the IL‐1AI, but not total IL‐1ra, were found to be correlated with alveolar bone loss score. Three IL‐1 molecules were also measured in the gingival tissue of patients with periodontitis. A similar progressive decrease of the IL‐1AI was detected in gingival tissue with periodontitis. These results suggest that the amounts of both crevicular IL‐1 and IL‐1AI are closely associated with periodontal disease severity.
Reliability of a self‐reported health questionnaire in a periodontal disease study
Authors: Ho Alex W. , Grossi Sara G. , Dunford Robert G. , Genco Robert J.
Published: July 2006

This study assesses the reliability of a self‐reported health questionnaire completed by 413 subjects aged 25–74 yr in the Erie County Periodontal Disease (ECPD) Study. Specific questions on general and oral health conditions were completed by each subject during a first visit and at a follow‐up examination 2 yr later, and the two compared. Results showed that the overall measure of agreement between the two visits is substantial (average kappa, k=0.80). Variation by gender and age were minimal. Questions regarding allergy to medications, oral treatment, reason for tooth extraction, health symptoms and history of systemic diseases exhibited high levels of agreement (kappa ranged from 0.71–0.90). Information on vitamin and mineral intake yielded k= 0.63. Oral conditions scored the lowest but were still acceptable (k= 0.57). These findings indicate that there were no significant discrepancies in self‐reported responses to the health questionnaire used in the ECPD Study. Although the information provided by the subject may not be as accurate as compared to laboratory testing, it is nevertheless a reliable source of information which can be utilized cost‐effectively in research studies.
The relationship between radiographic and clinical changes in the periodontium
Authors: Machtei Eli E. , Hausmann Ernest , Grossi Sara G. , Dunford Robert , Genco Robert J.
Published: July 2006

Change in clinical attachment level (CAL) and radiographic change in crestal bone height are often used to assess periodontal breakdown and disease progression. These two variables are also used to monitor the effect of treatment. The purpose of the present longitudinal study was to evaluate the correlation between changes in CAL and alveolar bone loss. Following initial screening, 79 subjects with established periodontitis were monitored quarterly for 1 yr, using a pressure‐sensitive automated probe. CAL and relative attachment level (RAL) were recorded at 6 sites for each tooth. Radiographs were obtained at baseline and 1 yr. Crestal bone changes were determined using an image enhancement technique. Mean change in attachment level was 0.16 mm. Similarly, mean proximal bone loss measured radiographically was 0.16 mm. In 6.9% of all the sites, and 13.7% of all pooled interproximal sites, AL loss was in excess of the threshold defined as 2 s.d. of repeated measurements (mean 1.54 mm). Similar percentages of sites (12.9%) had radiographic evidence of proximal bone loss exceeding the threshold (0.55–1.08 mm). A site‐based analysis of active sites revealed an overall poor correlation between the 2 variables (kappa value=0.03) which was the result of a very poor sensitivity (0.16) despite a relatively good specificity (0.81). A patient‐based comparison of clinical and radiographical changes revealed an overall kappa value of 0.08, with sensitivity and specificity of 0.51 and 0.56, respectively. However, baseline CAL and crestal bone height showed good correlation (r=0.73; p=0.0001). It is suggested that changes in CAL and radiographic bone level progress somewhat independently. Over a short‐term period of time they might not follow the same course; however, in the long term, these differences seem to level off. For longitudinal monitoring of disease progression and response to therapy both methods may be needed; while for cross‐sectional evaluation and long‐term prospective studies, either variable may be used alone.
Prevalence of Actinobacillus actinomycetemcomitans serotypes in Japanese patients with periodontitis
Authors: Yamamoto Matuo , Nishihara Tatsuji , Koseki Takeyoshi , He Tao , Yamato Kenji , Zhang Yi Jie , Nakashima Keisuke , Oda Shigeru , Ishikawa Isao
Published: July 2006

Oral Actinobacillus actinomycetemcomitans strains are serologically classified into 5 distinct groups, a to e. We examined the distribution of A. actinomycetemcomitans serotypes in Japanese patients with periodontitis. A total of 157 A. actinomycetemcomitans clinical isolates from diseased sites of 39 patients with periodontitis were serotyped by using serotype‐specific rabbit antisera against. A. actinomycetemcomitans serotypes a, b, c, d and e strains. In the immunodiffusion assay, autoclaved extracts of 42, 6, 39, 9 and 41 A. actinomycetemcomitans clinical isolates reacted with serotypes a, b, c, d and e antisera, respectively. Although 37 patients were infected with a serotype strain, 2 patients harbored 2 different serotype strains, b/e and b/untypeable. To establish a correlation between serotype and genotype of A. actinomycetemcomitans clinical isolates from 2 patients who had different serotype strains, we used arbitrarily primed polymerase chain reaction (AP‐PCR) to fingerprint clinical isolates of different serotypes. The AP‐PCR genotype among 4 clinical isolates (b/e and b/untypeable) were identical to that of A. actinomycetecomitans Y4 (serotype b), indicating the presence of multiple A. actinomycetemcomitans serotypes which are genetically homogenous in the periodontally diseased sites of patients with periodontitis.