Location of proliferating gingival cells following toothbrushing stimulation

OBJECTIVES: Mechanical stimulation by toothbrushing promotes healing of gingivitis through accelerating cell proliferation. Junctional epithelium proliferates at periodontal pocket formation. A question is arisen whether toothbrushing contributes to the repair of gingival inflammation or deterioration of pocket formation. The location of proliferating cells in gingiva stimulated mechanically by toothbrushing was investigated. MATERIALS AND METHODS: A total of 24 teeth of dogs underwent daily plaque removal with a curette (plaque removal) or both plaque removal and toothbrushing (toothbrushing). Proliferative activity of gingival cells in six individual zones was evaluated by assaying expression of proliferating cell nuclear antigen (PCNA). RESULTS: Toothbrushing increased densities of PCNA-positive basal cells in the junctional epithelium, connective tissues adjacent to the junctional epithelium, the alveolar bone of the oral epithelial side and the oral epithelium. However, the densities of PCNA-positive cells at the apical portion of the junctional epithelium, connective tissues adjacent to the cementum and the alveolar bone of the periodontal ligament side did not increase following toothbrushing. CONCLUSIONS: Toothbrushing promotes proliferation of gingival cells other than fibroblasts in periodontium and basal cells in the apical portion of the junctional epithelium. The repair of periodontal tissues might be promoted by toothbrushing within the limit of the direct mechanical stimulation.     

牙周炎牙龈组织基质金属蛋白酶活性及蛋白表达的研究

目的：探讨来源于宿主的基质金属蛋白酶（MMPs)在牙周炎发病机制中的作用。方法：利用明胶酶活性分析（zymography)和免疫组化方法，检测15例牙周炎患者牙龈组织中和4例健康牙龈中MMP－2、MMP－9的酶活性及蛋白表达。结果：牙周炎牙龈组织以及正常的牙龈组织中均能检测到MMP－2、MMP－9的前体形式（pro-MMP-2和pro-MMP-9)，只有在牙周炎牙龈组织中发现活化形式的MMP－2，没有见到活化形式的MMP－9。免疫组化结果显示：15例成人牙周炎牙龈组织中MMP－2在炎性结缔组织中较正常牙龈阳性表达，而且经图像分析较正常牙龈组织MMP－2阳性细胞表达显著增强（P＜0．05）。结论：提示基质金属蛋白酶MMP－2参与牙周组织破坏过程，在细胞外基质的降解、牙周组织破坏过程中起重要作用。     

Keratinocyte integrins in wound healing and chronic inflammation of the human periodontiurn

Periodontal epithelium plays a critical role in protection, destruction and repair of human periodontium. During optimal repair, epithelium migrates and covers the wound surface to prevent infection and damage of the vulnerable underlying connective tissue. During periodontal destruction, junctional epithelium undergoes transformation to pocket epithelium that has quite different characteristics from junctional epithelium. In the course of periodontal disease the epithelial attachment to the tooth surface is lost and the epithelium proliferates and extends pseudo-rete ridges deep into the inflamed connective tissue. Both scenarios, repair and destruction, involve active epithelial migration either in the wound provisional matrix or in the inflamed connective tissue matrix, respectively. This review covers recent research data on cellular receptors, integrins, that mediate epithelial cell migration during wound healing and destruction of human periodontiurn.     

Syndecan‐1 immunohistochemical expression in gingival tissues of chronic periodontitis patients correlated with various putative factors

Kotsovilis S, Tseleni‐Balafouta S, Charonis A, Fourmousis I, Nikolidakis D, Vrotsos JA. Syndecan‐1 immunohistochemical expression in gingival tissues of chronic periodontitis patients correlated with various putative factors. J Periodont Res 2010; 45: 520–531. © 2010 John Wiley & Sons A/S Background and Objective: Limited information is available on the expression and distribution of syndecan‐1 within human gingival tissues/cells and on putative factors that might affect its expression. Therefore, the objective of the present study was to determine immunohistochemically the expression and distribution of syndecan‐1 in the gingival tissues of patients with chronic periodontitis and to examine the correlation of syndecan‐1 expression with various putative factors (environmental, patient/systemic and local factors). Material and Methods: Gingival specimens were surgically excised from the area of the junctional/pocket epithelium (study group 1, including 30 chronic periodontitis patients) or the gingival oral epithelium (study group 2, comprising another 30 chronic periodontitis patients), adjacent to teeth with poor prognosis. Standard two‐step immunohistochemistry and semi‐quantitative evaluation of immunohistochemical staining were used to determine syndecan‐1 expression. Statistical analyses on the impact of various putative factors were performed. Results: In the junctional/pocket epithelium or the oral epithelium, syndecan‐1 expression was weak to moderate in the suprabasal and basal epithelial cells and absent to weak in the internal basal lamina, external basal lamina and gingival connective tissue matrix. Syndecan‐1 expression in the junctional/pocket epithelium was statistically significantly stronger than in the oral epithelium in inflammatory cells within the underlying gingival connective tissue (primarily plasma cells and lymphocytes) and in scattered fibroblast‐like cells. Conclusions: Syndecan‐1 expression in the junctional/pocket epithelium or the oral epithelium can exhibit a significant positive correlation with the severity/degree of histologically evaluated local gingival inflammation, but in general is not significantly correlated with age, smoking, full‐mouth and local clinical (probing pocket depth and clinical attachment level) and radiographical parameters (radiographical bone loss) of periodontal status.     

Immunohistochemical study of cathepsin G and medullasin in inflamed gingival tissues from periodontal patients

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.     

Periodontal probing: What does it mean?

Abstract The periodontal probe has been and continues to be used as an important diagnostic instrument by the dental profession. The measurements recorded with the probe have generally been considered to represent a reasonably accurate estimate of sulcus or pocket depth. Recent reports on the histopathology of the periodontal lesion and the histological features of a healing lesion, together with histological studies on the relationship of the probe to periodontal tissues, have shed some new light on periodontal probing. It is now apparent that probing depth measured from the gingival margin seldom corresponds to sulcus or pocket depth. The discrepancy is least in the absence of inflammatory changes and increases with increasing degrees of inflammation. In the presence of periodontitis the probe tip passes through the inflamed tissues to stop at the level of the most coronal intact dento-gingival fibers, approximately 0.3–0.5 mm apical to the apical termination of the junctional epithelium. Decreased probing depth measurements following periodontal therapy may be due in part to decreased penetrability of the gingival tissues by the probe. Following treatment aimed at obtaining new attachment in periodontal defects, wider variations may occur between the location of the probe tip and the most coronal dento-gingival fibers than in the case of untreated sites. This is due in part to the formation of a so-called “long” junctional epithelium. In the absence of inflammation this epithelium may not be penetrable during ordinary probing, but could account for a rapid increase in probing depth measurements when inflammatory changes allow the probe to traverse the epithelium and/or the adjacent infiltrated connective tissue. In view of the difficulty inherent in relating periodontal probing measurements to actual sulcus or pocket depth, the interpretation of periodontal probing in the practice of periodontics may need reappraisal.     

Age-dependent changes in the distribution of BrdU- and TUNEL-positive cells in the murine gingival tissue.

BACKGROUND: Age-dependent morphological and cell kinetic changes of the gingival tissue seem to be related to the occurrence of periodontal disease. The purpose of this study was to investigate the age-dependent changes in the distribution of BrdU- and TUNEL-positive cells in murine gingival tissue. METHODS: Gingival tissue of the lower first molar region of 2-, 3-, 5-, 7-, 10-, 15-, 20-, 30-, 40-, 50-, 60-, 70- and 80-week-old mice was used in this study. BrdU- and TUNEL-positive cells were evaluated at the following 4 sites: 1) gingival epithelium (GE); 2) junctional epithelium (JE); 3) submucosal connective tissue of the gingival epithelium (GECT); and 4) submucosal connective tissue of the junctional epithelium (JECT). RESULTS: No significant differences in the mean number of BrdU-positive cells at each site were demonstrated among the various age groups. No significant change in the mean number of TUNEL-positive cells was demonstrated in either the GE or JE groups among the various age groups. Meanwhile, a significant increase in the TUNEL-positive cells was observed in the GECT of mice 40 weeks or older, and in the JECT of mice 20 weeks or older. CONCLUSIONS: These results indicate that no age-dependent change in the cell proliferation or cell death occurred in the gingival and junctional epithelial layers as well as in the cell proliferation in the submucosal connective tissue. Meanwhile, a significant decrease in the cellular component of the submucosal connective tissue of both gingival and junctional epithelial layers caused by apoptosis occurred with aging. The decreased cellular component in the submucosal connective tissue thus seems to be related to either gingival recession or to the apical migration of the JE with aging. These morphological changes with aging possibly occur in humans and may be related to the susceptibility to periodontal disease in aged individuals.     

Bacterial invasion of gingiva in advanced periodontitis in humans

Using scanning electron microscopy, bacteria were found invading the epithelial wall of deep periodontal pockets in five out of eight cases. In one case, bacteria had reached the connective tissue. The following bacterial morphotypes were identified: cocci, short rods, filaments and spirochetes. A corncob-like formation with a few scattered spirochetes was found in one case between the junctional epithelial cells. Numerous cocci, rods and filaments were found in intercellular spaces of the epithelium in the lateral wall of the pocket and accumulating on the epithelial side of the basement lamina. Bacterial invasion of gingival tissue may have important pathogenic and therapeutic implications.     

The periodontal pocket: pathogenesis,histopathology and consequences

The conversion of junctional epithelium to pocket epithelium is regarded as a hallmark in the development of periodontitis. Knowledge of factors contributing to the initiation and progression of pocket formation is important and may result in the development of better preventive measures and improve healing outcomes after therapeutic interventions. The periodontal pocket is a pathologically deepened gingival sulcus. In healthy periodontal conditions, the defense mechanisms are generally sufficient to control the constant microbiological challenge through a normally functioning junctional epithelium and the concentrated powerful mass of inflammatory and immune cells and macromolecules transmigrating through this epithelium. In contrast, destruction of the structural integrity of the junctional epithelium, which includes disruption of cell‐to‐cell contacts and detachment from the tooth surface, consequently leading to pocket formation, disequilibrates this delicate defense system. Deepening of the pocket apically, and also horizontal expansion of the biofilm on the tooth root, puts this system to a grueling test. There is no more this powerful concentration of defense cells and macromolecules that are discharged at the sulcus bottom and that face a relatively small biofilm surface in the gingival sulcus. In a pocket situation, the defense cells and the macromolecules are directly discharged into the periodontal pocket and the majority of epithelial cells directly face the biofilm. The thinning of the epithelium and its ulceration increase the chance for invasion of microorganisms and their products into the soft connective tissue and this aggravates the situation. Depending on the severity and duration of disease, a vicious circle may develop in the pocket environment, which is difficult or impossible to break without therapeutic intervention.     

Localization of plasminogen activators and plasminogen-activator inhibitors in human gingival tissues demonstrated by immunohistochemistry and in situ hybridization

The plasminogen-activating system plays an important part in tissue proteolysis in physiological as well as pathological processes. Plasminogen activators u-PA (urokinase) and t-PA (tissue) as well as the inhibitors PAI-1 and PAI-2 are present in gingival crevicular fluid in concentrations significantly greater than in plasma. This fact, and the finding that the concentrations of t-PA and PAI-2 are higher in areas with gingival inflammation, indicate local production of these components. The present study describes, by means of in situ hybridization and immunohistochemistry, the localization of the plasminogen activators and their inhibitors in gingival tissues from patients undergoing periodontal surgery. t-PA mRNA and t-PA antigen were primarily found in the epithelial tissues, predominantly in the sulcular and junctional regions, although occasionally in the oral epithelium and in blood vessels of the connective tissue. u-PA and u-PA-receptor signals were seen in single cells within the junctional and sulcular epithelia and adjacent to blood vessels close to the junctional epithelium, but rarely in the oral epithelium. Similar to t-PA, the predominant location of PAI-2 mRNA was the gingival epithelia. In the junctional and sulcular epithelia, PAI-2 mRNA was seen throughout the thickness, while in the oral epithelium the strongest signals were seen in stratum granulosum and stratum spinosum. PAI-1 mRNA was invariably found in the connective tissue associated with blood vessels. The present study confirms earlier indications of local production of plasminogen activators and their inhibitors in gingival tissues. In addition, the results demonstrate that t-PA and PAI-2 in these patients are produced predominantly in the epithelial tissues. Furthermore, the presence of t-PA and PAI-2 seems to be most pronounced in the areas likely to be subjected to bacterial assault.     

MMP-2在吸烟牙周炎患者牙龈组织中的表达

目的:从MMP-2的酶活性和mRNA水平探讨吸烟与牙周病的关系。方法:利用明胶酶活性分析(zymog-raphy)和RT-PCR方法,分别检测6例吸烟牙周病、6例不吸烟牙周病、6例吸烟正常人、6例不吸烟正常人的牙龈组织中MMP-2的酶活性和mRNA表达。结果:吸烟和牙周病牙龈组织中MMP-2酶活性都较正常组有增加,但吸烟牙周病组的MMP-2的酶活性与吸烟无牙周病组和不吸烟牙周病组有明显差异(P<0.01),吸烟牙周病患者牙周组织中MMP-2的mRNA水平较正常组明显增加(P<0.01)。结论:MMP-2在吸烟牙周炎牙周组织的破坏中起重要作用。     

Localization of interleukin-1 (IL-1) mRNA-expressing macrophages in human inflamed gingiva and lL-1 activity in gingival crevicular fluid

The exact cell type and site(s) involved in interleukin-1 (lL-1) production during gingival inflammation was determined by combining immunohistochemistry and in situ hybridization. IL-1 messenger RNA (mRNA)-expressing cells in human inflamed gingiva were identified as macrophages. The rate of IL-α mRNA expression in these macrophages was the same as IL-1 β mRNA expression. The rate of IL-1 mRNA expression was higher in connective tissue furthest from the pocket epithelium, although more macrophages were present at the connective tissue subjacent to the pocket epithelium. The IL-1 activity in gingival crevicular fluid (GCF) obtained from inflamed gingiva was higher than that from healthy gingiva and decreased after periodontal therapy. The IL-1 activity in GCF was almost completely abolished by the addition of anti-IL-1α antibody but not by anti-IL-1 β antibody, indicating that IL-1α is the predominant form in GCF. However, the IL-1 activity in GCF was unrelated to the number of IL-1 mRNA-exprerssing macrophages in the same gingival site where the GCF was obtained at the same time. The results suggest that macrophages in the connective tissue subjacent to the oral epithelium contribute to the production of IL-1 but those in connective tissue subjacent to the pocket epithelium play a different role in the generation of gingival inflammation.     

The junctional epithelium: from health to disease

The junctional epithelium is located at a strategically important interface between the gingival sulcus, populated with bacteria, and the periodontal soft and mineralized connective tissues that need protection from becoming exposed to bacteria and their products. Its unique structural and functional adaptation enables the junctional epithelium to control the constant microbiological challenge. The antimicrobial defense mechanisms of the junctional epithelium, however, do not preclude the development of gingival and periodontal lesions. The conversion of the junctional to pocket epithelium, which is regarded as a hallmark in disease initiation, has been the focus of intense research in recent years. Research has shown that the junctional epithelial cells may play a much more active role in the innate defense mechanisms than previously assumed. They synthesize a variety of molecules directly involved in the combat against bacteria and their products. In addition, they express molecules that mediate the migration of polymorphonuclear leukocytes toward the bottom of the gingival sulcus. Periodontopathogens-such as Actinobacillus actinomycetemcomitans or, in particular, Porphyromonas gingivalis-have developed sophisticated methods to perturb the structural and functional integrity of the junctional epithelium. Research has focused on the direct effects of gingipains, cysteine proteinases produced by Porphyromonas gingivalis, on junctional epithelial cells. These virulence factors may specifically degrade components of the cell-to-cell contacts. This review will focus on the unique structural organization of the junctional epithelium, on the nature and functions of the various molecules expressed by its cells, and on how gingipains may attenuate the junctional epithelium's structural and functional integrity.     

Cytolethal distending toxin damages the oral epithelium of gingival explants

The cytolethal distending toxin (Cdt), expressed by the periodontal pathogen Aggregatibacter actinomycetemcomitans, inhibits the proliferation of cultured epithelial cells by arresting the cell cycle. The gingival epithelium is an early line of defense against microbial assault. When damaged, bacteria collectively gain entry into underlying connective tissue where microbial products can affect infiltrating inflammatory cells, leading to the destruction of the attachment apparatus. Histological evaluation of rat and healthy human gingival tissue exposed ex vivo to the Cdt for 36 and 18 hours, respectively, revealed extensive detachment of the keratinized outer layer and distention of spinous and basal cells in the oral epithelium. Treated human tissue also exhibited disruption of rete pegs and dissolution of cell junctions. Cells in the connective tissue appeared unaffected. Primary gingival epithelial cells, but not gingival fibroblasts, isolated from the same healthy human tissue were cell-cycle-arrested when treated with the toxin. These findings provide new evidence that the Cdt severely damages the oral epithelium, ex vivo, by specifically targeting epithelial cells, in situ. The Cdt shows preferential targeting of the epithelium as opposed to connective tissue in animal and human gingival explant models. Abbreviations: cytolethal distending toxin (Cdt), connective tissue (CT), 4',6-diamidino-2-phenylindole (DAPI), human gingival epithelial cells (HGEC), human gingival explants (HGX), human gingival fibroblasts (HGF), junctional epithelium (JE), oral epithelium (OE), rete pegs (RP), sulcular epithelium (SE).     

Patterns of cytokeratin expression in the epithelia of inflamed human gingiva and periodontal pockets

Fourteen specimens of periodontal pockets and the associated marginal gingiva were collected and either frozen for examination using antibodies against various defined cytokeratin specificities or processed for 2-dimensional gel electrophoresis. The epithelium forming the pocket lining typically extended into the connective tissue of the pocket wall in the form of a network of finger-like strips. Immunocytological staining indicated that keratins (K) 5, 6, 14 and 19 were expressed by almost all cells of the pocket lining and K13 and K16 by the suprabasal cells. The coronal region of the pocket lining showed some cells staining for K4, Staining for K8 and K18 was seen in the apieal region of the pocket lining and in the finger-like extensions of epithelium into the connective tissue. Compared with normal gingiva, the sulcular and the oral gingival epithelia showed a marked increase in staining for K19. Surprisingly, the pattern of keratin expression of the epithelium of the pocket lining was found to be essentially similar to that of normal junctional epithelium and the anatomical position of the boundaries between each epithelial phenotype were not significantly altered. These patterns of keratin expression were confirmed by the 2D electrophoretic analyses of microdissected regions of epithelium. The potential significance of inflammation to the epithelial changes associated with pocket formation is discussed.     

Hepatocyte growth factor (HGF) system in gingiva: HGF activator expression by gingival epithelial cells

Hepatocyte growth factor (HGF) acts as a mitogen, motogen, morphogen, and anti-apoptotic factor for various kinds of epithelial cells. We previously showed that periodontal ligament and gingival fibroblasts secreted an HGF-like chemoattractant for a gingival epithelial cell line and found that the HGF content of gingival crevicular fluid was well correlated with clinical parameters and interleukin-1beta level. Since HGF is secreted as an inactive form (proHGF), and converted to an active form by serine proteases such as HGF activator (HGFA), extracellular processing of proHGF is presumed to be critical in the regulation of HGF activity. To examine the role of the HGF system in epithelial invasion followed by loss of connective tissue attachment in periodontitis, mRNA expression of HGF, its receptor (c-met) and HGFA in gingival tissues was monitored. Ten gingival biopsies were obtained, and epithelium and connective tissues were separated by enzymatic digestion. The gene expression of HGF and keratinocyte growth factor (KGF) in gingival connective tissue, and c-met, HGFA and KGF receptor (KGFR) in gingival epithelial tissues was monitored using RT-PCR. Furthermore, HGFA protein in the conditioned medium of cultured primary gingival epithelial cells was examined using Western blotting. All the connective tissue samples expressed KGF, and 8 out of 10 samples expressed HGF. All the epithelial samples expressed KGFR and c-met, whereas 5 out of 10 samples expressed HGFA. Protein expression of HGFA by cultured primary gingival epithelial cells was also confirmed. In terms of local production and activation of HGF in gingival tissue, these results suggest that synergistic expression of HGF in connective tissue and HGFA expression in epithelium may contribute to disease progression in periodontitis.