Excessive fluoride intake alters the MMP-2, TIMP-1 and TGF-ß levels of periodontal soft tissues: an experimental study in rabbits.

OBJECTIVES: This study evaluated the influence of fluoride on periodontal soft tissues by investigating any alterations in their MMP-2, TIMP-1 and TGF-ß profiles secondary to excessive fluoride intake. MATERIAL AND METHODS: Fluorosis was induced in 18 rabbits (test group) through consumption of fluoride added to drinking water, whereas 10 rabbits consumed regular tap water as daily supply (control group). Following fluorosis verification, animals were sacrificed and their 1st mandibular molar teeth were utilized in the assessments. MMP-2, TIMP-1 and TGF-ß were separately investigated for gingival epithelium (GE), gingival connective tissue (GC) and periodontal ligament (PL) to evaluate periodontal soft tissues. Histological sections were prepared from the groups, the parameters were determined by immunohistochemistry, and their levels were calculated by quantification of the immunostainings. RESULTS: Staining intensity of MMP-2 in GC and PL (p < 0.01); TIMP-1 and TGF-ß of GE, GC and PL (p < 0.01) were higher in the test group compared to those of the control group. Intra-group staining of TIMP-1 was higher than MMP-2 in all test group compartments (p < 0.01) and in the control group GE (p < 0.01). TIMP-1 was also higher than TGF-ß in the GE and PL of the test group (p < 0.05) and in the GE of the control group (p < 0.01). CONCLUSION: These results suggest that excessive fluoride intake may affect periodontal soft tissues by increasing MMP-2, TIMP-1 and TGF-ß, and thereby altering the MMP-2/TIMP-1 and TIMP-1/TGF-ß ratios. CLINICAL RELEVANCE: Excessive fluoride consumption may alter the periodontal tissue homeostasis which may be detrimental in the maintenance of periodontal health.

Comprehensive analysis of gene expression in the junctional epithelium by laser microdissection and microarray analysis.

BACKGROUND AND OBJECTIVE: The junctional epithelium attaches to the tooth enamel at the dentogingival junction. The attachment mechanisms of the junctional epithelium have been studied histologically, but the molecular functions of the junctional epithelium have not been elucidated. The aim of this study was to perform a comprehensive analysis of gene expression in the junctional epithelium and to search for specific genetic markers of the junctional epithelium. MATERIAL AND METHODS: A comprehensive analysis of genes expressed in the mouse junctional epithelium and oral gingival epithelium was performed using laser microdissection and microarray analysis. To extract high-quality RNA from these tissues, we made frozen sections using a modified film method. Confirmation of the differential expression of selected genes was performed by quantitative real-time PCR and immunohistochemistry. RESULTS: The modified method produced RNA of sufficient quality for microarray analysis. The result of microarray analysis showed that 841 genes were up-regulated in the junctional epithelium compared with the oral gingival epithelium, and five were increased more than 50-fold in the junctional epithelium. These five genes were secretory leukocyte protease inhibitor (Slpi), keratin 17 (Krt17), annexin A1 (Anxa1), myosin light peptide 6 (Myl6) and endoplasmic reticulum protein 29 (Erp29). In particular, Slpi expression in the junctional epithelium was approximately 100-fold higher than in the oral gingival epithelium by real-time PCR. Additionally, immunohistochemistry indicated that the Slpi protein is highly expressed in the junctional epithelium. CONCLUSION: We developed a method for generating fresh-frozen tissue sections suitable for extraction of good-quality RNA. We determined that Slpi is characteristically expressed in the junctional epithelium. Our results provide a substantial advance in the analysis of gene expression in the junctional epithelium.

Muscarinic acetylcholine receptor activation increases transcellular transport of macromolecules across mouse and human intestinal epithelium in vitro.

The intestinal epithelium acts as a barrier restricting uptake of luminal macromolecules such as dietary antigens and microbes. Here, we examined the role of cholinergic signalling in the regulation of permeability to macromolecules. Mouse jejunum was mounted in Ussing chambers and permeability was determined by measuring the flux of the antigen-sized protein, horseradish peroxidase (HRP), across the tissue. Baseline HRP permeability was significantly reduced by neural blockade with tetrodotoxin or cholinergic muscarinic antagonism with atropine, suggesting that ongoing release of endogenous acetylcholine from enteric nerves regulates barrier function. Exogenous addition of the muscarinic agonist bethanechol caused significant increases in both HRP flux and the area of HRP-containing endosomes in enterocytes. Bethanechol-enhanced HRP flux was abrogated by the M3 receptor antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), the phospholipase A(2) inhibitor quinacrine, and the cyclooxygenase inhibitor indomethacin. Complementary in vitro studies showed direct effects of bethanechol on T84 epithelial cells, where increased HRP uptake was associated with increased F-actin, and increased cytosolic phospholipase A(2) (cPLA(2)) phosphorylation. Taken together, these results provide evidence for cholinergic regulation of transepithelial transport of macromolecules, mainly mediated by activation of M3 receptors with subsequent involvement of phospholipase A(2) and cyclooxygenase products.

Histochemical examination of periodontal junctional epithelium in p21/p27 double knockout mice.

The periodontal junctional epithelium (JE) is maintained in a steady state through a dynamic process that balances proliferation and exfoliation of epithelial cells. However, mechanisms that regulate JE are not well understood. To better understand how proliferation of the JE is controlled in healthy gingiva, we have studied functional roles of the CDK (cyclin dependent kinase) inhibitors p21 and p27 in JE using knockout mouse model systems. Image analysis of the dentogingival junction in p21 or p27 single knockout mice as well as p21/p27 double knockout mice (dKO) was performed. The analysis revealed enlarged JE in p21/p27 dKO mice due to an increase in the area of the epithelium and associated connective tissue 'islands'. Immunohistochemistry was performed for p21, p27, cyclin D1, and proliferating cell nuclear antigen (PCNA). The highest levels of PCNA-positive cells were detected in the p21/p27 dKO mice, reflecting increased cell turnover. Lower levels of cyclin D1 were detected in the JE of p21/p27 knockout mice, suggesting that p21 and p27 regulate stability of cyclin D1 in oral epithelium. These data suggest that p21 and p27 have a critical role in controlling epithelial cell proliferation in the JE and thus function to maintain the JE at a normal size.

In situ detection of matrix metalloproteinase-9 (MMP-9) in gingival epithelium in human periodontal disease.

BACKGROUND AND OBJECTIVE: As the periodontal lesion develops, the junctional epithelium migrates apically in conjunction with the dissolution of the most coronal Sharpey's fibers. Because matrix metalloproteinase-9 (MMP-9) has been identified in migrating epithelial cells and invading tumors, we propose that this enzyme is produced by gingival keratinocytes in advanced periodontal lesions. METHODS: To test this idea, biopsies of inflamed gingival tissues were obtained from patients with advanced periodontitis. Healthy gingival tissue samples were utilized as controls. The presence and activity of MMP-9 was evaluated by combining indirect immunofluorescence of gingival tissue samples and gelatin zymography of gingival epithelium separated from connective tissue. RESULTS AND CONCLUSIONS: The staining pattern showed the presence of MMP-9 in junctional and pocket gingival epithelial cells, polymorphonuclear neutrophils (PMNs) and as a scattered deposit along connective tissues of periodontitis-affected gingival tissues. Gelatin zymography permitted the identification of pro-MMP-9 in surcular/pocket epithelium derived from inflamed gingival tissues. Lower levels of MMP-9 were detected in epithelium not exposed to inflammation. These observations suggest a role for MMP-9 in gingival epithelial response to periodontal infection.

Matrilysin (matrix metalloproteinase-7) expression in human junctional epithelium.

Matrilysin is a matrix metalloproteinase expressed in exocrine and mucosal epithelium in many human tissues. Immunohistochemical staining showed that matrilysin is expressed in suprabasal cells of junctional epithelium facing the teeth and in epithelial cell rests of Malassez. No matrilysin expression was seen in the periodontal pocket tissue. In a tissue culture model mimicking junctional epithelium, matrilysin expression was also observed in suprabasal epithelial cells. Of 13 anaerobic oral bacterial species tested, F. nucleatum, F. necrophorum, P. endodontalis, and P. denticola stimulated matrilysin expression in porcine periodontal ligament epithelial cells from 2.5- to 5.7-fold, compared with untreated cells. The enzyme was localized in intracytoplasmic vesicles that also reacted with antibodies against lysosomal membrane protein h-lamp-1. The results indicate that matrilysin may play an important role in the normal physiology of junctional epithelium.

A fluid-phase endocytotic capacity and intracellular degradation of a foreign protein (horseradish peroxidase) by lysosomal cysteine proteinases in the rat junctional epithelium.

We investigated the co-localization of lysosomal cathepsins B, H and L, and horseradish peroxidase (HRP) in junctional epithelial (JE) cells both as a fluid-phase endocytotic marker to demonstrate the fluid-phase endocytotic capacity of JE cells, and to understand the morphological relationships of the endocytosed foreign substances to lysosomal cathepsins in these cells. The diaminobenzidine (DAB) histochemical and cytochemical methods and immunohistochemical avidin-biotin-peroxidase complex and immunocytochemical post-embedding colloidal gold methods were used. Under light microscopy, DAB reaction products based on HRP were found in JE but were rare or absent in the oral sulcular epithelium and oral epithelium. Immunolabeling for cathepsins B and H was found in the granular structures of the cells, but no cathepsin L was identified. With electron microscopy, DAB reaction products, which indicated both HRP and the azurophil granules of neutrophils, were endocytosed into JE cells. Using a post-embedding technique, gold particles indicating HRP were present on the plasma membrane of JE cells, at the periphery of electronlucent vacuoles, and in the electrondense granules. Gold particles indicating cathepsin B or H were found in the electrondense granules. With different sizes of colloidal golds, the co-localization of cathepsin B or H with HRP was indicated only in the electrondense portion of the larger vacuoles consisting of electronlucent and -dense parts. This study provided the first morphological data which indicate that JE has a fluid phase endocytotic capacity, and which suggest that the lysosomal cathepsins B and H are involved in the intracellular degradation of foreign substances invading through the gingival sulcus in JE cells.

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.

Possible roles of medullasin in nifedipine-induced human gingival overgrowth.

In order to clarify a possible pathophysiological role of medullasin, a neutrophil elastase-like proteinase, in nifedipine (NF)-induced gingival overgrowth, the distributions of medullasin-positive cells immunostained in specimens from patients with NF-induced gingival overgrowth and chronic marginal gingivitis were compared in three different biopsy areas. Twenty gingival biopsies were obtained from five patients with gingival overgrowth and 20 biopsies from another five patients with chronic marginal gingivitis. In the marginal gingivitis group, the mean percentage of positive cells in the vicinity of pocket epithelium (zone I) was significantly higher than in the areas of connective tissue of the mid-portion (zone II) and adjacent to oral epithelium ( zone III) (p < 0.05). In the gingival overgrowth group, on the contrary, the positive cells significantly increased in zone II as compared with zones I and III (p < 0.05). Further, medullasin-positive cells of zones II and III in the overgrowth group had infiltrated more extensively than those in the gingivitis group (p < 0.001), indicating the participation of this enzyme in the mechanism of NF-induced gingival overgrowth. These observations suggest that medullasin may play a part in NF-induced overgrowth both in host defence and in immunoregulation, possibly cytotoxically.

Loss of attachment in the marginal periodontium of the rat incisor under non-inflammatory conditions. Expression of alkaline phosphatase activity. Experimental Oral Biology Group.

Alkaline phosphatase (ALP) has been suggested to play a role in acellular cementum formation and maintenance of periodontal attachment. In an attempt to determine whether changes in attachment level are associated with altered expression of ALP-activity in the periodontium we induced natural loss of attachment in rats by pinning the lower incisor to the jaw bone. Previous studies have shown that this procedure results in regressive changes in the marginal periodontium without any inflammatory response. Six months after blockage of eruption the attachment level on the experimental (right) side had shifted about 700 microm in the apical direction. On the control (left) side the apical termination of the junctional epithelium had remained stationary with respect to the alveolar crest. Our observations have shown that during the first few weeks of the experiment loss of attachment is accompanied by considerable reduction of ALP-activity in the supracrestal part of the periodontium. At later time intervals, however, no distinct relation was found between apical migration of junctional epithelium and loss of ALP-activity in the supracrestal region, indicating that the two phenomena are not directly related to each other. The domain of the ALP-positive fibroblasts in the supracrestal extension of the periodontal ligament decreased in size and was replaced by ALP-negative connective tissue cells probably coming from the outer gingival domain. Since at all time intervals a distinct demarcation could be observed between the ALP-positive and ALP-negative areas, we interpret our data as indicating that ligament and gingival cells do not mix.

Laminin localization and gelatinolytic activity of epithelial rest of Malassez grown on titanium.

The purpose of this study was to examine laminin localization in epithelial rests of Malassez grown on titanium and their gelatinase activity. Laminin localization was confirmed by immunocytochemistry in all epithelial cells grown on titanium. The conditioned medium from the epithelial cells grown on titanium exhibited 72kD- and 92 kD-gelatinase activities by gelatin zymography. Those results suggest that laminin contributes to the epithelial cell attachment to titanium and that gelatinases may be mobilized for epithelial cell movement on titanium.

Inhibition of epithelial cell matrix metalloproteinases by tetracyclines.

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-dimethylaminotetracycline in concentrations ranging from 1 to 50 micrograms/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 intracellular 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.

Epithelial alpha-naphthyl acetate esterases in the green vervet monkey gingiva before and after periodontal surgery and during tooth eruption.

To provide enzymatic information on de novo formed junctional (JE) and sulcular epithelium (SE), we performed periodontal surgery on 24 teeth. Ten to 14 days postoperatively, all experimental and 16 control teeth were extracted with adjacent buccal gingiva. In addition, specimens from unerupted and partly erupted teeth containing enamel epithelium (EE) were examined. Fixed cryostat sections were cut in series, stained with HE, or incubated with and without substrate for demonstration of alpha-naphthyl acetate esterase activity and for control purposes, respectively. The distribution and intensity of the alpha-naphthyl acetate esterase activity of newly reformed JE and SE was identical to that of the original JE and SE, i.e. suprabasal and very strong. In contrast, both the oral gingival epithelium (OGE) and the EE displayed a very weak enzyme reaction. These observations indicate that the presence of alpha-naphthyl acetate activity of original and reformed JE and SE is probably site specific and of nondevelopmental origin. Heavy inflammation after healing was associated with enhanced epithelial proliferation of OGE and, in addition, marked esterase activity of these proliferations and corresponding OGE. This points at a possible inflammatory induction of the marked esterase activity seen in JE and SE as well as site-specific, connective tissue influences. Further investigation is needed to elucidate the effect of inflammation on the esterase activity.

Plasminogen activator in human gingival tissue adjacent to dental implants.

The installment of endosseous dental implants has become an accepted treatment procedure, and the long-term clinical results appear excellent. The composition of the soft tissue environment, however, is different from that around natural teeth. One characteristic of original junctional epithelium is its association with plasminogen activator (PA) activity. In 11 patients with a total of 30 ITI hollow-screw titanium dental implants, 16 biopsies were taken. Histologic cryostat sections were assayed for the presence of PA in the junctional epithelium. The results demonstrated that junctional epithelium around titanium implants yields PA activity in a manner very similar to that of natural teeth. The ability to produce this enzyme activity is not related to the developmental origin of the junctional cells, but to their position and function at the base of the gingival sulcus.

[A cytochemical study of lysosomal system in rat junctional epithelium after intravenous horseradish peroxidase injection].

Lysosome formation was studied in rat junctional epithelium (JE) by detection of acid phosphatase (ACPase) using the cerium reaction method after intravenous injection of horseradish peroxidase (HRP). Intravenously injected HRP was endocytosed by the whole JE, especially at the coronal portion. ACPase activity was also observed in numerous organellae (vacuoles, dense bodies, multivesicular bodies and Golgi cisternae) in the junctional epithelial cells. These ACPase positive organellae often fused with each other, and their contents were mixed in the fused structures. The numbers of the ACPase-positive organellae in the HRP-exposed JE were more numerous than those in the JE without HRP, especially at the coronal portion. Thus, the distribution of the ACPase-positive products in the JE was closely related to that of HRP-positive products. These findings indicated that the whole JE has not only endocytotic ability but also intracellular digestion ability by the lysosomal enzymes.

[Difference in uptake of junctional epithelium using microperoxidase and horseradish peroxidase as tracers in healthy rat gingiva].

A microperoxidase (MP, molecular weight 1,900; molecular diameter 20 A) or a horseradish peroxidase (HRP, molecular weight 40,000; molecular diameter 40 A) was intravenously injected into healthy rat junctional epithelium (JE) to investigate the endocytosis of foreign substances. By light microscopy, intravenous HRP was taken up throughout the JE, and the uptake was marked at the coronal portion of the JE. On the other hand, MP was taken up by only the coronal portion of the JE. At the electron microscopic level, the tracers were taken up by endocytotic organellae (phagosomes and micropinocytotic vesicles) of junctional epithelial cells (JE cells). HRP-positive endocytotic organellae in the JE cells were more numerous than MP-positive organellae. Thus, JE, especially its coronal portion, exhibited strong endocytotic activity for HRP compared with that for MP. These findings suggest that the JE has selective endocytotic ability for foreign substances, and plays an important role in protecting periodontal tissue.