Inflamed Gingiva Research Articles

The effect of chronic inflammation on gingival connective tissue proteoglycans and hyaluronic acid.

Proteoglycans have been isolated and analysed from extracts of normal and chronically inflamed human gingiva in order to determine the effects of chronic inflammation on these important soft connective tissue extracellular macromolecules. The uronic acid content of glycosaminoglycans isolated by papain digestion of normal and inflamed gingiva did not differ significantly. Likewise, electrophoretic analysis revealed that the content of hyaluronic acid, heparan sulfate, dermatan sulfate and chondroitin sulfate was similar. The sulfated glycosaminoglycans from both sources eluted from a Sepharose C1-6B column with a Kav of 0.45 (approximate Mr 25,000). However, hyaluronic acid from normal gingiva was predominantly of a large size eluting in the void volume of a Sepharose. CL-6B column, while that isolated form inflamed tissue was mostly a small molecular weight species which eluted in the included volume of a Sepharose CL-6B column. Using dissociative conditions, intact proteoglycans could be more readily extracted from inflamed tissues (90% of the total tissue uronic acid) than from normal tissues where only 80% of the total tissue uronic acid was extractable. Even though DEAE-Sephacel ion-exchange chromatography revealed no differences in charge between normal and inflamed gingival proteoglycans, Sepharose CL-4B chromatography revealed more molecular size polydispersity in samples from inflamed tissue than from normal tissue. Taken together, these results indicate that while hyaluronic acid is depolymerized in inflamed tissue, no evidence of sulfated glycosaminoglycan degradation was found. Therefore, the most likely cause for disruption to the molecular integrity of the proteoglycans is via proteolytic alteration to the proteoglycan core protein.

Proteoglycans synthesized by cultured fibroblasts derived from normal and inflamed human gingiva.

The in vitro proliferations rates and proteoglycans synthesized by adult human gingival fibroblasts derived from six age- and sex-matched donors of healthy and chronically inflamed gingiva were analyzed. Fibroblasts from inflamed gingiva demonstrated a slower growth rate than cells from healthy tissue. The rate of incorporation of [35S]sulfate into cell layer-associated proteoglycans and the release of these macromolecules into the culture medium did not differ appreciably between the two groups of cells. Similarly, no detectable differences in the overall charge of the proteoglycans synthesized by normal and inflamed gingival fibroblasts, as assessed by their elution from DEAE-Sephacel, were noted. However, Sepharose CL-4B chromatography revealed that the medium-associated proteoglycans made by the inflamed tissue fibroblasts were depleted in one species of chondroitin sulfate proteoglycans and contained more dermatan sulfate than did control cells. In addition, the intracellular proteoglycan pool was found to be greatly diminished in the inflamed tissue fibroblast cell layers. Glycosaminoglycan analysis of the proteoglycans confirmed these observations. Compared to normal gingival fibroblasts, the inflamed tissue fibroblasts released less heparan sulfate into the medium. Additionally, increased levels of dermatan sulfate and depleted amounts of chondroitin sulfate in the medium of inflamed gingival cells were noted. The observed changes were stable through several transfers in culture and indicate that chronically inflamed tissue may contain fibroblasts manifesting a heritable phenotype differing from fibroblasts in normal connective tissue.

Inhibition of superoxide generation by human polymorphonuclear leukocytes with chlorhexidine. Its possible relation to periodontal disease.

During the acute phase of periodontal disease, as many as 60% of the cells of the junctional epithelium may be accounted for by polymorphonuclear cells (PMNs). It is generally accepted that these cells play a dominant role in the destruction of connective tissue by virtue of the proteinases they release and the free oxygen radicals they generate. Modulation of the proteolytic activity and free radical production seems to be essential for the inhibition of tissue destruction. We have therefore studied the effect of chlorhexidine on the generation of free oxygen radicals and luminol-dependent chemiluminescence (LDCL) by stimulated human PMNs. Nontoxic chlorhexidine concentrations (0.1-1 microgram/ml) were found to inhibit superoxide production but did not affect LDCL. We therefore suggest that in addition to its antiseptic effect, chlorhexidine may also modulate the generation of free radicals by activated PMN cells in the inflamed gingiva.

Immunocytochemical demonstration of S-100 protein-containing dendritic cells in the normal and inflamed human gingiva.

Langerhans cells functioning as epidermal macrophages are known to contain S-100 protein, a peculiar brain-derived protein. The present study demonstrates the occurrence and ultrastructure of S-100-immunoreactive dendritic cells in normal and inflamed gingiva in human subjects. A few S-100-immunopositive cells were found scattered in the normal gingival epithelium, but these greatly increased in number in the inflamed gingiva. By electron microscopical observation, the S-100-immunopositive cells in the normal gingiva were confirmed as containing Birbeck granules characteristic of Langerhans cells, while the cells multiplying in the inflamed gingiva corresponded to indeterminate dendritic cells, which have been regarded as immature Langerhans cells in the skin. The close juxtaposition of lymphocytes to the S-100-immunopositive indeterminate dendritic cells was frequently recognized in the inflamed gingiva, a finding which suggests that these cells, and not the typical Langerhans cells, may function as antigen-presenting cells.

Metabolism of progesterone by healthy and inflamed human gingiva in vitro

[4- 14C]Progesterone was incubated with homogenate and mitochondrial, microsomal and soluble fraction preparations of healthy and inflamed gingiva from human subjects of both sexes. The subcellular preparations were supplemented with an NADPH-regenerating system and incubated for 2 h at pH 7.4 and 37°C. The metabolites were identified by column, multiple TLC and radioautography and quantified with liquid scintillation counting. In inflamed tissue the metabolic activity was higher than in healthy gingiva. On the basis of the identified metabolites it can be concluded that the human gingiva of both sexes contains marked 3α-, 3β- and 20α-hydroxysteroid dehydrogenase, Δ 4-5α- and Δ 4-5β-steroid hydrogenase activities, and less 20β-hydroxysteroid dehydrogenase activity.

Characterization of Collagens in Phenytoin-Enlarged Human Gingiva

The collagen content and composition of human gingivae enlarged due to phenytoin medication were compared to normal and inflamed gingival tissues. Phenytoin-enlarged gingiva contained significantly higher amounts of collagen. The ratio of type I to type III collagen was different in enlarged tissue, with less type I and an elevated amount of type III collagen present. The amount of type V collagen did not differ among normal, inflamed and phenytoin-enlarged gingivae.

Localization of fibronectin in gingival connective tissue of the beagle dog. Immunofluorescent light microscopic findings.

Affinity purified antibodies to plasma fibronectin were used to localize fibronectin in the connective tissues of inflamed and noninflamed beagle gingiva. In noninfiltrated gingival connective tissue, fibronectin was demonstrated in the basement membrane beneath gingival epithelium and around blood vessels as a uniform and intensely stained band about 3 to 10 micron thick. Fibronectin was also distributed throughout the connective tissue in association with collagen fibrils as a more diffuse, less intensely stained pattern. The inflamed gingiva included in this study was characterized by proliferation of epithelial pegs, heavy infiltration of plasma cells and loss of collagen within the subepithelial connective tissue. In these sites, fibronectin was present as an intensely stained band around blood vessels and at the crest of connective tissue papillae nearest the sulcular space. The fibronectin in the basement membrane beneath the epithelium appeared diminished and less uniformly distributed. A delicate network of fibronectin was present around plasma cells and the remaining collagen fibers.

Histochemical demonstration of lectinbinding sites and keratin in inflamed human gingiva

Histochemical demonstration of lectin‐binding sites and keratin peptides in gingival epithelia was reported and differences in staining and distribution were compared to inner and outer gingival epithelia.Gingival epithelia on the outer side exhibited zonal or regional distributions of lectin‐binding, and the cytochemical staining was generally found in the cell coat and intercellular materials. Keratin protein was found frequently in the spinous cell, infrequently in the basal cells, and not at all in the superficial layer. The sugar residues in the cell coat of gingival epithelia probably were mannose, galactose, N‐acetyl‐galactosamine, and N‐acetyl‐glucosamine.The inner side of the epithelia, crevicular, and pocket epithelia were characterized by irregular and incomplete staining for lectins. These epithelia also displayed less keratin staining compared to the outer gingival epithelia. These findings suggest the possibility of depolymerization of glycosaminoglycans in the cell coat by enzymes of either pocket bacterial origin or host tissue origin.

Effect of Inflammation on the of Collagen Types, I, III, IV, and V and Type I Trimer and Fibronectin in Human Gingivae

The effect of inflammation on the distribution of collagen types I, III, IV, and V and type I trimer and fibronectin in human gingivae was studied after staining them with antibodies to these proteins, Gingival tissues were obtained after periodontal surgery and incubated with antibodies, and staining was visualized by indirect immunofluorescence using FITC- or rhodamine-conjugated second antibodies. The results showed that antibody to type I collagen stained normal gingival connective tissue uniformly and revealed the presence of thick fiber bundles, The staining was sparse at areas of inflammation and leukocytic infiltration. Anti-type-III antibody revealed a fine fibrillar netWork in the normal gingivae, especially near the epithelium; the type III was also lost at sites of inflammation. Type V collagen antibody also stained the gingival connective tissue intensely but, unlike the types I and III staining, it was retained in inflamed areas. Normal gingivae were not stained by the type I trimer antibody; however, staining occurred at inflamed sites. Both normal and inflamed tissues were stained by type IV collagen antibody. and the staining was restricted to basement membrane structures, The normal and inflamed gingivae contained a thick fiber network which bound unti-fibronectin antibody. We conclude that the various ginglvai collagen fibers are made up of collagen types I, III, and V, that the types I and III collagens are preferentially lost during inflammation, and that type I trimer appears at Inflamed sites.

Cathepsin D, L and B in inflamed human gingiva.

Cathepsin D, cathepsin L, and cathepsin B activities were detected in homogenates of human gingival tissue from 20 patients with different degrees of periodontal disease and in one patient with juvenile periodontitis. Clinical plaque index (PI), gingival index (GI), and pocket depth were determined. Patients were divided into three groups with regard to the severity of the disease. Averaged values for the activities of all three lysosomal proteinases increased from the group with less damaged tissue to the group with more advanced periodontal disease. When the correlations between the specific activities of the cathepsins and pocket depth in each of 20 patients were considered by the linear regression analysis, the correlation coefficient for cathepsin D was much higher than for the other two cysteine proteinases. Cathepsin D was also extracted from the homogenates by specific immuno‐adsorbtion on anticathepsin ‐ D antibody ‐ Sepharose and quantitatively determined; the average amount of enzyme ranged from 118.5 to 184.5 μg per mg of total protein extracted from the tissues and was positively correlated to pocket depths.

Synthesis of Type V Collagen by Fibroblasts Derived from Normal, Inflamed and Hyperplastic Human Connective Tissues

Increased type V collagen content is a feature of many inflammatory and fibro-proliferative diseases. In order to understand the mechanism responsible for this increase we have studied type V collagen synthesis by fibroblasts obtained from normal, inflamed and phenytoin-hyperplastic human gingivae. Cultures were immunostained with antibodies to type V and I collagens to determine the proportion of cells producing type V collagen. In addition, cells were metabolically labeled with radioactive amino acids, and type V procollagen and collagen were fractionated and quantitated. Results showed that all the cells were stained by both anti-type V and type I antibodies. Anti-type V staining was observed outside the cells whereas anti-type I staining was localized intracellularly. The proportion of radioactive type V collagen in the individual cell strains ranged from 0–5.2% of the total collagens; however, differences among the three cell types were not statistically significant. No differences were observed in the procollagen [V] levels, which accounted for 6.5–15.1% of the total procollagens. We conclude that changes in fibroblast population may not be a significant factor contributing to increased type V collagen in inflammation and hyperplasia, and that the increase may be due to other factors such as modulation of synthesis activities by environmental ligands and resistance of type V collagen to degradation.

Mechanisms involved in the binding of IgG immune complexes to sections of inflamed gingiva.

The binding of particulate (sensitized sheep erythrocytes= EA) and soluble (horse radish peroxidase (HRP)‐anti‐HRP) IgG immune complexes to cryostat sections of inflamed human gingiva was studied. EA bound preferentially to inflammatory cell infiltrates in all biopsy specimens, and HRP‐anti‐HRP complexes were found to bind to the surface of mononuclear cells. Binding of EA was abolished by treatment of cryostat sections with 1 mM periodic acid (30 min) or 4% formaldehyde (10 min). Binding was also abolished by exposure of sections to cold ethanol for 5 min. No binding of fluorescein labelled, heat aggregated human IgG to sections of washed and ethanol‐fixed biopsies of inflamed human gingiva was observed. Results of inhibition experiments with rabbit and human native or heat aggregated IgG, rabbit and human IgG Fc and IgG F(ab′)2 fragments, human IgG 1 and IgG2, mildly reduced and alkylated human IgG and with rabbit anti‐human Clq, indicated that the binding is not due to activated C1 or to the presence of rheumatoid factors. Our findings are consistent with the concept that the binding is due to the presence of IgG Fc receptors (IgGFcR) on mononuclear cells. Ethanol fixation of specimens compromises any conclusions regarding IgGFcR activity in the tissues.

The effect of alloxan diabetes on prolyl and lysyl hydroxylase activity in uninflamed and inflamed rat gingiva

The influence of diabetes on gingival inflammation was studied through its effect on prolyl and lysyl-hydroxylase activities and on tissue-collagen content. Inflammation induced for 7 days with either endotoxin or antigen-antibody complexes reduced the activity of both enzymes by about 45 per cent, and decreased the concentration of soluble and insoluble collagens. Diabetes alone decreased the enzyme activity by more than 50 per cent but prevented the loss of the soluble and insoluble collagens which occurs during inflammation. The complex interaction of diabetes and inflammation on collagen metabolism in gingival tissue may be explained in part by abnormalities of collagen synthesis; alterations in collagen maturation and degradation, and in leukocyte function, may also occur.

Identification of Langerhans Cells in Human Gingival Epithelium

The purpose of this study was to qualitatively compare three recent techniques of Langerhans cells detection in oral epithelium and to quantitatively compare Langerhans cells in clinically normal and clinically inflamed human gingival biopsies. Eleven subjects were selected who displayed chronic periodontitis and moderate gingival inflammation. A quadrant associated with clinically inflamed tissues was not treated, while the remaining teeth were scaled and root-planed. Two gingival biopsies were taken: clinically normal, treated tissue; and clinically inflamed, untreated tissue. Langerhans cells were stained using HLD-DR, S-100 and OKT6. They were quantitated using a standard grid for OKT6-stained sections only. Approximately 5 times as many Langerhans cells were identified in the biopsy specimens of clinically inflamed human gingiva as in clinically normal gingiva of the same patient. Of the methods studied, OKT6 was qualitatively determined to be the best for visualization of these cells. An immunologic role in the host response to chronic periodontal disease is postulated for Langerhans cells.

The effects of carthamus tinctorius L, .BETA.-glycyrrhetinic acid, carbazochrome, and chlorhexidine in inflamed gingiva induced by activating plaque accumulation in beagle dogs.

We evaluated the effects of drugs that included water extracts of Carthamus tinctorius L (E-C), β-glycyrrhetinic acid (Gly.), carbazochrome (Carz.), alone or in combination with (Mx.) and chlorhexidine (CHX) on the inflamed gingiva induced by accelerated plaque accumulation in Beagle dogs. After a clinically healthy gingiva was obtained by means of scaling and tooth brushing for 4 to 6 weeks, gingival inflammation was induced by the placement of silk floss ligatures around the premolars and molars and by feeding a soft diet for one week. The drugs were topically applied with a silicon gum tray for 5 and 10 days after inducement of gingivitis. A significant reduction of the number of infiltrated PMNs in the connective tissues adjacent to the upper part of the sulcular epithelium was observed with all drugs applied to gingiva at 10 days after the application. These findings suggested that E-C, Gly., Carz., as well as CHX are effective in the treatment of gingivitis singly or in combination.

T lymphocyte subsets in the inflamed gingiva of human adult periodontitis.

T lymphocyte subsets in the inflamed gingiva of human adult periodontitis.

Free amino-acid composition of wax-stimulated whole saliva in human subjects with healthy periodontium, severe chronic periodontitis and post-juvenile periodontitis

The free amino-acid composition of wax-stimulated whole saliva of 3 subjects with different periodontal status was determined. The amino acids were analysed by gas chromatographic separation on a packed column and determined as their N-heptafluorobutyrylisobutyl esters. In 2 patients with periodontal diseases, the salivary concentrations of proline and glycine were significantly elevated, suggesting increased collagen and protein degradation and their increased release from the inflamed gingiva. δ-Amino- n-valeric acid is related to bacterial metabolism so that the changes found in its concentrations could suggest an altered plaque metabolism and different bacterial contents in gingival pockets of patients with chronic and post-juvenile periodontitis.

T lymphocyte subsets in the inflamed gingiva of human adult periodontitis

T lymphocyte subsets in the inflamed gingiva of human adult periodontitis

Observations on Temperature and Temperature Patterns of the Gingiva: I. The Effect of Arch, Region and Health

The purpose of this investigation was to study the pattern and constancy of temperatures recorded in the gingival crevices and some factors affecting them. The temperatures of the interproximal crevices in the maxillary and mandibular arch of 16 female dental hygiene students were evaluated at the time of examination, immediately afterwards and following a 14-day interval. The temperature difference between each site and the subject's sublingual temperature were calculated. In the analysis, a comparison of sites with a confirmed status of bleeding tendency and pocket depth was carried out. The results show temperature differences between arches (mandible vs maxilla) and regions (posterior vs anterior). The differences are independent of the health status. Inflamed gingiva generally has a higher temperature than clinically healthy gingiva. There is an indication that healthy tissue, more than inflamed tissue, reacts to an outside stimulus with an increase in temperature. It is necessary that environmental and stimulative factors are strictly controlled if temperature gradients are to be applied for diagnostic purposes in periodontics.

Cell populations associated with gingival bleeding.

The presence of crevicular bleeding after probing is an objective clinical sign of gingival inflammation. It has been associated with a plasma cell-dominated inflammatory infiltrate in deep pockets and has been suggested as an indication of active periodontitis. The purpose of this study was to characterize the cellular composition of gingival connective tissue associated with shallow pockets which bled after probing. Prior to biopsy, the mid-facial gingiva associated with 30 teeth from 26 patients was assessed for the presence or absence of visual inflammation, pocket depth and bleeding after probing with a standardized force of 25 g. A horizontal reference incision was made on the facial aspect of the gingiva to demarcate for histologic analysis the specific gingival area probed and evaluated for inflammation. Cell populations were determined from histological sections using morphometric point counting techniques in six standard fields at and coronal to the level of the reference incision. The percentage of cell types and the per cent volume densities of all tissue components were compared between clinically normal and inflamed gingiva. In clinically inflamed gingiva there was a significantly greater percentage of lymphocyte/macrophage/monocyte cells and a smaller percentage of fibroblast/endothelial cells. The percentage of plasma cells was only a fraction of that found for other inflammatory cells. The results of this study indicated that a lesion in the gingiva associated with bleeding after probing can consist of an inflammatory infiltrate which is not dominated by plasma cells.