Experimental gingivitis in the monkey

Rhesus monkeys receiving an oral hygiene program which included brushing, interdental cleansing and topical applications of chlorhexidine gluconate demonstrated a clinically normal gingiva for periods of up to 3 months. Wide fluctuations within individual dental units were noted with respect to histological sulcus depth, degree of connective tissue infiltration with lymphocytes and plasma cells, and total leukocyte or polymorphonuclear leukocyte (PMN) counts in the Junctional epithelium, even when the plaque and gingivitis index scores were 0. Of 18 clinically normal dental units sampled, only 6 appeared free of connective tissue inflammation. However, more than half of the tissue blocks obtained from dental units with a gingivitis and plaque index of 0 also showed scores of 0 with respect to the connective tissue inflammation (CTI) score and the number of PMNs in the Junctional epithelium. Within three days following discontinuation of oral hygiene procedures, rhesus monkeys developed a clinically noticeable gingivitis which began in the interdental papillae. Increases in CTI scores and number of leukocytes in the Junctional epithelium were evident after 2 days without oral hygiene. These values tended to increase further during the experimental period. A slight, but significant increase in sulcus depth was also noted during this time period. Regardless of the clinical state of the gingiva, a positive correlation was established between CTI scores and the number of PMNs and leukocytes in the Junctional epithelium. In early gingivitis, the plasma cells did not appear to outnumber lymphocytes, as has been reported for chronic gingivitis of longer duration.     

Variability of histologic criteria in clinically healthy human gingiva

After prophylaxis, 5 dental hygienists performed optimal oral hygiene under supervision for 6 months. At months 0, 1, 4 and 6, Plaque Index, Gingival Exudate Flow Rate and Gingival Index were assessed, and a buccal biopsy of their gingiva taken. Point counting procedures were performed at 2 different levels of magnification to estimate the volume densities of epithelium, infiltrated and non-infiltrated connective tissue, and collagen. The percentages of fibroblasts, polymorphonuclear neutrophilic granulocytes, lymphocytes, plasma cells and macrophages were estimated by counting the number of profiles of these cells in the connective tissue area close to the apical end of the junctional epithelium. All the changes inside the tissue occurred slowly. During the 6-month period there was a continuous increase of the volume density of the epithelium in the gingiva. An increase in the percentage of fibroblasts was observed between months 1 and 4 together with a decrease in the percentage of lymphocytes and in the volume density of the infiltrated connective tissue. Between months 4 and 6 an increase of the volume density of the collagen was found together with a further increase in the percentage of fibroblasts and a further decrease in the percentage of lymphocytes. After 6 months of perfect oral hygiene no more plasma cells were visible. This study has shown that, even in presence of clinically healthy gingiva, subclinical changes may take place. It appears realistic to accept the presence of a very mild gingivitis localized in an area adjacent to the attachment as compatible with gingival health.     

Observations on the initial stages of healing following human experimental gingivitis A clinical and morphometric study

The aim of the present study was to investigate stereologically the histologic alterations occurring during gingival healing after experimental gingivitis and to compare clinical parameters with histological findings. 8 dental students volunteered for the investigation. After a prophylaxis, they performed optimal oral hygiene to reach mean plaque and gingival indices approaching zero. They then abolished all oral hygiene procedures for a period of 21 days. After this experimental gingivitis phase, they again performed optimal oral hygiene for 8 days to restore gingival health. At days 0, 1, 2, 4 and 8 after experimental gingivitis, the plaque index (PlI), the gingival index (GI) and the gingival exudate flow rate (GEFR) were assessed and their buccal gingiva was biopsied. Point counting procedures were performed at 2 different levels of magnification on light microscopic sections to estimate the volume fractions of epithelium, infiltrated and non-infiltrated connective tissue, and collagen. The relative numbers of fibroblasts, polymorphonuclear neutrophils, lymphocytes, plasma cells and macrophages were estimated by counting the number of profiles of these cells in a specific connective tissue area adjacent to the apical end of the junctional epithelium. A rapid drop in the PlI was noted with increasing time after oral hygiene, followed by a slower decrease in the GI and GEFR scores. The histological picture during the entire experiment was that of an initial gingival lesion. At day 0, no chronic inflammation of the gingiva characterized by a predominance of plasma cells was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison between histological and clinical parameters during human experimental gingivitis

The purpose of this investigation was to study stereologically the histopathologic alterations occurring during a human experimental gingivitis, and to establish a relationship between clinical parameters and histologic findings. Eight dental students volunteered for the study. After a prophylaxis they performed optimal oral hygiene for 3-4 weeks to reach mean plaque and gingival indices approaching zero. They then abandoned all oral hygiene procedures for a period of 21 days. At d 0, 4, 7, 14 and 21, Plaque Index (PII), Gingival Index (GI) and Gingival Exudate Flow Rate (GEFR) were assessed, and a buccal biopsy of their gingiva was taken. Point counting procedures were performed at 2 different levels of magnification to estimate the volume densities of epithelium, infiltrated and non-infiltrated connective tissue, and collagen. The percentages of polymorphonuclear neutrophilic granulocytes, lymphocytes, plasma cells, macrophages and fibroblasts were estimated by counting the number of profiles of these cells in the connective tissue area close to the apical end of the junctional epithelium. The histological picture during the entire experiment was one of an early lesion (Page & Schroeder 1976). The clinically healthy gingiva did not correspond to a histologically healthy gingiva containing only a few inflammatory cells, probably because the 3-4 wk of perfect oral hygiene were not sufficient to generate histological health. Furthermore, no chronic inflammation of the gingiva, as characterized by a predominance of plasma cells, was observed after 3 wk without oral hygiene. Thus, more than 3 wk of no oral hygiene are necessary to obtain an established gingival lesion. With increasing gingivitis scores between GI = 0 and GI = 2 there was a significant increase in the percentages of lymphocytes and a significant decrease in the percentages of fibroblasts. With increasing GEFR similar trends in percentages were observed for lymphocytes and fibroblasts. It was concluded that GI scores and GEFR reflect histologic changes in tissue and, hence, are valid indicators of gingivitis development.     

Mast cells of the human gingiva

One-side gingivitis was induced in 21 individuals by local abolition of tooth cleansing for 15–17 days. Adjoining sections of biopsies from the non-cleansing side as well as from the control side with good tooth cleansing were stained with toluidine blue (pH 1.0), acridine orange (pH 0.5), astra blue (pH 0.2—0.3), alcian blue-safranin and the EACNAS-GBC technique in order to demonstrate mast cells specifically. The clinically normal gingiva was found to be rich in mast cells. In the inflamed tissue, the number was increased, especially in the pocket area of the connective tissue. The difference was due to an increase of both mature and, particularly, of immature mast cells. In those cases, where the inflammatory infiltrates were heavy, however, the number of mast cells was reduced in the area of cellular infiltration. The results indicate that products from the bacterial plaque at the gingival margin may directly or indirectly induce proliferation of mast cells in the adjacent connective tissue. Above a certain degree of stress, however the mast cells may respond by degranulation and release their active substances, which by contributing to the inflammatory reaction may enhance the local resistance.     

Histopathology of initial gingivitis in humans

Abstract The present study concerns the inflammatory alterations in the gingival margin during initial gingivitis in 11–13 year old human subjects. At day 0 of the experiment, all participants had clean teeth and healthy gingiva. All active oral hygiene measures were excluded for 4 days. From upper and lower premolars, which were extracted for orthodontic reasons, contralateral gingival biopsies, including the tooth and the adjacent gingiva, were obtained on days 0 and 4. The presence of inflammatory cells in the junctional epithelium and the adjacent connective tissue was determined quantitatively in semi-thin sections. The collagen content of the gingival margin was also determined. From day 0 to day 4 there was only a slight increase in the number of neutrophilic granulocytes in the junctional epithelium and adjacent connective tissue, while a more pronounced increase was found in the number of mononuclear leukocytes. A loss of collagen was noticed in 4 of the subjects, while 2 did not show any changes in collagen content. The inflammatory reaction seen in the present study differs somewhat from that observed in adult humans and adult dogs. The results correspond more to the reaction seen in juvenile dogs.     

Periodontal response after tooth movement into intrabony defects

The present study was undertaken since conflicting evidence exists regarding the effect of such tooth movement on levels of connective tissue attachment. Localized intrabony pockets were produced around isolated incisors in four rhesus monkeys. The root surfaces were planned to the level of the bone at the base of the angular bony defects. An oral hygiene regime was begun and continued for the remainder of the study. The experimental teeth were moved orthodontically into, and through, the original area of the intrabony defect. Two months after cessation of active tooth movement, block specimens were removed for histologic analysis. Control specimens comprised those teeth with induced periodontal defects, but without tooth movement. In specimens not subjected to tooth movement, angular bony defects were present and epithelium lined the root surface to the apical extent of instrumentation. The alveolar bone adjacent to the orthodontically moved teeth no longer had angular defect morphology. On the pressure side, epithelium lined the root surface, was interposed between root surface and bone and terminated at the apical limit of root instrumentation. On the tension side, the crest of the bone was located apical to the level of root planing, and epithelium lined the instrumented portion of the root surface. It was concluded that orthodontic tooth movement into intrabony periodontal defects was without effect upon the levels of connective tissue attachment.     

An ultrastructural study of proliferative gingivitis

Proliferative gingivitis was studied by electron microscopy. The crevicular epithelium adjacent to inflamed gingival connective tissue was primarily characterized by its relatively larger intercellular spaces which contained a variety of emigrating cells and granular precipitates resembling plasmic substances. Neutrophils and lymphocytes migrated between epithelial cells so that the intercellular spaces enlarged. The interruption of basement membrane was associated with inflammatory cells. Among the inflammatory cells plasma cells were predominant and displayed a variety of morphological characteristics. They consisted primarily of tightly packed, flattened cisternae of rough endoplasmic reticulum (rER) and a prominent paranuclear golgi zone. Fragments of cytoplasm apparently originating from the plasma cells were lying free in the surrounding intercellular space, in which collagen fibrils were impaired, disrupted and dissolved. All of these changes in plasma cells may relate to the synthesis and release of antibody. It is suggested that the inflammatory destructive features in connective tissue were mainly contributed to plasma cells.     

The use of extracted teeth to evaluate clinical measurements of periodontal disease.

Clinical indicators of periodontal disease, Gingivitis Index, Gingival crevicular fluid and pocket depth measurements were obtained from the gingiva surfaces of 30 teeth. The gingival margins were marked on the surfaces of the teeth prior to extraction. The extracted teeth were stained with hematoxylin and air dried, and the distances from the groove to the base of the calculus, plaque, and connective tissue attachment were obtained. The plaque-free zone was also measured. Comparisons were made between clinical and tooth surface measurements. A high correlation was found between clinical pocket depth measurements and tooth surface parameters. The correlations between all tooth surface parameters and GCF were statistically significant. The G.I. was significantly correlated only with the penetration of calculus into the pocket. The clinical pocket depth was statistically the same as the distance from the gingival groove to the coronal connective tissue attachment. The plaque-free zone appeared to represent the junctional epithelium.     

Stereological observations on long-term experimental gingivitis in man

The purpose of the present investigation was to study stereologically the histopathologic changes in the gingiva during 6 months of abolished oral hygiene and to study the development of chronic gingivitis in man. After a thorough prophylaxis procedure, 5 dental students performed optimal oral hygiene under supervision for a period of 3 weeks. At the end of this pre-experimental phase, they were asked to abolish all oral hygiene procedures for 4 (2 individuals) to 6 months (3 individuals). At day 21, and after 2, 3, 4, 5 and 6 months, the gingival exudate flow rate and the gingival index were assessed, and buccal gingival biopsies taken. Semi-thin histologic sections were stained with basic fuchsine and methylene blue. By point counting at 2 different levels of magnification, the volume densities of epithelium, infiltrated (ICT) and non-infiltrated connective tissue, and collagen were estimated. The %s of fibroblasts, PMN's lymphocytes, plasma cells and macrophages were estimated in a predetermined standardized area close to the apical termination of the junctional epithelium. With increasing time, the volume densities of the ICT rose concomitantly with a decrease in the volume densities of the collagen. In spite of great interindividual variations, a slow shift in the proportions of some cell populations was consistently observed. While the fraction of PMN's, lymphocytes and macrophages remained stable, a decrease of fibro-blasts (57 to 39%) and an increase of plasma cells (0.2 to 10%) was observed. This study has, therefore, demonstrated that, in 6 months of plaque accumulation, a chronic gingivitis with a predominance of PMN's and lymphocytes develops.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Morphological studies on periodontal disease in the cynomolgus monkey

Electron microscopic observations were made on naturally occurring gingivitis, on gingivitis at non-ligated sites of an experimental animal, and on ligature-induced periodontitis in a periodontal disease model using female cynomolgus monkeys.
For both types of gingivitis specimens, a plaque associated chronic inflammatory reaction was observed, comparable to that described for the human established lesion. Bacterial accumulations on tooth surfaces were separated from the epithelium by neutrophils which exhibited variations in fine structure and extent of bacterial phagocytosis related to their proximity to the plaque. In the inflamed connective tissue of the papillae, collagen was reduced to strands extending between the cellular elements. The plasma cell was the most common inflammatory cell and had three major variations in form.
With ligature-induced periodontitis, similar inflammatory features were observed in the gingiva. A complex flora like that encountered in human periodontitis was observed around and within the ligature as well as more apically in the pocket. As compared to the flora in gingivitis, more spirochetes were encountered, a larger proportion of the microorganisms appeared to be in a living state, and bacteria were in contact with the epithelial surface, rather than being walled off by the neutrophils. This seemingly more aggressive plaque was associated with an advanced lesion characterized by changes that indicated sequential destruction of the collagenous attachment to cementum and concommitant apical migration of the epithelial attachment. No bacteria were observed in the soft tissues, even in the wide intercellular spaces between the epithelial cells.     

Histologic characteristics of experimental gingivitis in the juvenile and adult beagle dog

Abstract An earlier study (Matsson & Attström 1979) revealed an unexplained difference between juvenile and adult dogs in the propensity to develop clinical signs of gingivitis. The aims of the present investigation were to depict the structural composition of clinically normal gingiva and to analyze the histologic changes in the gingiva during plaque development in juvenile and adult dogs. Six beagle dogs were used. Two periods of discontinued oral hygiene were studied, the first at 3 and the second at 12 months of age. Biopsies were sampled on days 0, 4, 7, 14, 21 and 28 of each period. Sections from the biopsies were analyzed at two levels of magnification. Compared to adult dog gingiva, juvenile gingiva seemed to display: 1) a thicker keratinized layer of the oral epithelium, 2) a junctional epithelium that structurally resembles the oral epithelium, 3) a cuticular structure at the surface of the junctional epithelium, 4) a limited mononuclear inflammatory cell response during experimental gingivitis, and 5) a delayed establishment of an infiltrated connective tissue portion during experimental gingivitis. In addition, during experimental gingivitis, subgingival plaque formed along the tooth surfaces to a lesser extent in the juvenile stage compared to adult dogs.     

Initial gingivitis in dogs

The gingival characteristics after four days of gingivitis development have been studied in three Beagle dogs. Gingival normality was induced by regular tooth cleaning and gingivitis was provoked by abolishing oral hygiene procedures and giving the animals a soft diet. Measurements of the gingival fluid flow and the amount of crevicular leukocytes served to clinically assess the gingival condition. Semi- and ultra-thin sections from contralateral biopsies of buccal gingival tissues from premolars obtained on day 0 and day 4 respectively were subjected to stereologic analysis based on morphometric point counting procedures. On day 0 of the experiment two dogs revealed minute amounts of gingival fluid and crevicular leukocytes and the gingival tissues of these dogs were normal. The third dog had somewhat higher amounts of the gingival parameters and the connective tissue adjacent to the junctional epithelium harboured a small lymphoid infiltrate occupying 8.3 ± 2.8% of the gingival tissues. On day 4 the gingival fluid flow and the amounts of crevicular leukocytes were increased in all dogs compared to their values on day 0. The coronal con nective tissue in the two dogs with initially normal gingiva revealed on day 4 an infiltrated portion which occupied 6.1 ± 2.4% of the gingival tissues. The infiltrate was predominated by neutrophilic granulocytes, monocytes, macrophages, immunoblasts and small lymphocytes. A 60% loss of the collagen density in the infiltrated fraction was noted between biopsies from day 0 and day 4 in these two dogs. The tissues of the dog which entered the experiment with a preestablished lymphoid infiltrate reacted with similar but more intense inflammatory processes on day 4 than those of the dogs with normal gingiva on day 0. The results indicate that initial gingivitis in dogs is characterized by acute exudative inflammation and immunopathological processes as well as by a marked loss of collagen fibers in the connective tissue located beneath the coronal part of the junctional epithelium. Possible mechanisms responsible for the observed alterations have been discussed.     

Histological and clinical parameters of human gingiva following 3 weeks of chemical (chlorhexidine) or mechanical plaque control

The aim of the present study was to compare stereologically the histopathologic variations following 3 weeks of chemical (chlorhexidine) or mechanical plaque control. 18 students and dental hygienists volunteered for this investigation. After prophylaxis, they performed optimal oral hygiene to reach mean plaque and gingival indices approaching 0. Six of them then performed mechanical plaque control of 3 weeks (control), while the other 12 rinsed 3 times daily with a 0.12% chlorhexidine solution (test). At days 0 and 21, the plaque index (PlI), the gingival index (Gl) and the gingival exudate flow rate (GEFR) were assessed and biopsies were obtained from buccal sites. Point-counting procedures were performed at 2 different levels of magnification on light microscopic sections to estimate the volume fractions of epithelium, infiltrated and non-infiltrated connective tissue, and collagen. The relative numbers of fibroblasts, polymorphonuclear neutrophils, lymphocytes, plasma cells, macrophages and mast cells were estimated by counting the number of nuclear profiles of these cells in a specific connective tissue area adjacent to the apical termination of the junctional epithelium. After 21 days, the PlIs of the test subjects were significantly higher than the PlIs of the controls, but their Gl were similar. At the end of the experimental period, the various volume fractions and %s of cell profiles remained stable with the exception of an increase in the %s of lymphocytes in the test group. This study has shown that, clinically as well as histologically, the daily use of chlorhexidine for a 3-week period is equally efficient as optimal mechanical tooth cleaning in maintaining a healthy gingiva in the buccal sites investigated.     

Histopathological study of experimental periodontitis in rats--ultrastructures, permeability, immunohistochemistry, and morphometric analysis in both pocket and long junctional epithelial

To elucidate the biological characteristics of both pocket and long junctional epithelia in experimental periodontitis, elastic rubber was inserted between the first and second molars of the left maxilla in rats. The rubber was removed after a week. An immunohistochemical study using anti-laminin and permeability experiments using peroxidase were conducted; examinations were made of ultrastructures and lanthanum; and a morphometric analysis was made of the distribution of capillaries immediately below the epithelium. Results: 1. Periodontal pockets formed from 5 days to 2 weeks after removal of the rubber. The long junctional epithelium was appearent from the fourth week after removal. Immunohistochemical study showed that laminin was located at the internal and external basal laminae in the long junctional epithelium but not on the surface of the pocket epithelium. 2. Electron microscopy showed the pocket epithelium to consist of flattened cells aligned parallel to the tooth surface in the coronal portion. The epithelium, which included numerous vacuoles, manifested especially wide intercellular spaces in which a large number of polymorphonuclear leukocytes were evident. Bacteria surrounded by neutrophils could be seen on the surface of the cementum in the pocket. Invading neutrophils split the epithelium at the central portion of the pocket. In the apical portion, the epithelium formed spindles that adhered to the cementum by means of half-desmosomes. Many neutrophils and fenestrated capillaries were observed in connective tissue immediately below the epithelium. 3. The long junctional epithelium consisted of 2 or 3 cell layers aligned parallel to the tooth surface. Intercellular spaces in the long junctional epithelium were as wide as those in the pocket epithelium and contained a small number of lymphocytes and a few neutrophils. Half-desmosomes were detected between the epithelial cells and the cementum. From the central to the apical region, epithelial tissues assumed a knife-edge form. Desmosomes and gap junctions occurred among these cells. Dense granules containing a limiting membrane and homogeneous electron-dense material were observed int he peripheral cytoplasm. Although fibroblasts aligned parallel to the epithelium occurred in them, few collagen fibers, inflammatory cells and capillaries were to be recognized in the connective tissue. 4. Permeability experiments in the pocket and the long junctional epithelium produced analogous results. Light microscopy showed that, in both epithelia, peroxidase penetrated into intercellular spaces and leaked into the pocket. Electron microscopy showed that, as an electron-dense material, lanthanum was detected in intercellular spaces and connective tissues.(ABSTRACT TRUNCATED AT 400 WORDS)     

In situ hybridization study on tissue inhibitors of metalloproteinases (TIMPs) mRNA-expressing cells in human inflamed gingival tissue

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.     

Fibrinolytic activity of gingival epithelium

Specimens of both human and animal gingivae were examined by fibrinolytic autography and all showed lysis over the gingival pocket epithelium, lysis never occurring over oral gingival epithelium. Epithelium separated from the connective tissue also showed fibrinolytic activity confined to the pocket epithelium. Examination of human gingival crevice washings showed fibrinolysis over some nucleated squamous epithelial cells as well as over anuclcated cell fragments. Although ihe neutrophil leucocytes in crevicular washings have fibrinolytic activity, this activity does not compare with that of the epithelium. The fibrinolytic activity of gingival pocket epithelium may be important in the pathogenesis of gingivitis and periodontitis, particularly due to the ability of plasmin lo activate complement.     

Plaque formation at healthy and inflamed gingival sites in young individuals

Abstract The present investigation was performed to evaluate the influence of gingivitis on the amount of de novo plaque that forms during a 24-h period of no oral hygiene. 292 fully dentate subjects participated in the study. The condition of the gingiva and the presence of supragingival plaque were examined at 4 surfaces of each tooth at a baseline examination. Following this examination, the participants were subjected to a comprehensive mechanical tooth cleaning and instructed to refrain from tooth cleaning measures during the subsequent 24 h. The plaque examinations were repeated at the end of the 24-h period. The results from the clinical trial revealed that during a 24-h period of no tooth cleaning, subjects with naturally occurring overt gingivitis, in general, formed more plaque than young individuals with healthy gingivae. Furthermore, plaque in all parts of the dentition, formed more frequently on tooth surfaces adjacent to sites with gingivitis than at healthy sites. It was concluded that the condition of the gingiva plays an important role for de novo plaque formation.