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)     

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.     

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)     

Experimental gingivitis in young and elderly individuals

Abstract The development of experimental gingivitis was studied in young and elderly humans during a 21-d period of oral hygiene abstention. The state of the gingiva was assessed by the Gingival Index and by measurements of the amount of gingival exudate on filter paper strips placed at the entrance of the gingival sulcus of the lower lateral incisors and cuspids. Soft deposits were assessed by the Plaque Index and by differential counts of microorganisms in gram stained smears of dento-gingival plaque. At the end of the plaque growth period, the patients were given a thorough dental prophylaxis. Gingival condition and plaque were assessed at regular intervals during a subsequent period of controlled oral hygiene. The development of gingivitis during the oral hygiene abstention period was more rapid and more severe in old than in young individuals. Plaque accumulation was greater in the older persons. A definite difference in plaque consistency was also observed. However, microscopic counts of various types of microorganisms did not reveal any differences throughout the period of plaque accumulation. When active oral hygiene was reinstituted, the state of the gingiva rapidly returned to pre-experimental levels in both groups. The findings of this study indicate that with age there is an altered host response to the microorganisms of the plaque.     

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.     

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.     

Phenotypic dynamics of macrophage subpopulations during human experimental gingivitis

The purpose of the present study was to investigate whether functionally different macrophages are present in clinically healthy gingiva and during human experimental gingivitis. Eight male probands were introduced to an oral hygiene program until all reached mean Plaque and Gingival Index scores approaching zero. During the following 19 days all oral hygiene was abandoned. At d -14, 0, 2, 4, 7, 11 and 19 clinical indices and gingival biopsies were taken. Cryostat sections were incubated with monoclonal antibodies against mature macrophages (25F9), inflammatory macrophages (27E10) and anti-inflammatory macrophages (RM 3/1). Positive cells were counted in the inflammatory infiltrate (IF) and the connective tissue (CT). At d -14 elevated numbers of 27E10-positive cells were observed which decreased significantly at d 0 (p less than 0.018) and increased again at d 19 (p less than 0.026). Significant differences in the number of RM 3/1-positive cells were found between d 0 and d -14, 2, 4 and 7 (p less than 0.05) while no differences in the number of 25F9-positive cells were observed throughout this study. It was concluded that experimental gingival inflammation is characterized by the appearance and disappearance of functionally different macrophage subpopulations.     

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.     

Clinical and histologic characteristics of normal gingiva in dogs

The aims of the present study were to establish normal gingiva in dogs, to characterize the clinical conditions prevailing and to stereologically describe the structural composition of the normal gingival tissues. Three beagle dogs were subjected to regular oral hygiene procedures during 15 weeks. Measurements of gingival fluid flow and the amounts of crevicular leukocytes served to clinically assess the gingival conditions. Semi-and ultrathin sections from biopsies of normal buccal gingival tissues from premolars were subjected to stereologic analysis based on morphometric point counting procedures. Gingival normality was achieved in two of the dogs. The normal gingival tissue in these dogs was characterized clinically by abscense of gingival fluid flow and by a minute amount of crevicular leukocytes. A gingival sulcus was most often absent. The junctional epithelium was without rete pegs, and the entire gingival connective tissue was densely filled by homogeneous collagen fiber bundles. A few isolated inflammatory cells were present in the junctional epithelium and the adjacent connective tissue. No clusters of inflammatory cells forming an infiltrate could be observed.
Stereologically, the gingival tissue comprised 48% epithelium and 52% connective tissue. The junctional epithelium occupied 10% of the gingival tissue and included a fraction of 2.8% occupied by leukocytes. The latter by volume comprised 50% neutrophilic granulocytes and mononuclear cells each. The connective tissue was composed of 67% collagen fibers, 14% free cells and 19% residual tissue. The composition of the connective tissue adjacent to the junctional epithelium differed somewhat from that of more central connective tissue fractions.     

Interepithelial lymphocytes in experimental gingivitis in young and elderly individuals

Experimental gingivitis was induced and monitored in young and elderly subjects. After 9 days of oral hygiene abstention, a gingival biopsy was obtained from the labial surface of the lower right cuspid. The distribution of lymphocytes within the crevicular epithelium was assessed, and the ultrastructural morphology of the lymphocytes was studied. From day 0 to day 9 the increase in gingival inflammation (Gingival Index) was greater in elderly than in young subjects. The number of lymphocytes per 100 epithelial cells was greater within the crevicular epithelium of elderly subjects. However, this difference was not statistically significant. The ratio of lymphocytes was significantly higher in the coronal than in the apical portion of the epithelium in both age groups. On an ultrastructural level two types of interepithelial lymphocytes, inactive and intermediate, were recognized. The intermediate cell types displayed morphological characteristics typical of cells responding to antigen stimulation.     

Influence of experimental neutropenia in dogs with chronic gingivitis

The influence of a four day period of experimental neutropenia on the clinical state and the structural constituents of chronically inflamed gingiva has been studied in four beagle dogs. Neutropenia was induced by heterologous anti-neutrophil serum. The effects on the gingiva were evaluated by Gingival Index (GI) and Gingival Exudate measurements and by morphometric analysis of various tissue components in sections from biopsies of buccal gingiva sampled on days 2, 3, and 4. Control biopsies were obtained before induction of neutropenia. The GI did not markedly change during the observation period. The amount of Gingival Exudate, however, significantly decreased following the induction of neutropenia and remained low throughout the experiment. Neutrophilic granulocytes disappeared in the gingival tissues, while, apart from a relative increase in plasma cells, no other tissue components seemed to change. No bacterial invasion of the junctional epithelium or of the gingival connective tissue could be observed. Thus during a four day period the bacterial defense mechanisms at the dentogingival junction seem to be able to prevent penetration of microorganisms into the gingival tissues despite the absence of neutrophilic granulocytes.     

Clinical and light microscopic observations of gingivitis and early ligature-induced periodontitis in the cynomolgus monkey.

Clinical and histological observations were made on gingivitis and ligature-induced periodontitis in 4 adult female cynomolgus monkeys (Macaca fascicularis) to define the changes occurring in the early periodontitis lesion. Silk ligatures were tied around selected posterior teeth and replaced weekly for 4 weeks. The changes from gingivitis to periodontitis induced by ligation, and back to a state of clinical health after ligature removal, scaling and polishing, were characterized by Plaque Index, Gingival Index, probing pocket depth, attachment loss, and histologic evaluation. A mild, chronic marginal gingivitis was the normal finding in the gingiva of posterior teeth. The inflammatory infiltrate in the connective tissue contained primarily lymphocytes. Hygienic measures once a week reduced the amount of infiltrate and the epithelial proliferation, but did not eliminate it. Placement of silk ligatures rapidly changed the clinical picture to a moderate or severe gingivitis, which presented an almost bizarre response of sulcular and oral epithelium, with an increase in polymorphonuclear neutrophil infiltration. Within 2 weeks there was significant probing attachment loss. The clinical response on removal of ligatures and plaque was almost as rapid as the onset. The animal model is useful for manipulating variables in ways not possible in the study of human periodontitis.     

Correlations among gingival indices: a methodology study

Using an experimental gingivitis model, 99 subjects completed a 3-week study to determine the correlations between a visual index of gingivitis, the Modified Gingival Index (MGI), and the Gingival Index (GI), the Interdental Bleeding Index (IBI), and the Papillary Bleeding Index (PBI). Following a baseline examination consisting of the MGI and either the GI, IBI, or PBI, each subject received a full mouth scaling and rubber cup polishing to render the teeth plaque and calculus free. Subjects then rinsed twice daily for 30 seconds with 20 ml of either an active antimicrobial or control mouthrinse for 3 weeks while abstaining from all other oral hygiene. The MGI correlated significantly with the GI, IBI, and PBI, both at baseline and at 3 weeks for all subjects.     

Microvascular volumes in healthy and inflamed gingiva in humans

It has been suggested that the vascular alterations found in inflamed gingiva may be of significance in the enhanced extension of the pathological process into the periodontium. The purpose of this investigation was to measured the changes in blood vascular volume occurring in gingiva with the onset of gingivitis and its resolution. Twenty-six individuals participated in this study. Gingival biopsies were taken following a 21-day experimental gingivitis, following resolution of a 21-d experimental gingivitis and during a 6-month experimental gingivitis and a 6-month period of optimal oral hygiene. A total of 126 biopsies was obtained, from which 378 sections were cut at 2 microns for stereological analysis. At low magnification, reference volumes were estimated using point counting procedures and expressed as mm3 of gingiva per mm length of vestibular gingiva, in a vestibulo-lingual plane. At higher magnification the ratio between the volume of vessels and connective tissue was calculated. The final results were expressed as mm3 of vessels per mm length of vestibular gingiva, in a vestibulo-lingual plane. The mean vessel volume expressed per unit length of vestibular gingiva ranged from 0.010 to 0.024 mm3/mm. No statistically significant differences in vascular volumes were found between inflamed and non-inflamed gingiva. It was concluded that the changes in vascular architecture during early gingivitis described in the literature had either taken place in the subjects prior to the time of experimentation or that any vascular changes (cytologic or functional) which had taken place may be compensatory for the changes in architecture described in the literature.     

The effect of topical application of dextran on the gingiva of the beagle dog.

Dextrans derived from Leuconostoc mesenteroides were placed on clinically healthy gingiva of Beagle dogs once a day for 21 days. Control gingival tissues received saline. Both healthy controls and dextran-treated tissues were brushed daily. Inflamed control tissues were obtained by allowing plaque to accumulate for 21 days. Tissues receiving daily application of dextran solutions developed chronic gingival inflammation but displayed no clinical signs of gingivitis. Healthy control gingival tissues showed no clinical signs of gingivitis and minor histologic inflammatory changes. Tissues exposed to dental plaque showed the typical clinical and histological inflammatory changes of gingivitis. Thus dextran, a substance similar to the extracellular polysaccharide found in dental plaque, was able to penetrate the sulcular epithelium, enter healthy gingival connective tissue and cause chronic inflammation. This connective tissue inflammation occurred without inducing any of the clinical signs of gingivitis. Therefore, it is concluded that dextran produced one component of the gingivitis response, chronic histologic inflammation, independent of another major component of the disease, clinical inflammation.     

Non-surgical periodontal treatment of cyclosporin A-induced gingival overgrowth: immunohistochemical results

Aim:  The present study was planned to analyze the effects of a 12-month non-surgical periodontal treatment on histologic and immunohistochemical features of cyclosporin A (CsA)-induced gingival overgrowth (GO).
Materials and methods:  Gingival samples were collected from 21 liver transplant subjects exhibiting CsA-induced GO prior to, and 12 months after non-surgical periodontal therapy including oral hygiene instructions, scaling and 2-month recall appointments, and also from 18 healthy control subjects. Gingival biopsy specimens were stained with hematoxylin–eosin and monoclonal antibodies for vimentin, CD3 (T-lymphocytes), CD20 (B-lymphocytes), CD34 (endothelium) and Ki-67 (fibroblasts proliferation rate), using a streptavidin-biotin-peroxidase complex method.
Results:  Total inflammatory cells, gingival vessels and fibroblast proliferation rate demonstrated significant reduction after non-surgical periodontal treatment ( P  < 0.0001) in overgrown gingiva, while B- and T-lymphocytes remained nearly unchanged ( P  = 0.61 and 0.33, respectively). At the 12-month evaluation no significant differences were found when comparing the gingival biopsies from CsA-treated patients and those from healthy controls ( P  > 0.05).
Conclusions:  Control of clinical inflammation by means of non-surgical periodontal treatment results both in lowering of inflammatory infiltrate and in changes in connective tissue composition. Thus, plaque-induced inflammation would seem to modulate the drug-gingival tissue interaction.
Clinical relevance:  A strict plaque control program play a pivotal role in the management of transplant patients exhibiting cyclosporin A-GO.     

Cell populations associated with conversion from bleeding to nonbleeding gingiva

The purpose of this study was to observe changes in cell populations of the interdental gingival tissue, which accompanied the conversion of a bleeding to a nonbleeding state induced by scaling and improved oral hygiene. Fifteen bleeding and 18 stopped-bleeding interproximal gingival biopsies were obtained from 33 patients and processed for light microscopic evaluation. The morphometric analysis of eight connective tissue components revealed that the percentage volume density of all inflammatory cells decreased, and the percentage of fibroblasts and collagen increased, when the gingiva changed from a bleeding to a nonbleeding state. Furthermore, the inflammatory cell infiltrate in bleeding and stopped-bleeding specimens was dominated by mononuclear cells of the lymphocyte/macrophage/monocyte group, while plasma cells and polymorphonuclear leukocytes comprised only a small fraction of the inflammatory cells present. Significant repair of gingival connective tissue had occurred in the stopped-bleeding specimens.     

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.     

The reversal of localized experimental gingivitis

Twenty dental students randomly divided into four groups of five participated in this trial. Three weeks of supervised oral hygiene preceded the study in order to ensure a optimum state of gingival health. The gingival condition was assessed by means of the Gingival Index and measurements of gingival exudate. Plaque accumulation was assessed by means of the Plaque Index. Using plaque-guards, two 21-day periods were alllowed for the induction of localized experimental gingivitis around a lateral incisor and adjacent canine in each jaw. The contralateral areas served as controls. Habitual oral hygiene procedures were maintained in all other areas of the mouth throughout the experimental periods. Following 21 days of localized plaque accumulation in the mandibular experimental areas, mechanical toothcleaning procedures were introduced at intervals of once a day (Group A), once every second day (Group B), once every third day (Group C) and once every fourth day (Group D). Groups A and B regained gingival health in 10 days. The experiment was then repeated in the maxillary experimental areas. Group A, rinsing once daily with chlorhexidine solution regained gingival health in 4 days. The results obtained in the localized experimental gingivitis model were similar to those reported when totally withdrawing oral hygiene.