Bacterial invasion in experimental gingivitis in man.

Gingival biopsies of the mesial papilla area of the first molar were obtained from each patient at 0-, 14- and 21-day intervals during plaque formation. The biopsies were fixed, serially sectioned, and Gram-stained. The incidence and distribution of the bacteria-like structures were studied by microscopy. In all the specimens the bacterial nature of Gram-stained material was substantiated. In the epithelium the highest number of bacteria was found at the outer layer of marginal oral epithelium, sulcular epithelium and apical oral epithelium along with a decreasing pattern of penetration progressing deeper into the layers of tissue. For junctional epithelium the situation was just the opposite. Each subject had significantly higher counts at Day 21 than at Day 14 for both epithelium and connective tissue. Also significantly higher counts were found in connective tissue compared with epithelium. The higher bacterial density of intragingival bacteria was associated with the higher gingival and plaque indices. This study suggests that early stages of gingival inflammation may be mediated by invasion of bacteria.

Recurrent periodontal disease and bacterial presence in the gingiva.

Previous investigations have shown that, in biopsies taken from untreated sites of periodontitis, bacteria were present between the epithelial cells and within the connective tissue. In the present study we have examined Gram-stained sections of diseased gingival sites where the disease had recurred after surgical periodontal treatment. The six subjects chosen for the study were patients who had undergone surgical therapy for the treatment of periodontitis and who, upon subsequent recall visits, showed evidence of at least one site recurrent after treatment as detected by bleeding on probing and increased pocket depth of 5 mm or more. A normal control site from the same patient was chosen, preferably contralaterally, showing positive response to treatment with no signs of disease. Sections were stained with either hematoxylin and eosin for tissue survey or Gram for assessment of bacteria and examined by light microscopy. In many specimens, the bacterial nature of Gram-stained material was substantiated. Preliminary results showed a significantly increased number of bacteria in the refractory sites when compared with control sites which responded positively to treatment. The results of this investigation provided further evidence that bacterial presence inside the periodontal tissue may be an important pathogenic factor in periodontal disease.

The presence of bacteria in the oral epithelium in periodontal disease. III. Correlation with Langerhans cells.

Langerhans cells (LC) are cell types found in the skin and gingiva. LC have immunological functions as phagocytic cells and as antigen-presenting cells for T and B lymphocytes. Sections from biopsies of the gingiva in cases of periodontal disease were found to have increased numbers of LC. These biopsies also contained intragingival bacteria. Serial sections of frozen specimens of human gingiva were prepared for staining. Hematoxylin and eosin were used for tissue survey, the Gram stain for assessment of bacterial invasion, anti-Leu-6 monoclonal antibody associated with peroxidase technique (PAP) to identify LC, antibacterial sera to Bacteroides gingivalis and Actinobacillus actinomycetemcomitans associated with peroxidase to specifically identify these two common periodontopathogenic bacteria. Additional positive identification of bacteria was performed by preparing the same histological section containing gram-stained particles for scanning electron microscope and transmission electron microscope LC confirmation. The results suggest that the increased number of LC seen in diseased sites of oral epithelium containing intragingival microorganisms may be one of the host immune mechanisms to penetration by bacteria.

The presence of bacteria in the oral epithelium in periodontal disease. II. Immunohistochemical identification of bacteria.

Serial histological sections of gingiva obtained from each of six advanced adult periodontitis, two localized juvenile periodontitis and two periodontally healthy patients were used for specific identification of bacteria within the oral epithelium and adjacent connective tissue. Healthy gingival biopsies served as controls. Sections from patients and control biopsies were Gram-stained and also screened with antibacterial sera associated with the peroxidase immunocytochemical technique for specific bacterial identification. The "Pop-off" electron microscopic technique was also used to further demonstrate the bacterial nature of peroxidase-stained material. In addition, the possible correlation between bacteria and areas of possible reduced keratinization was investigated. The results showed that sections of orthokeratinized healthy gingiva did not contain bacteria. Gram-stained sections from diseased sites contained large numbers of bacteria in the oral epithelium and adjacent connective tissue. Bacteroides gingivalis and to a lesser extent Capnocytophaga gingivalis were found in periodontitis, and Actinobacillus actinomycetemcomitans was found in juvenile periodontitis when the immunoperoxidase technique was used. The bacterial nature of peroxidase-stained material was confirmed by the "pop-off" technique. In the disease biopsies, bacterial presence was correlated with areas of reduced amounts of keratin suggesting that the oral epithelium may be a portal of entry for bacteria into gingival tissues.