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The activity of elastase increases significantly in the gingival sulcus during inflammation. The release of this enzyme from crevicular and peripheral polymorphonuclear leucocytes (PMNs) was studied in seven patients with gingivitis, six with adult periodontitis, seven with rapidly progressive periodontitis and in nine healthy subjects. Peripheral PMNs were isolated from blood and crevicular PMNs from gingival washings. After preincubation with cytochalasin B, the same numbers of crevicular and peripheral cells were incubated either in phosphate-buffered saline (spontaneous release) or in the same buffer containing formyl methionyl-leucyl-phenyl-alanine (stimulated release). Elastase activity was measured in the supernatant by a fluorimetric technique. The results confirm that compared to peripheral PMNs, crevicular cells show a higher spontaneous release of elastase and a lower stimulated release. The activity of elastase released either spontaneously or after stimulation of crevicular cells was significantly lower in the controls as compared with cells obtained from patients with gingivitis or periodontitis. Crevicular PMNs collected from patients with deeper pockets appear to release larger amounts of elastase than those from controls.

The techniques presently available for counting crevicular polymorphonuclear leucocytes (CPMNs) require special equipment and are only semi-quantitative. The present new and simple method is based on the property that Durapore strips have to harvest a maximum number of crevicular cells and to release them when shaken in a suitable buffer. Durapore strips were inserted into periodontal crevices or pockets, shaken in PBS and the liberated PMNs counted in a Neubauer chamber. The validity of the method was ascertained in 5 experiments. First, it was shown that 90% of the harvested cells were released from Durapore strips after being shaken in buffer. Second, decreasing numbers of CPMNs were found when performing sequential samplings. Third, when counting CPMNs before and after therapy, a significant decrease in the number of cells was found. Fourth, we could confirm that sites with bleeding on probing did contain a much higher number of CPMNs as compared to non-bleeding sites. Finally, our counting technique confirmed that deeper pockets contain a significantly higher number of CPMNs.

Human gingival keratinocytes were cultured and, after the first passage, subjected to cell detachment assays with polymorphonuclear leucocytes (PMNs) and/or sonic extracts from Actinobacillus actinomycetemcomitans Y4, and Eikenella corrodens 1073. The effector-to-target cell ratio was 30:1. Bacterial extracts alone caused no disruption of keratinocyte monolayers. PMNs alone also caused only minimal detachment after 14 h incubation. Adding A. actinomycetemcomitans to the PMN-keratinocyte co-cultures at the concentration of 100 micrograms/ml caused dramatic cell detachment. The effect of A. actinomycetemcomitans was heat labile and not inhibited by polymyxin B. Cell detachment was inhibited by alpha 1-antitrypsin, whereas catalase and superoxide dismutase could not prevent it. No lysis of keratinocytes was observed after incubation, as judged by 51Cr release. E. corrodens had little effect even at the concentration of 1000 micrograms/ml. H2O2 and partially purified PMN elastase also caused detachment of keratinocytes. These data indicate that PMNs can cause non-lytic detachment of keratinocytes when interacting with certain bacteria.

Albumin and alpha 2-macroglobulin were analysed in gingival fluid collected by filter paper strips from 6 volunteers before, during and after 10 days of dental plaque accumulation. The two proteins were also determined in samples of venous blood at the beginning of abstention from tooth brushing and 10 days later. A nephelometric technique was used for the analysis of albumin, but alpha 2-macroglobulin was determined by ELISA. The plaque and gingival indices, as well as the flow of gingival fluid, increased significantly during the period of plaque accumulation and then returned to baseline values. The concentrations of the two proteins in blood remained within normal limits. In gingival fluid, however, the concentration of albumin increased about two-fold at the peak of inflammation, while that of alpha 2-macroglobulin increased as much as 6 times. The enhanced passage of alpha 2-macroglobulin into the gingival sulcus in comparison to the entirely plasma-derived albumin was consistent with the concept that the greater excretion of globulin during inflammation must be due to an increased local synthesis.

In a group of 5 women taking oral contraceptives, the concentration of polymorphonuclear leucocytes along the gingival margin and the activities of cathepsin D, beta-glucuronidase and elastase were found to increase significantly during the intermenstrual phases.

In the course of the extensive tissue remodeling occurring after flap surgery and during the subsequent gingival reattachment in man, numerous intracellular vacuoles were observed in fibroblasts of the lamina propria. These vacuoles contained one or several typical collagen fibrils and had either an electron-lucent matrix or contained an electron-dense material obscuring the fibrillar outlines and cross-striations.

Free gingival grafts were placed in the lower anterior vestibular region of 30 patients each of whom had a gingival recession. According to the width of the recession and to the surgical procedure, the patients were divided into four groups. Group 1 showed a "narrow" (less than 3 mm) type of recession, while Group 2 had a "wide" type. In both of these groups the graft was placed over the denuded root. In Group 3 (narrow recession) and Group 4 (wide), a collar of vestibular gingiva was left in place on the lower margin of the denuded root at the moment of grafting. Measurements and photographs taken before and 1 month after surgery, 6 months and 2 years postoperatively, showed varying degrees of root covering by the phenomenon of creeping attachment. An optimal recovery of the gingival profile was obtained in the three patients of Group 1. In the other categories of patients the creeping of the attachment apparatus was relatively small (between 0 and 33%) within the 2 years of observation. The factors which seem to have a definite influence upon the phenomenon of creeping attachment are the following: width of the recession, position of the graft, bone resorption, position of the tooth and hygiene of the patient.

Most of the previous histological studies devoted to free gingival grafts in humans were limited to the description of the various phases of healing between the graft and the subjacent tissues. The present long-term study illustrates the histological findings 1 to 36 months after grafting along the zone of passage between graft and surrounding tissues. In the transitional zone between graft and alveolar mucosa it was found that the structural characteristics of the palatal mucosa, with its dense network of collagen fibres and its orthokeratinized superficial layer were conserved even after an interval of time as long as 16 months. These features contrasted with the loose network of collagen fibres and the presence of numerous elastic fibres in the alveolar mucosa. As for the transitional zone between graft and gingiva the findings confirmed that these two types of mucosa are similar, except that the gingival epithelium showed, in epon embedded sections, a layer of parakeratinization, as opposed to the dense layer of orthokeratin found on the grafted palatal epithelium. A constant feature in the present investigation was the change in orientation of the collagen fibres along the zones of healing, both between graft and alveolar mucosa and between graft and gingiva.

The concentration of alpha 2-macroglobulin (alpha 2-M) in human gingival sulci has been investigated in two studies: first, in gingival washings during a 21-day period of experimental gingivitis in eight human volunteers and, second, in crevicular fluid collected with filter paper strips before and after initial periodontal therapy in 11 patients. The concentration of total alpha 2-M was found to increase in the washings of the volunteers throughout the period of experimental gingivitis. In the group of patients receiving periodontal therapy, the absolute amount of alpha 2-M in the fluid showed a significant decrease after therapy. The gingival index of inflammation and the crevicular fluid flow also decreased significantly. The specific content of the inhibitor (micrograms of alpha 2-M per mg of fluid per min), however, was found to increase in the fluid with decreasing inflammation. As detected by crossed immunoelectrophoresis, the fluid collected in these patients before therapy, in the presence of severe inflammation, invariably showed peaks of both free and complexed alpha 2-M. In contrast, the fluid collected from the same sites after healing of the inflammation contained no detectable free alpha 2-M.

The gingival sulcus is formed by the space between the sulcular epithelium, the junctional epithelium, and the wall of the tooth. The functioning of its different parts is important in the early changes taking place when marginal plaque is accumulating. The cellular turnover of the junctional epithelium is very high, which could explain its high healing potential. The intercellular spaces are wider in the junctional epithelium, when compared to those of the sulcular or oral epithelia. Polymorphonuclear leucocytes can migrate through these spaces, attracted toward the sulcus by chemotactic substances. Numerous substances have been shown to permeate the junctional epithelium, both toward the sulcus and from the sulcus within the connective tissue. The sulcus is normally sterile and will tend to eliminate any foreign particle. Leucocytes and desquamated epithelial cells will also tend to leave the gingival sulcus and will be found in saliva. Various instruments, such as paper or celluloid strips of platinum loops, can be used to collect the content of the gingival sulcus. Quantitative investigations can be performed by analyzing crevicular fluid or the products of washings of the marginal gingiva.

Samples of hydroxyapatite (HA) beads smaller than 1 mg were coated with 10 microliter of either saliva, serum, or human crevicular fluid before being added to a suspension of Streptococcus sanguis or Bacteroides gingivalis. In some assays, preparations of a granular fraction, elastase, or cathepsin G from human leukocytes were used to coat HA or to treat saliva-coated HA (SHA) before mixing with bacteria. The number of cells adhering to the beads was then counted under a scanning electron microscope by a standardized procedure. More cells were found to adhere to SHA in this assay than in the conventional large-scale assay. Human crevicular fluid, even when diluted up to three times, completely inhibited the adherence of S. sanguis to HA. A 100% inhibition of S. sanguis adherence was also observed when HA was coated with the granular fraction of leukocytes, and a 65% inhibition observed when SHA was treated with the enzyme preparation. When used to coat HA, elastase and cathepsin G reduced the adherence of S. sanguis by 30 and 50%, respectively. The binding of S. sanguis to elastase- or cathepsin G-treated SHA was also reduced. B. gingivalis 33277 was found to adhere in high numbers to SHA. Coating HA with crevicular fluid or with the lysosomal enzyme preparation had a limited negative effect. We postulate that crevicular fluid prevents the adherence of S. sanguis by virtue of either its enzyme content or its albumin content or both.

The granule fraction of human polymorphonuclear leucocytes (PMNs), the concentrated product of gingival washing from 2 human volunteers and the culture fluid of samples of human gingiva were incubated with neutral salt soluble collagen from rat skin and the patterns of collagen degradation were studied by SDS polyacrylamide gel electrophoresis. Collagenase from human gingiva cleaved the collagen molecules in a fashion similar to that of the PMN granule fraction. Collagen was also attacked by elastase from human PMNs and, to a lesser extent, by elastase from the gingival washings.

Most of the investigations reviewed in the present paper show that the connective tissue has an inductive potential upon epithelial differentiation. As for the investigations showing an epithelial predetermination, one must admit that they have been limited to particular tissue systems (for instance feathers and hairs) and that they could imply errors of manipulation or interpretation. As stated by Gilette: "Mesenchyme as the determinant is the favorite today, but perhaps only because experiments to demonstrate the opposite would be harder to assess" (Gilette, 1971). On the clinical point of view, one could draw an important conclusion from these investigations: at the present time, only the free or pedicle mucosal grafts including epithelium and connective tissue, or only connective tissue, are indicated to obtain a satisfactory increase of the width of attached gingiva.

Cells from explants were cultured in the presence of [L-35S]-methionine for 24 h. Cultures containing cycloheximide or cultures frozen immediately after addition of labelled methionine served as controls. The medium was removed and, after washing, cells were disrupted by repeated freezing and thawing. Synthesis of alpha 2-macroglobulin (alpha 2-M) was evaluated by autoradiography of the SDS-PAGE patterns obtained with the culture medium, and by autoradiography of the crossed immunoelectrophoretic patterns obtained with both the cell extract and the culture medium. There was incorporation of labelled methionine in newly-synthesized alpha 2-M. No autoradiographic bands were found in the frozen preparations, and there was a progressive inhibition of globulin synthesis in the presence of increasing concentrations of cycloheximide. This synthesis of alpha 2-M by human gingival fibroblasts may explain the presence of free alpha 2-M in gingival fluid and might be of significance in the control of proteolysis during gingivitis and periodontitis.

Sections of human gingiva obtained in a cryostat have been treated with the granular fraction of human polymorphonuclear leucocytes (PMNs) at pH 3,6 and 7,3, as well as with the enzyme elastase (neutral protease) purified from the same cells. The sections treated by the enzymes have been compared to control serial sections incubated in the absence of enzyme or with the enzyme in the presence of a specific inhibitor. The results showed that the granular fraction at an acid pH seems to affect primarily the connective tissue, while the same fraction at a neutral pH, like the enzyme elastase exerts a preferential action upon epithelium structures.

In periodontal surgery, healing after Guided Tissue Regeneration (GTR) may be explained by differences in functional activities of gingival and periodontal ligament fibroblasts (GF and PDLF). Several studies in vitro have supported this hypothesis, but much remains to be defined. In the present work, gingival and periodontal ligament fibroblasts derived from five healthy subjects were isolated and compared in vitro. The morphology of the cells was observed under scanning electron microscopy (SEM). Several extracellular matrix components (ECM) were studied to compare the effects on fibroblast attachment, proliferation, and protein synthesis. Several biochemical markers were examined in both cellular extract (CE) and conditioned medium (CM). We also examined the muscle differentiation markers alpha-smooth muscle actin, desmin, and smooth-muscle myosin. Finally, we studied the effects of epithelial cells on the proliferation and protein synthesis of the two types of fibroblasts. GF and PDLF appeared identical under the SEM. All ECM components enhanced attachment; however, while collagen types I and IV promoted the attachment of GF, gelatin, laminin, and vitronectin promoted that of PDLF. Most ECM components increased the proliferation rate of GF and the biosynthetic activity of PDLF. The biochemical markers were similarly distributed between the two cell types, except for alkaline phosphatase, which was detected only in the CE of PDLF. Both GF and PDLF strongly expressed alpha-smooth-muscle actin and were negative for desmin; only PDLF were positive for smooth-muscle myosin. Epithelial cells increased the proliferation of both GF and PDLF but had no effect on their biosynthetic activity. These in vitro results may better explain the in vivo functional differences between GF and PDLF. [ABSTRACT FROM AUTHOR]