Dissolution of type I collagen fibrils by gingival fibroblasts isolated from patients of various periodontitis categories

The classification of periodontitis in various disease categories, including juvenile periodontitis, rapidly progressive adult periodontitis and slowly progressive adult periodontitits is based mainly on differences in disease progression and age group susceptibility. Because dissolution of collagen fibers is an integral part of periodontal attachment loss, we investigated whether the clinical differences among these periodontitis/control groups are reflected in the collagen-degrading activity of gingival fibroblasts isolated from affected tissues. All fibroblast strains isolated from the 4 groups (n=48) displayed cell-associated collagenolytic activity when seeded in contact with a reconstituted film of type I collagen fibrils. Cells from the control group (n = 14) dissolved the collagen fibril film twice as fast as those from each of the 3 disease groups (juvenile periodontitis (n = 13), rapidly progressive adult periodontitis (n = 7), and slowly progressive adult periodontitits (n = 14)). Both interleukin-1β and phorbolester accelerated the rate of dissolution 2-4-fold, but even after cytokine or phorbolester stimulation control cells were still considerably more effective in dissolving the collagen fibrils than cells from the disease groups. The observation made in this study, that dissolution of collagen fibrils by gingival fibroblasts from periodontally diseased individuals is significantly slower than by cells from healthy control subjects, challenges disease paradigms based on a direct relationship between collagenolytic potential and disease activity.     

The in vivo expression of the collagenolytic matrix metalloproteinases (MMP-2, -8, -13, and -14) and matrilysin (MMP-7) in adult and localized juvenile periodontitis

Periodontal inflammation is characterized by irreversible degradation of periodontal ligament collagen fibers leading to loss of tooth attachment. Cultured gingival keratinocytes and fibroblasts express, in vitro, various matrix metalloproteinases (MMPs) which can degrade fibrillar collagens. We hypothesized that several MMPs are also synthesized in vivo by sulcular epithelium, and analyzed the collagenolytic MMPs (MMP-2, -8, -13, and -14) and matrilysin (MMP-7) in gingival tissue specimens and gingival crevicular fluid from adult and localized juvenile periodontitis patients by in situ hybridization, immunohistochemistry, and Western immunoblotting. MMP-2, -7, -8, and -13 were expressed in gingival sulcular epithelium. MMP-7 and -13 were also located in fibroblasts and macrophages, and MMP-8 in neutrophils. MMP-8- and -13-positive cells/mm2 were higher in periodontitis gingiva when compared with healthy control tissue (p < 0.01). In periodontal diseases, gingival sulcular epithelium expresses several, rather than a single, collagenolytic MMPs, and this proteolytic cascade is evidently responsible for the tissue destruction characteristic of adult and juvenile periodontitis.     

Collagen degradation by interleukin-1beta-stimulated gingival fibroblasts is accompanied by release and activation of multiple matrix metalloproteinases and cysteine proteinases

Objective: Several collagenolytic matrix metalloproteinases (MMPs) have recently been identified in gingival fibroblasts, while secreted cysteine proteinases could also participate in connective tissue destruction in periodontitis. To clarify their involvement, we examined enzyme release during collagen breakdown by cultured cytokine-stimulated fibroblasts. Materials and methods: Gingival fibroblasts were derived from four chronic periodontitis patients and cultured on collagen gels in serum-free medium for 1-4 days. Collagenolysis was measured by hydroxyproline release into the medium. Proteinases were assessed by electrophoresis and immunoblotting. Results: Adding interleukin-1beta resulted in progressive gel breakdown. This was associated particularly with a shift in MMP-1 band position from proenzyme to active enzyme and the appearance of active as well as proenzyme forms of cathepsin B. There was also partial processing of pro-MMP-13 and increased immunoreactivity for active cathepsin L. In addition, both pro-forms and active forms of MMP-8, membrane-type-1-MMP and MMP-2 were present in control and treated cultures. Conclusions: Fibroblast MMP-1 was most likely responsible for collagen dissolution in the culture model, while cathepsin B may have been part of an activation pathway. All studied proteinases contribute to extracellular matrix destruction in inflamed gingival tissue, where they probably activate each other in proteolytic cascades.     

Tetracycline inhibition identifies the cellular origin of interstitial collagenases in human periodontal diseases in vivo

Mammalian interstitial collagenases (E.C.3.4.24.7) are considered as key initiators of collagen degradation in periodontal diseases. However, the cellular sources of collagenases present in gingival crevicular fluid have not been completely clarified. Resident fibroblasts and epithelial cells as well as infiltrating neutrophils and monocyte/macrophages are potential sources of the enzymes. We have recently found significant differences in tetracycline inhibition between human neutrophil and fibroblast interstitial collagenases. To address the cellular source of collagenase present in gingival crevicular fluid in 2 distinct periodontal diseases, we studied the tetracycline inhibition of collagenase in gingival crevicular fluid of patients with localized juvenile periodontitis and adult periodontitis. Gingival crevicular fluid samples were collected from deep (greater than 5 mm) periodontal pockets and assayed for collagenase in the presence of 0-1000 microM doxycycline as well as a chemically modified tetracycline devoid of antimicrobial activity (4-de-dimethylaminotetracycline). The drug concentration required to inhibit 50% of collagenase activity (IC50) in localized juvenile periodontitis gingival crevicular fluid was 280 microM for doxycycline and 470 microM for 4-de-dimethylaminotetracycline. Significantly lower values, 10-20 microM, were obtained for collagenase in gingival crevicular fluid of patients with adult periodontitis. We propose that systemic tetracycline levels are efficient inhibitors of collagenase in gingival crevicular fluid in affected sites of patients with adult periodontitis but not of patients with localized juvenile periodontitis and that the fibroblast type interstitial collagenase is the predominant collagenase type in gingival crevicular fluid in affected sites of patients with localized juvenile periodontitis and the neutrophil collagenase in adult periodontitis gingival crevicular fluid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Collagenase activity in gingival crevicular fluid of patients with juvenile periodontitis

The nature and origin of collagenases in gingival crevicular fluid of juvenile periodontitis patients was investigated. Gingival crevicular fluid collected from deep untreated periodontal pockets of juvenile periodontitis patients was found to contain only vertebrate collagenase (EC 3.4.24.7) activity that cleaved soluble type-I and -III collagens into 3/4 and 1/4 length fragments, as analyzed by SDS-polyacrylamide gel electrophoresis. Type II collagen was degraded at a markedly slower rate. This substrate specificity is indicative of collagenases produced by fibroblasts, epithelial cells and macrophages. We have previously found that collagenase in gingival crevicular fluid of adult periodontics patients appears to be mainly derived from polymorphonuclear leukocytes (PMN). The reasons for the apparent difference in collagenase source between the groups were investigated. We examined whether the pathogen characteristic for juvenile periodontitis, Actinobacillus actinomycetemcomitans, can release collagenase from normal human PMNs. All 10 A. actinomycetemcomitans strains tested, freshly isolated from the subgingival plaque of juvenile periodontitis patients, caused release of collagenase from PMNs in vitro. These results suggest that the lack of normally functioning PMNs in the periodontium of juvenile periodontitis patients may result in a colonization of bacteria that activate the resident periodontal cells to produce increased amounts of collagenase.     

老年牙周炎牙龈组织诱导型一氧化氮合酶分布研究

目的 :观察慢性老年牙周炎患者牙龈组织中诱导型一氧化氮合酶 (iNOS)分布。方法 :采用免疫组织化学方法对 10例慢性老年牙周炎患者、10例慢性成人牙周炎患者、10例青少年牙周炎患者和 10例健康老年人牙龈组织中诱导型一氧化氮合酶分布进行了检测并比较研究。结果 :(1)牙周炎时牙龈组织中诱导型一氧化氮合酶主要在鳞状上皮细胞胞浆核周区颗粒状阳性表达 ,毛细血管壁内皮细胞、老化的胶原纤维及上皮下基底膜共同形成了一种乳头状轮廓样阳性表达形态 ,结缔组织和肉芽组织中各类炎症细胞也显阳性表达 ;(2 )慢性老年牙周炎组血管壁内皮细胞、结缔组织内炎症细胞、上皮乳头阳性表达例数明显低于青少年牙周炎组和慢性成人牙周炎组 (P <0 .0 5 )。血管壁内皮细胞和胶原纤维阳性表达例数低于健康老年人组 (P <0 .0 5 )。结论 :慢性老年牙周炎患者牙龈组织中诱导型一氧化氮合酶的表达明显降低 ,造成了局部一氧化氮(NO)合成减少 ,引起了局部牙龈组织免疫功能降低和免疫调节功能紊乱     

Expression of membrane alkaline phosphatase activity on gingival fibroblasts in chronic inflammatory periodontal disease

We investigated the expression of membrane alkaline phosphatase (ALP) activity on fibroblasts in inflamed gingiva from 7 patients with adult periodontitis. ALP activity was ultrahistochemically detected by a cerium-based capture method. The degree of ALP activity was estimated by morphometric analysis of the percentage of the perimeter on which ALP reaction product was deposited. Fibroblasts in the non-inflammatory connective tissue were surrounded by bundles of collagen fibrils, and the majority of these fibroblasts showed ALP-negative or weakly ALP-positive reaction. By contrast, fibroblasts in the inflammatory connective tissue were either surrounded by a non-collagenous substance or in contact with inflammatory cells, and the majority of these fibroblasts showed a strong ALP-positive reaction. These results suggest that the expression of membrane ALP activity on gingival fibroblasts is induced by microenvironmental changes associated with the loss of contact between the cells and the extracellular collagenous matrix during inflammatory reactions.     

Identification of polymorphonuclear leukocyte collagenase and gelatinase activities in mouthrinse samples: Correlation with periodontal disease activity in adult and juvenile periodontitis

In previous studies, elevations in the levels of active and latent collagenase in gingival crevicular fluid (GCF) have been correlated positively with periodontal disease activity. To provide a simple diagnostic approach for testing collagenolytic activity, the feasibility of using a 3.0 ml water mouthrinse to collect GCF simultaneously from all sites in the mouth was assessed. Patients with adult periodontitis (AP, n = 23) and local juvenile periodontitis (LJP, n = 7) were sampled before periodontal therapy and some (12 AP, 4 LJP) were also assessed longitudinally after scaling and root planing, administration of antibiotics, and following periodontal surgery. Healthy patients (n = 19) were used as controls. The levels of active collagenase, procollagenase, and collagenase inhibitor activity were determined by functional assays and quantitated after SDS-PAGE and fluorography. Gelatinase and progelatinase were assayed by enzymography on gelatin-substrate gels. Active collagenase levels were found to be significantly higher (14- to 20-fold) in AP and LJP patients compared to controls, whereas matrix metalloproteinase activity was not detected in mouthrinses from edentulous patients. Collagenase inhibitor levels were generally low in all groups of subjects tested. Following clinical treatment the levels of active collagenase and gelatinase were reduced; the reduction was significant for active collagenase after tetracycline treatment and scaling in LJP patients. Of the clinical indices recorded (gingival index, plaque index, and pocket depth) there were no significant correlations with enzyme activity but similar trends were observed between the changes in active collagenase and gingival index. In patients with untreated periodontal disease, collagenase occurred predominantly in the active form. N-ethylmaleimide (NEM) and p-aminophenylmercuric acetate (AMPA) were equally effective as activators of the latent collagenase, indicating that the collagenase was derived from PMNs, which were also the source of gelatinase. The results of these studies indicate that measurement of active collagenase and gelatinase in mouthrinse samples is potentially useful in the diagnosis and assessment of periodontal disease activity.     

Collagenase and collagenase inhibitor activities in crevicular fluid of patients receiving treatment for localized juvenile periodontitis

Collagenolytic activity was monitored in crevicular fluids of 3 patients receiving treatment for localized juvenile periodontitis (LJP) and in 3 control, clinically healthy patients to study relationships between mammalian collagenase and disease activity. It was found that tissue collagenase activity initially was significantly higher in diseased patients compared to controls, but that in II of 13 diseased sites the activity decreased substantially following clinical treatment. In control sites, active collagenase was generally low or absent. Latent collagenase concentrations tended to remain high in diseased sites and in sites with gingival inflammation. Collagenase inhibitor activity was found only in clinically healthy sites sampled from both control subjects and LJP patients throughout the experiment, and in diseased sites following various phases of treatment. The collagenolytic activity observed was typical of mammalian collagenase in that the primary degradation products of collagen were 3/4- and 1/4-fragments. However, additional specific fragmentation of native collagen 3/4-fragments was also observed, which could be attributed to a novel collagenolytic activity generally present in crevicular fluid in a latent form. The results of this study suggest that the determination of active collagenase and collagenase inhibitor in crevicular fluid might be useful in assessing both the disease status of periodontal tissues and the efficacy of clinical treatment.     

In vitro phagocytosis by crevicular phagocytes in various forms of periodontitis.

Phagocytes from the gingival crevice fluid (CF-cells) of 11 patients with localized juvenile and post-juvenile periodontitis (LJP/PJP), 14 with rapidly progressive periodontitis (RPP), 11 with adult periodontitis (AP), and 14 controls without periodontal disease were examined. Phagocytic activity in vitro was assessed. Crevicular washings were obtained from healthy sites of controls and diseased sites of patients after completion of the oral hygiene phase (professional and home care). The cells were carefully processed to avoid mechanical damage. The in vitro phagocytosis by uptake of opsonized C. albicans was performed in a moist chamber (30 minutes, 37 degrees C) and examined by light microscopy. CF-cells were differentiated on the basis of their morphological appearance. The majority of cells in crevicular washings were PMNs, some macrophages, and few lymphocytes. Phagocytic activity in patients with LJP/PJP and RPP was significantly decreased in comparison with that from AP and the control group. The decreased percentage of cells phagocytosing opsonized C. albicans was associated with the enhanced adherence of opsonized C. albicans. Moreover cell viability of CF-cells from LJP/PJP sites was significantly reduced. The data from the present study suggest that the in vitro phagocytosis of crevicular phagocytes in juvenile and rapidly progressive periodontitis lesions is diminished.     

The nature of the inflammatory infiltrates in childhood gingivitis, juvenile periodontitis and adult periodontitis: immunocytochemical studies using a monoclonal antibody to HLA Dr

Routinely fixed and processed gingival biopsies from childhood gingivitis, juvenile periodontitis and chronic adult periodontitis patients have been stained immunocytochemically with a monoclonal anti HLADr antibody to aid in the identification and quantification of cell types in the inflammatory infiltrates. Using immunoperoxidase staining and morphological criteria, 9 cell types were quantified in 30 patients. Lesions in the 3 groups were found to differ widely both in size and composition. In the small childhood gingivitis lesions, most cells were small lymphocytes, over half of which were HLADr positive, whereas in juvenile periodontitis biopsies, well over half the infiltrate was plasma cells. The chronic adult periodontitis samples showed greater variability in composition between these 2 extremes, perhaps reflecting differences in disease activity. These results suggest that, when disease is quiescent, the volume of inflamed gingival connective tissue is small and is dominated by B-small lymphocytes, whilst on activation, the lesion increases in size and much of the B-lymphocyte population is transformed to plasma cells. This view corroborates the results of other workers with regard to juvenile periodontitis, but suggests a different interpretation of the quiescent lesion of childhood gingivitis from that current in the literature.     

Assessment of complement cleavage in gingival fluid in humans with and without periodontal disease

The activation of the complement system may be an important immunopathologic mechanism in the initiation and progression of periodontal disease. The purpose of this study was to assess cleavage of complement components C3 (terminal pathway), C4 (classical pathway) and B (alternative pathway) in gingival fluid obtained from patients with varying types and severities of periodontal disease. Gingival fluid samples were obtained on filter paper strips from 18 healthy sites, 16 gingivitis, 59 chronic adult periodontitis, 45 rapidly progressive periodontitis, and 11 juvenile periodontitis lesions. Each patient was categorized on the basis of age and clinical indices, including Gingival Index, Plaque Index, measurement of pocket depth and loss of periodontal attachment in millimeters, presence of suppuration and bleeding on probing. Cleavage of C3, C4, and B from each site was assessed simultaneously by multilayer crossedimmunoelectrophoresis using solid phase absorbed specific antisera. The mean percentage C3 conversion ranged from a low of 12.6% in the healthy to 90.2% in the juvenile periodontitis group. Statistically significant differences, as determined by the Mann-Whitney U-Test, were observed between healthy sites and all other groups, gingivitis and all periodontitis groups, and juvenile vs. chronic periodontitis. C4 was present in all sites examined, but its cleavage product C4c was only observed in sites with juvenile periodontitis. B and its cleavage product Bb were consistently present in gingival fluid from inflamed lesions. The percentage of C3 cleaved to C3c correlated significantly (p < 0.001) with pocket depth (rho=0.58), gingivitis (rho=0.68) and bleeding on probing (rho=0.63). These results suggest that 1) increased complement cleavage is associated with increased severity of inflammation and periodontal destruction, and 2) classical pathway activation does not appear to occur in gingivitis and adult periodontitis, but may occur in juvenile periodontitis.     

Tissue localization of Actinobacillus actinomycetemcomitans in human periodontitis. I. Light, immunofluorescence and electron microscopic studies

Invasion of periodontal tissues by different bacterial morphotypes has been reported in human periodontitis; however, limited information is available as to prevalence, localization and the bacterial species involved. The present study determined prevalence and gingival localization of Actinobacillus (Haemophilus) actinomycetemcomitans in periodontal lesions of juvenile periodontitis patients. Thirty-five gingival biopsies were obtained from 12 juvenile periodontitis patients at the time of periodontal therapy. One additional control biopsy was obtained from each of two adult periodontally healthy subjects, one adult periodontitis patient and one periodontally healthy monkey (Macaca fosibolius). The biopsies were carefully processed to avoid mechanical introduction of bacteria into the tissues and were examined using light and electron microscopy. Rabbit antisera specific for the three A. actinomycetemcomitans serotypes were used for immunofluorescence microscopic localization of A. actinomycetemcomitans antigens in the gingival sections. Immunofluorescence microscopy showed A. actinomycetemcomitans specific antigens in the gingival tissues of 11 of the 12 juvenile patients examined. None of the control specimens showed evidence of A. actinomycetemcomitans antigens in the gingival connective tissue. One specimen from a periodontally healthy subject and the monkey biopsy, however, showed A. actinomycetemcomitans antigens in bacterial plaque on the surface of the crevicular epithelium. Transmission electron microscopic examination showed microcolonies of small gram-negative rods in the connective tissue, as well as single bacterial cells between collagen fibers and in areas of cell debris. In addition to these extracellular bacterial cells, evidence of bacterial cells was also found within gingival connective tissue phagocytic cells. The data from the present study suggest that the gingival tissue in juvenile periodontitis lesions harbors A. actinomycetemcomitans.     

The biologic character of gingival fibroblasts from normal and periodontitis sites]

Histological and clinical studies have found that periodontal ligament fibroblasts can produce connective attachment,while gingival fibroblasts cannot yet.The mechanism for gingival fibroblasts losing their ability of new attachment is unknown.We investigated the function of colagen synthesis by gingival fibroblasts from periodontitis sites,and normal sites and their response to TGF-beta,and Nicotinamide and Lactate,the inhibitors of poly(ADP-ribosyl)ation.We found that gingival fibroblasts from diseased sites produce much less collagen than from control sites (73.6+/-8.8vs.107.1+/-12.3,P<0.01),and these cells show unresponsive to TGF-beta,Nicotinamide and Lactate on collagen synthesis.These results may partly explain why gingival fibroblasts from periodontitis site lose their ability of new attachment.     

Enhanced collagen phagocytosis by rat molar periodontal fibroblasts after topical application of lipopolysaccharide – Ultrastructural observations and morphometric analysis

To investigate the effect of lipopolysaccharide (LPS) on phagocytic activity of collagen fibrils by periodontal fibroblasts, we studied rat molar gingival connective tissue and periodontal ligament under light and electron microscopy after topical application of LPS (5 mg/ml in physiological salt solution (PS)) on the gingival sulcus. Phagocytic activity of collagen fibrils by fibroblasts was evaluated by counting the number of collagen-containing vacuoles inside fibroblasts that were present within a defined area (1200 microns2). Values obtained from fibroblasts in the subepithelial connective tissue, the region near the alveolar crest, and the middle region of periodontal tissue were compared. Periodontal ligament fibroblasts showed increased phagocytosis of the collagen fibrils from 3 hours to 1 day after topical LPS application, but no differences were observed in the gingival tissue. The intracytoplasmic vacuoles containing collagen fibrils were of various sizes and shapes, showing positive for acid phosphatase and/or alkaline phosphatase reaction. Collagen phagocytic activity of the fibroblasts in the middle region of the periodontal ligament also increased after PS treatment. However, this was significantly less than that observed in LPS-treated animals (p less than 0.01). This study indicates that LPS may enhance the degradation of collagen by stimulating the phagocytic activity of the periodontal ligament fibroblasts.     

Nature of collagenolytic enzyme and inhibitor activities in crevicular fluid from healthy and inflamed periodontal tissues of beagle dogs

We have previously described the occurrence of collagenolytic enzyme activity in crevicular fluids from beagle dogs in which experimental conditions of gingivitis and periodontitis had been induced (Kryshtalskyj, Sodek & Ferrier 1986). In general, active collagenase activity was demonstrable in fluids from periodontitis sites, whereas in control and gingivitis sites various levels of latent collagenase and collagenase inhibitor activity were present. Analysis of the collagen digestion patterns by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) has shown that crevicular fluid samples with low collagenolytic activity generated ¾-α and ¼-α collagen fragments, typical of vertebrate collagenase. However, higher collagenolytic activity associated with periodontitis sites produced further degradation of the native collagen substrate. This activity was characterized by specific degradation products migrating between the ¾-α and ¼-α fragments. Similar digestion patterns are generated by enzymes in crevicular fluid from periodontitis sites in humans and by partially purified collagenase from osteoblastic cells (Otsuka, Sodek & Limeback 1984), but not by bacterial collagenase. Analysis of plaque, crevicular cell debris, saliva and serum indicated that the collagenolytic activity is derived from connective tissue and/or inflammatory cells, whereas collagenase inhibitor could also be derived from saliva and serum.     

Collagenolytic fragments and active gelatinase complexes in periodontitis

Background: Periodontal tissues remodel rapidly, which enables quick adaptation to mechanical changes. Matrix metalloproteinases (MMPs) are involved in these remodeling processes under control of tissue inhibitor of metalloproteinases (TIMPs). In periodontitis, overactivity of MMPs results in pathologic tissue degradation. The aim of this study was to analyze MMPs and TIMPs in healthy and diseased gingiva, periodontal ligament (PDL), and gingival crevicular fluid (GCF). Methods: Samples of gingiva, PDL, and GCF were obtained from healthy controls (gingiva: n = 18; PDL: n = 15; GCF: n = 8) and subjects with periodontitis (gingiva: n = 11; PDL: n = 18; GCF: n = 12). MMPs and TIMPs were analyzed by gelatin-, collagen-, and reverse zymography and by Western blotting. Total MMP activity was analyzed using a fluorogenic substrate. Results: TIMP-1 and -2, active and pro-MMP-2 and -9, and active MMP-1 and -8 were present in all samples. Large amounts of active MMP-2 complexes and collagenolytic fragments were also found. Their levels were higher in PDL and GCF from subjects with periodontitis. In general, TIMP levels were lower in diseased periodontal tissues. Especially diseased GCF contained more MMPs. Surprisingly, some MMPs were more abundant in healthy gingiva and PDL than in diseased tissue. Conclusions: Unexpected variations in MMP and TIMP levels in gingiva, PDL, and GCF may result from differences in subject characteristics and disease activity. The levels of active MMP-2 complexes and collagenolytic fragments are higher in the periodontium of subjects with periodontitis and might contribute significantly to periodontal destruction.     

Preparation and characterization of human gingival cells

A procedure was developed for the preparation of single cell suspensions from gingival and periodontal tissues using collagenase digestion followed by gentle mechanical disruption. The procedure was evaluated on rat gingival tissues. One-hour incubation was found to produce the greatest number of cells with the highest viability, and the largest recovery of mononuclear cells. Collagenase did not appear to effect recovery, viability or B cell surface markers on human peripheral blood cells. Human gingival and periodontal tissues were obtained from 35 patients: 20 with adult periodontitis; 8 with juvenile periodontitis; 7 with normal gingivae or chronic gingivitis. Scaling, followed by probing measurements and then surgery of the affected sites was routinely performed. In single cell suspensions from tissue obtained from surgery, significantly fewer mononuclear cells were recovered from the tissues of normal/chronic gingivitis patients than cells/mg of tissue recovered from adult periodontitis or juvenile periodontitis groups. The maximum contribution of blood mononuclear cells to the gingival cells averaged 0.5+0.2%. This method is useful for recovering gingival (periodontal) cells for phenotypic and functional studies.     

Thiol-dependent collagenolytic activity in culture media of Bacteroides gingivalis

Twenty strains of anaerobic bacteria including some oral species from Bacteroides, Eikenella, Fusobacterium, Capnocytophaga , and Actinobacillus were used for the detection of collagenolytic activity in their culture media (supernatants). Organisms were essentially grown in complete anaerobic basal broth, and collagenolytic activities in the culture media were measured using ¹⁴C-acetylated soluble collagen from rat skin. When the enzyme reaction was carried out in the standard reaction mixture for 60 min, very low collagenolytic activities were found in culture media of Bacteroides gingivalis 381, 1021, Bacteroides intermedius 163, K l–8, Bacteroides asaccharolyticus ATCC 27067, and Fusobacterium nucleatum ATCC 25586. However, the activity in the media of all species of B. gingivalis tested was enormously enhanced by the addition of reducing reagents having thiol-groups such as dithiothreitol, L-cysteine, etc. This stimulated activity was nearly completely inhibited by some thiol-blocking reagents. In contrast, these reagents had no effect on the collagenolytic activity in the media of the other anaerobes and in the collagenase preparation of Clostridium histolyticum . These results suggest that B. gingivalis produces thiol-dependent collagenolytic enzyme which may play an important role in the degradation of collagen fibrils in a reduced environment like the deep gingival pocket.     

Eosinophils during developing periodontal disease of rats

Experimentally provoked gingival inflammation in rats presented eosinophils in the cellular infiltrate. Eosinophil numbers increased progressively to a peak at 30 days, and subsequently there was no quantitative alteration. Initially eosinophils were found in inflammed areas underneath the gingival epithelium and in later periods were more numerous in the deep corium among the collagen fibers. The extracytoplasmic granules were among the collagen fibrils and close to fibroblasts which phagocytosed collagen. It is suggested that eosinophils participate in the experimentally induced periodontitis in rats, and that they can act on the metabolism of rat gingival collagen.