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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    ISSN: 1600-051X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract Host responses to periodontal infections include the production of several families of enzymes that are released by stromal, epithelial or inflammatory cells. Study of these enzymes in gingival crevicular fluid may lead to insights into pathogenesis and may provide a rational basis for the development of novel diagnostic tests. However, analogous to other diagnostic interventions in dentistry and medicine, validation of host enzymes as diagnostic indicators is dependent on clear-cut demonstrations of the identity of the enzyme, reproducibility, diagnostic accuracy and clinical utility. The enzyme of interest should be readily measured over a broad range of disease severity and in varied clinical settings. Ideally, the enzyme should also be an essential component of proposed pathogenic mechanisms. In this context, the connective tissue matrix degrading enzymes elastase, collagenase and gelatinase are promising because of their apparently central role in periodontal attachment loss and disease progression. Sensitive and specific assays are also available to quantify these enzymes. Other work on enzymes associated with cell death (aspartate aminotransferase, lactate dehydrogenase) and several neutrophil lysosomal enzymes (beta glucuronidase, arylsulphatase, cathepsins) has demonstrated positive associations between enzyme levels and attachment loss and inflammation. While numerous cross-sectional studies have indicated that the levels of hydrolytic enzymes in gingival crevicular fluid parallel the severity of periodontal lesions, there are much less data on reproducibility, diagnostic accuracy and clinical utility in longitudinal studies. As appropriate study design is an essential prerequisite for establishing the efficacy of host enzymes as diagnostic tests, future clinical investigations should include: (1) individuals who would most likely benefit by early diagnosis, i.e., rapidly progressive and recurrent periodontitis cases; (2) longitudinal, cohort study designs to show that attachment loss is temporally linked with large increases in enzyme activity; (3) the use of a battery of tests to overcome intrinsic problems of low predictive values when prevalence of active disease is low. In the final analysis, the utility of host enzymes as diagnostic indicators will need to be examined in randomized controlled trials in which the question is asked: are patients better off as a result of testing?
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1600-051X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract 82 patients with a recent history of periodontal abscesses and/or loss of gingival attachment (GAL) despite active periodontal therapy were enrolled in a double-blind, randomized, placebo-controlled trial. Clinical measurements and subgingival scaling were performed every 2 months. If any site exhibited ≥ 2 mm loss of GAL or a periodontal abscess, patients were administered either 100 mg Doxycycline per day for 3 weeks or placebo. During 12 months of monitoring, 55 patients exhibited recurrent active disease and were then randomly assigned to either the Doxycycline or placebo groups. Clinical measurements of GAL and microbiological culture of subgingival bacteria were made at intervals between 1 week and 7 months after completion of the drug regime. Within 7 months, 15 out of 19 patients on placebo exhibited recurrent disease compared to 13 out of 29 patients on Doxycycline, a relative risk reduction of 43% (p 〈 0.05) for Doxycycline compared to placebo. Minimal inhibitory concentrations of Doxycycline for subgingival plaque samples from active sites ranged between 25–100 μg/ml, which are several fold higher than reported crevicular fluid concentrations for this drug. However gingival crevicular fluid collagenase was inhibited in vitro at concentrations of 5-10 μg/ml Doxycycline. These data indicate that Doxycycline provides significant risk reduction of recurrent periodontitis in patients with active disease.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1600-0765
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The survival rate of avulsed permanent teeth following replantation is affected primarily by the duration of the extra-alveolar period and the nature of the storage conditions. These factors are believed to strongly affect the viability of periodontal ligament (PL) cells but in vitro assays of cell viability based on vital dye assays are only weakly correlated with the tooth survival rate after replantation. The aim of the present study was to examine the relative dependence of cell membrane integrity, attachment and clonogenic capacity of human PL cells on the temperature and duration of the extra-alveolar period and the type of storage medium. Twenty-four premolar teeth were extracted for orthodontic reasons from 9 patients 11–18 years of age. Teeth were maintained at 4°C or 23°C for 15, 30, 60 or 120 min in either milk or dry conditions. Cell membrane integrity was determined by BCECF/AM dye inclusion. Plating efficiency was determined by measurement of cell attachment at 3 and 6 h. The clonogenic capacity of progenitor cells was estimated by limiting dilution and colony counts. For all assays teeth stored in milk at 4°C showed the highest percentages of BCECF positive, attached cells with clonogenic capacity. Increased storage time (15–120 min) was associated with a 50% relative reduction of BCECF staining and a 5-fold relative reduction of cell attachment regardless of storage conditions. However, the clonogenic capacity of progenitor cells decreased 25-fold over the same duration of storage. These data demonstrate that in vitro assays of clonogenic capacity are much more sensitive to extra-oral storage time and storage conditions than dye inclusion or cell attachment. We suggest that in comparison with in vitro measures of cell membrane integrity, the clonogenic capacity of PL cells is more closely linked to tooth survival rate, probably reflecting the capacity of PL progenitor cells to recolonize the root surface after replantation.
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  • 4
    ISSN: 1600-0765
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: To assess the temporal relationship between periodontal tissue destruction and the activity of collagenase, exudate from inflamed periodontal tissues was collected and latent and active collagenase activities were measured by a functional assay in a longitudinal cohort study. Comparisons were made between human subjects with either: 1) inflammation with a previous history of progressive loss of connective tissue and bone support (n=14); 2) inflammation and previous history of bone loss but now clinically stable (n=27); or 3) inflammation and no loss of bone support (n=17). Experiments using specific enzyme inhibitors, blocking antibodies and SDS-PAGE fluorograph to identify the pattern of collagen substrate degradation demonstrated that the collagenase activity was derived from neutrophils and not from bacteria or other host cells. Active collagenase activity pooled from 6 sites per subject was respectively 5 and 6-fold higher in the group with progressive loss of connective tissue compared to the groups with either inflamed tissues alone or with inflammation and previous bone loss. In contrast, latent collagenase was increased up to 2 fold higher in the group with inflammation but no bone loss compared to the group with progressive lesions. Moreover, the ratio of active to total collagenase activity was 50% higher in the group with progressive lesions. Although in all subjects successive measurements of site-specific active collagenase 1 month apart demonstrated wide variation (r〈0.50), only in sites with progressive periodontal destruction was there significant increase of active collagenase with time (1.28×l0−4 collagenase units per day). There were also sharp elevations in active enzyme level at the time of detection of loss of connective tissue attachment in specific sites of 8 subjects. At the time of detection of connective tissue attachment loss, there was an overall 40% increase of pooled active collagenase activity in all subjects with progressive loss of connective tissue compared to pre-breakdown sampling times. These data provide strong in vivo evidence for a direct role of active neutrophil collagenase in the pathological destruction of periodontal connective tissue.
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  • 5
    ISSN: 1600-0765
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: We have studied the relationship between perturbations of fibroblast turnover in inflamed gingiva of different severities. To perform detailed spatial analyses of gingival fibroblast progenitor cells, inflammatory cell infiltrates and blood vessels, 3 Cynomolgus monkeys with healthy periodontium and 2 with naturally occurring gingivitis and ligature-induced periodontitis were pulse-labeled with 3H-thymidine. Morphometric analyses of radioautographs from mid-sagittal supra-alveolar gingival connective tissues of incisors were performed in sites subjacent to junctional, sulcular and oral epithelium, in the body of the lamina propria and just superior to the alveolar crest. The percentage of fibroblasts incorporating 3H-thymidine label, expressed as the labeling index (LI), was higher subjacent to the sulcular epithelium in periodontitis (1.73±0.37) than in healthy sites (1.06±0.22). This was not statistically significant (0.05 〈 p 〈 0.1) due to the small number of animals used. The sites subjacent to the sulcular epithelium also exhibited the largest increase in lymphocyte density from health to gingivitis (p 〈 0.01). In contrast, the LI of fibroblasts subjacent to the oral epithelium was 5-fold higher in healthy (0.82±0.17) compared to periodontitis sites (0.13 ± 0.09; p 〈 0.05). Labeled fibroblasts were found close to blood vessels in all compartments and in all disease states; distance to blood vessels was reduced in inflamed sites (p 〈 0.10). There were increased numbers of blood vessels per unit area in the lamina propria of gingivitis compared to healthy sites. However, there were no regional differences with respect to blood vessel numbers or area in sites subjacent to junctional epithelium with different disease states. The results indicate that: 1) experimentally-induced inflammation in the gingiva of Cynomolgus monkeys is associated with site-specific perturbations of cell turnover; 2) fibroblast progenitors are preferentially situated adjacent to blood vessels as in the periodontal ligament; 3) the vascular response to inflammation is a generalized increase in blood vessel numbers, but not their size; 4) reactive proliferation of fibroblasts may compensate for cell death in the lamina propria but is not detectable at the site of connective tissue attachment loss subjacent to the junctional epithelium. Failure to maintain the fibroblast progenitor population may be an important component of attachment loss in progressive periodontitis lesions.
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  • 6
    ISSN: 1600-0765
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Collagenolytic enzymes released by neutrophils are associated with the destruction of periodontium in periodontal diseases. Measurement of these enzymes in gingival crevicular fluid (GCF) could be used to test for periodontal diseases and thereby simplify diagnosis. To test this hypothesis, gelatinase (MMP-9) was analyzed in GCF samples with a simple assay system. GCF was collected by a mouthrinse method from 10 patients with gingivitis (G); 10 well-treated and maintained periodontitis patients (TP) without detectable loss of attachment; and 9 patients with recurrent loss of periodontal attachment (〉2 mm) and/or abscess formation (RP). Clinical measurements including tooth mobility (MOB) and gingival attachment level (GAL) were made monthly for a maximum of 10 months. Active and latent forms of gelatinase were measured by a functional assay using gelatin substrate-gel enzymography and the activities were quantified by laser densitometry. Reproducibility analysis demonstrated that the assay (inter-gel, inter-assay, inter-scan) and diurnal variations were small compared to biological variation. The presence of active gelatinase was detected in 97.8% of TP samples, 86.4% of RP samples, but in only 11.4% of G samples. In addition, the mean active gelatinase activity was found to be significantly higher (p〈0.001) in the RP (71 006 U) than the TP (43814 U) groups, both of which were higher (p〈 0.001) than the G group (2824 U). During periods of attachment loss, samples from the RP group exhibited a 2-fold increase of mean active gelatinase activity (129414 U). Correlation and regression analyses demonstrated that active gelatinase activity was most strongly associated with loss of GAL (r = 0.52, p〈0.0001) and to a lesser degree with mean MOB (r = 0.35, p〈0.03). However, neither total (latent plus active) nor latent gelatinase levels were associated closely with any clinical parameters of periodontitis. Metronidazole treatment (250 mg, tid, for 7 days) of RP patients significantly reduced the level of active and latent gelatinase 4- to 6-fold (p〈 0.002). These data indicate that measurement of active gelatinase in mouthrinse samples may provide a simple and robust diagnostic test for periodontal diseases and for assessment of treatment response.
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  • 7
    ISSN: 1600-0765
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
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  • 8
    ISSN: 1600-0765
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Existing enzymatic and mechanical techniques of cell release from human gingiva were quantitated in regard to: cell yield; viability; freedom from cell clumping; and debris formation. To optimize these parameters methodologies were modified and combined with new procedures which included incubation with a variety of enzymes. Fibrous and inflamed tissues from 17 patients were examined. The optimal procedure that was selected included thorough mincing of the tissue plus the addition of DNA-ase, elastase and hyaluronidase to a standard bacterial collagenase mixture. Consistently, up to 3.4 × 106 cells per 100 mg of tissue were released after tissue digestion, with a mean viability of 85%. The cell suspensions were relatively free of debris and cell clumps. Data from Coulter particle counting was in close agreement with hemocytometer counts. The composition of the cell suspension was not significantly different from the original tissue sample. Tissue remnants following the digestion procedure only contained an estimated 1 % of the original number of cells in the sample. The single cell suspensions produced using this method were found to be suitable for analysis by flow cytometry. The possible selective cytotoxicity of the enzymatic technique on cell populations in the DNA-synthesis phase of the cell cycle was examined in healthy gingival tissue of a Cynomolgus monkey. The gingiva was injected with 3H-thymidine and the percentage of labeled fibroblasts was enumerated in radioautographs either of sections prepared from undigested tissue or in smears of single cell suspensions. There was no significant difference in the percentage of labeled cells between sections or smears. The data suggest that the method reported here can be applied to flow cytometry analysis of progenitor cell populations in gingival tissues.
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  • 9
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    ISSN: 1600-0765
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Labeling index and total grain count data of periodontal ligament cells after either continuous infusion or single injection with tritiated thymidine were compared with values predicted by two different models of cell proliferation. One model assumes that all proliferating cells are homogeneous and proceed through the cell cycle at a rate described by an exponential distribution. In the second model, it is assumed that cell division is asymmetric. Mitosis produces one proliferating and one differentiated cell, each with a characteristic and distinct lifetime and proliferative capacity. The predictions of the first model deviated widely from the continuous labeling and total grain count data. In comparison, the second model predicted a linear relationship between labeling index and time which is consistent with data from mouse, hamster and rat periodontal ligament cells. The model also accurately predicted the total grain count after a single injection of tritiated thymidine. The data suggest that the periodontal ligament contains renewing and differentiated populations of cells which can be described in terms of proliferative potential and lifetime.
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  • 10
    ISSN: 1600-0765
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: An improved understanding of the differentiation of periodontal ligament cells could facilitate the development of new treatment approaches for overcoming the loss of specialized cell types caused by periodontitis. To study healing of wounded periodontal tissues and the differentiation of mineralizing connective tissue cells in periodontal ligament, we have examined the influence of wound size and collagen implantation on the regeneration of periodontium and on immunohistochemical staining for osteopontin and bone sialoprotein. Four groups of Wistar rats were wounded by drilling through the alveolar bone and by extirpation of the periodontal ligament. Wounds were 0.6 or 1.8 mm in diameter and defects were either implanted with collagen gels or were treated without implants. Rats were killed at 1 wk or 2 months after wounding and tissue sections were stained with monoclonal antibodies against rat osteopontin and bone sialoprotein. Collagen implants strongly increased staining for osteopontin and bone sialoprotein in defects at 1 wk. By 2 months alveolar bone healed completely regardless of the wound size but in large defects, periodontal ligament width was significantly reduced with or without implants. In large wounds at 2 months, collagen implants inhibited bone regeneration and there was stronger staining for osteopontin and bone sialoprotein in the bone replacing the implant, indicating that collagen prolonged bone remodelling. We conclude that implantation of exogenous collagen affects alveolar bone healing but does not preserve the width of the regenerated periodontal ligament. Therefore collagen does not appear to contribute to homeostasis in the periodontium following wounding.
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