Use of a fluorogenic septapeptide matrix metalloproteinase assay to assess responses to periodontal treatment.

BACKGROUND: Quantification of gingival crevicular fluid matrix metalloproteinase activity may provide improved assessment of periodontal disease status and response to treatment. A fluorogenic matrix metalloproteinase substrate assay (FSA) has been developed using a methoxycoumarin-containing septapeptide analog of the alpha2(I) collagen cleavage site. This substrate exhibits increased fluorescence following cleavage by many matrix metalloproteinases, and the enzyme activity can be readily estimated with a fluorimeter. Here we compared this assay with classical methods of periodontal assessment including bleeding on probing, crevicular fluid flow, and probing depth to assess its utility as an indicator of changes in periodontal status and treatment response. METHODS: Complete measurements of probing depth were obtained for Ramfjord teeth on subjects who had been previously treated for periodontitis. Subjects were subsequently divided into groups based on existing periodontal disease severity: gingivitis (n = 21), stable periodontitis (n = 41), and severe periodontitis (n = 50). Crevicular fluid volume, bleeding on probing, and FSA were measured at each Ramfjord tooth or substitute. After baseline measurements, subjects received subgingival scaling and prophylaxis; 3 months later, they were reassessed. RESULTS: FSA measurements were positively associated with severity of disease at baseline. After treatment there were substantial reductions of FSA in gingivitis (approximately 51%; P <0.01) and severe periodontitis (approximately 45%; P <0.001), but not in stable periodontitis (13%; P >0.2). All groups showed a positive association between FSA measurements and higher bleeding scores at individual sites. FSA measurements were also positively associated with crevicular fluid flow at baseline, but after treatment there was a approximately 67% decrease (P <0.01) in the highest crevicular fluid flow class. There were significant reductions of FSA at follow-up for sites with probing depths between 0 to 3 mm (23%; P <0.05) and 4 to 6 mm (31%; P <0.05). However, the largest reduction was for sites with probing depth between 7 to 9 mm (49%; P <0.001). CONCLUSIONS: These results indicate that monitoring patients by measurement of matrix metalloproteinase levels in gingival crevicular fluid with the quenched fluorescent substrate assay provides estimates of inflammatory status, periodontal destruction, and response to treatment, especially in more severe periodontitis lesions.

A new bioerodible polymer insert for the controlled release of metronidazole.

This study evaluates a new class of bioerodible polymers as periodontal inserts for the controlled release of metronidazole. The system is based on association polymers formed from compatible blends of cellulose acetate phthalate (CAP) and a hydrophobic block copolymer of polyoxyethylene and polyoxypropylene, Pluronic L101. In addition to characterizing these polymers by thermal analysis, their erosion and metronidazole release characteristics were determined both in vitro, and in vivo using a rat model. The results show that increasing the concentration of Pluronic L101 in the blend to 50% and above leads to a sharp reduction in the rates of polymer erosion and metronidazole release. The characteristics of these slowly eroding films are potentially suitable for use as periodontal drug inserts with an effective duration of up to several days. Depending on the blend composition, the mechanism of metronidazole release was found to range from a surface erosion-controlled process to an erosion-modulated diffusion process. In all in vivo experiments, no signs of adverse tissue reactions were detected. Based on these results, prototype delivery inserts were designed and subsequently evaluated in volunteer patients. Preliminary results from this pilot study show that the metronidazole concentration in the gingival crevicular fluid was significant throughout the sampling period of up to 3 hr and remained well above the minimum inhibitory concentration for most periodontal pathogens. In addition, no discomfort or irritation was reported by the test subjects.

Impact of microbiological consultation on clinical decision making: a case-control study of clinical management of recurrent periodontitis.

Data obtained from diagnostic tests may influence the clinician's perception of the patient's state and in some instances may alter subsequent choices of therapeutic interventions. To determine if microbiological consultation influences the clinical management of patients with recurrent periodontitis, an observational, case-control study was conducted to measure the amount and type of periodontal treatment provided by periodontists (n = 13) who had referred patients with recurrent periodontitis for microbiological consultation. The control group consisted of periodontists (n = 10) who had not referred recurrent periodontitis patients for testing. Patients (n = 31; 20 females, 11 males; mean age 49.8 +/- 10.0 years) treated by the case group of periodontists were matched for age and sex to patients (n = 48; 22 females, 26 males; mean age 49.9 +/- 8.5 years) treated by the control group of periodontists. Questionnaires were administered to quantitatively assess the amount and type of treatment before and after receiving the microbiological report. Specific analyses were performed as a function of the time of receipt of the microbiology report. Case-control differences prior to the receipt of the report indicated that the amount of surgery/year was 43% greater for controls (P < 0.04), in spite of control patients exhibiting fewer deep pockets than case patients (P < 0.05). Case-control differences after the receipt of the report indicated that case patients were provided with 45% greater number of appointments/year (P < 0.005), 46% greater scaling/year (P < 0.02), and 79% greater antibiotics/year (P < 0.01) compared with controls. The report influenced 9 (69%) case periodontists to change treatment. Case patients who received a change in treatment (n = 21) exhibited greater number of deep pockets at the time of entry into the study (P < 0.05) as compared with case patients who did not receive a change in treatment. Paired t-tests of differences within groups before and after the report demonstrated that case patients had a significant increase in treatment after the report as shown by 22% greater number of visits/year (P < 0.05) and 54% greater number of antibiotic prescriptions/year (P < 0.01). In contrast, controls demonstrated no significant change in treatment. Further, contrasts of change in treatment before and after the report again indicated that case patients exhibited a significantly higher number of visits/year (P < 0.04) and number of antibiotic prescriptions/year (P < 0.02) compared with control patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Relationship between periodontal probing velocity and gingival inflammation in human subjects.

Little is known about the biophysical characteristics of the dentogingival junction in response to the development or resolution of inflammation. The Toronto Automated Periodontal Probe (TAPP) provides an estimate of the integrity of the dentogingival junction by measuring intrapocket probing velocity. The aim of this study was to measure changes of probing velocity in inflamed human periodontium before and after subgingival debridement. 32 subjects exhibiting gingival inflammation were selected; 29 completed the study. Gingival index (GI), plaque index (PLI), bleeding index (BI) and the rate of gingival crevicular fluid flow (CFF) were measured as concomitant variables. The experimental group (N = 16) received scaling, root planing and oral hygiene instruction at baseline. The control group (N = 13) received no treatment until after 28 days. Subjects were seen at baseline, day 14, 21 and 28 for measurement of probing velocity and concomitant variables on 6 index teeth. At day 28, the control group was treated and then reassessed 28 days later. The experimental group showed a reduction of 51.6% for mean crevicular fluid flow (p < 0.0001), 79.7% for mean plaque index (p < 0.0001), 58.0% for mean gingival index (p < 0.0001), and 72.0% for mean bleeding index (p < 0.01) at day 28, confirming that inflammation was reduced compared with baseline. No significant changes were observed in the control group until after treatment. The velocity of probing and the formation of a plateau in the velocity profile were recorded.(ABSTRACT TRUNCATED AT 250 WORDS)

Serial doxycycline and metronidazole in prevention of recurrent periodontitis in high-risk patients.

The efficacy of metronidazole and doxycycline in preventing recurrent periodontitis was studied in 23 patients. After treatment in the previous 7 months with either bimonthly scaling and 3 weeks of systemic doxycycline (11 subjects) or scaling and placebo (12 subjects), patients were monitored for recurrent periodontitis and were scaled every 2 months. When either a periodontal abscess or greater than 2 mm loss of gingival attachment was observed, metronidazole was administered (250 mg every 8 hours) for 10 days. In the placebo plus metronidazole group, 5 patients (42%) exhibited recurrent periodontitis after the metronidazole regimen compared with only one (9%) in the doxycycline plus metronidazole group (P less than 0.096). Subgingival plaque samples at study and healthy control sites were screened for the presence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Eikenella corrodens, and Fusobacterium nucleatum by immunofluorescence and for spirochetes using Ryu's stain. Presence/absence analysis of the sum of scores of the 6 individual pathogens demonstrated large reductions (P less than 0.005) in the frequency of detection of pathogens in the former doxycycline compared with the placebo plus metronidazole group at both study and control sites before and one month after metronidazole. By 7 months after metronidazole, there was no detectable difference between groups. These results indicate that prevention of recurrent periodontitis with metronidazole may be enhanced by previous treatment with doxycycline.

c-fos oncogene expression in dexamethasone stimulated osteogenic cells in chick embryo periosteal cultures.

Although the complex effects of glucocorticoids on bone cells have been studied extensively in vitro, little is known about the molecular mechanisms of glucocorticoid responses in osteogenic cells. As c-fos and its protein product are believed to play a key role in intracellular signal transduction, and since their role in regulation of bone formation is well-recognized, we studied the effect of the glucocorticoid analogue dexamethasone (DEX) on the expression of c-fos oncogene in the chick periosteal osteogenesis (CPO) model. C-fos mRNA expression was determined by in situ hybridization at various time points after 10(-7) M DEX treatment. Prior to DEX treatment, the cultures had been synchronized with 2 mM thymidine. The mean area of positively hybridized cells in experimental (DEX-treated) and control (DEX-free) cultures was quantitated by computer assisted morphometry. In DEX-treated cultures c-fos mRNA could be detected transiently and mainly in the osteogenic layer at 30, 45 and 60 min after treatment whereas no c-fos expression could be detected above background level in the control groups. Differences between experimental and control groups were significant (P less than 0.01) as determined by a general linear model (GLM) analysis of variance. These data indicate that in the CPO culture system, DEX (10(-7) M) induces c-fos expression. The findings are compatible with the hypothesis which states that glucocorticoid-induced phenotypic changes in osteogenic cells may be mediated by c-fos.

Evidence for polymorphonuclear leukocyte collagenase and 92-kilodalton gelatinase in gingival crevicular fluid.

Analysis of inflammatory exudate collected from sites of experimental periodontitis in cynomolgus monkeys has revealed the presence of collagenase and a 92-kDa gelatinase that comigrated after electrophoresis with the 92-kDa gelatinase released from polymorphonuclear leukocytes. Since neutralizing antibodies to fibroblast collagenase had no effect on the collagenase activity and bacterial collagenases could not be detected, polymorphonuclear leukocytes appear to be the major source of collagenolytic proteinases in inflammatory fluid from gingiva.

Collagenase activity in recurrent periodontitis: relationship to disease progression and doxycycline therapy.

Previous reports have suggested that active progression of periodontitis may be correlated with increased collagenolytic activity, and that improved clinical conditions after tetracycline treatment may be explained by inhibition of host collagenase. Eighty-two patients with a recent history of periodontal abscesses and/or loss of gingival attachment level (GAL) despite active periodontal therapy were enrolled in a double-blind, randomized, placebo-controlled trial. Clinical measurements, sampling of gingival crevicular fluid (GCF) and subgingival scaling were performed every 2 months. If any site exhibited greater than 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 (n = 30) or placebo (n = 25) groups. Analysis of active collagenase and latent collagenase in GCF samples were determined by functional assays and quantitated after SDS-PAGE and fluorography. Collagenase activities were assayed at sites exhibiting active destruction (study site), at sites with pocket depth comparable to the study site but without active destruction, and at healthy sites. Clinical measurements of GAL and collagenase activity were made at intervals between 1 wk 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. Collagenase activity exhibited large variations among patients and was analyzed as presence or absence of active collagenase with a logistic model.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of self-instruction methods for teaching diagnostic testing.

In a randomized controlled trial, self-teaching booklets and computer media were evaluated for teaching diagnostic testing to dental students as a foundation for further development of clinical decision making skills. Effectiveness was assessed by pre- and post-tests. Reliability of these test instruments was examined by analyzing the pre- and post-test scores of 49 first year dental students who received no instruction. Forty-one second year dental students were exposed to clinical epidemiological principles applied to endodontic diagnosis through either self-teaching booklets or computer media. No statistically significant difference was found between the mean test scores of the students through self-teaching booklets and computer media. Although first and second year students showed a statistically significant improvement between the pre-test and post-test, the improvement in the second year class was greater. An addendum was later made to the main trial to compare the self-teaching booklet to the traditional lecture format in teaching endodontic diagnosis. Seventy-one third year dental students were exposed to these same materials through either a lecture or the self-teaching booklet and then similarly tested. There was no significant difference between the self-teaching booklet and traditional lecture for the third year students.

A randomized, placebo-controlled trial of doxycycline: effect on the microflora of recurrent periodontitis lesions in high risk patients.

Twenty-seven patients with a recent history of periodontal abscesses and/or loss of gingival attachment level (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. When a site exhibited greater than or equal to 2 mm loss of GAL or a periodontal abscess, patients were administered either doxycycline at a dosage of 200 mg to start and 100 mg per day for 3 weeks, or a placebo. Clinical measurements of GAL and microbial analysis of subgingival plaque at study and control sites were made at the time of active disease and at intervals of 1 week and 7 months after completion of the drug regime. Plaque samples were screened for the presence of Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Bacteroides intermedius (Bi), Eikenella corrodens (Ec) and Fusobacterium nucleatum (Fn) by indirect immunofluorescence antibody technique and for spirochetes (Sp) using Ryu's stain. Based on presence or absence analysis of the sum scores of the 6 pathogens, both the placebo (n = 10) and the doxycycline groups (n = 17) exhibited similar scores at the time of detection of active disease (mean placebo = 2.38 +/- 0.32; mean doxycycline = 2.95 +/- 0.27; P = 0.18). One week after treatment, the probability of detection was unchanged in the placebo group (mean placebo = 3.14 +/- 0.47), but was significantly reduced in the doxycycline group (mean doxycycline = 1.77 +/- 0.26; P = 0.0002). Study (active) sites exhibited scores 2 to 3 times higher than control (inactive) sites before doxycycline treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Randomized controlled trial of doxycycline in prevention of recurrent periodontitis in high-risk patients: antimicrobial activity and collagenase inhibition.

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 greater than or equal to 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 less than 0.05) for Doxycycline compared to placebo. Minimal inhibitory concentrations of Doxycycline for subgingival plaque samples from active sites ranged between 25-100 micrograms/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 micrograms/ml Doxycycline. These data indicate that Doxycycline provides significant risk reduction of recurrent periodontitis in patients with active disease.

Controlled force measurements of gingival attachment level made with the Toronto automated probe using electronic guidance.

Precise reproduction of probe placement, probing force and probing angulation may influence the reliability of replicate gingival attachment level measurements (GAL). Reproducibility of GAL measurements was determined with the Toronto automated periodontal probe (TAPP) in which a novel electronic guidance system was incorporated to improve the precision of probing angulation. Bench testing of forces produced at the probe tip was performed with an electronic balance. Reproducibility and precision of force generation were tested in a variety of instrument configurations using laboratory models. The data indicated that highly precise and reproducible probing forces can be generated with the TAPP over a large (10 to 90 g) probing range. Instrument precision was +/- 0.27 mm when probing forces of 40-60 g were used. The guidance system provided accurate and reproducible measurements of the probe handle in both roll and pitch axes when compared with precisely machined wedges (R = 0.99, roll; R = 0.98, pitch). Deviation of probing angulation greater than 5 degrees on replicate measurements of GAL was associated with significant alterations in the location of circumferential probe placement and in the size of the difference between GAL measurements (delta GAL). Clinical assessment of probing angulation in 6 patients demonstrated that delta GAL was 0.40 +/- 0.05 mm when probing angulation deviation was less than 5 degrees but increased to 0.96 +/- 0.11 mm when deviations exceeded 5 degrees. Taken together, these data indicate that the regulation of probing angulation in clinical measurement of GAL with the TAPP is an important determinant of the reproducibility of periodontal probing.

Measurements of probing velocity with an automated periodontal probe and the relationship with experimental periodontitis in the Cynomolgus monkey (Macaca fascicularis).

The Toronto Automated Periodontal Probe allows simultaneous measurements of gingival attachment level and probing velocity. The efficacy of probing velocity in measuring the integrity of the dento-gingival junction was studied and compared with loss of gingival attachment level, gingival inflammation, tooth mobility and gingival crevicular fluid collagenolytic activity. The gingiva around the maxillary and mandibular incisor teeth of 2 monkeys were first brought to clinical health by daily prophylaxis. Baseline measurements were made and inflammation was then induced around the maxillary incisors by withdrawing plaque control and by placing silk ligatures subgingivally. The mandibular incisors served as controls. After 10 weeks, the ligatures were removed and the teeth scaled and polished daily for 1 week. The plaque index and gingival index reached maximum values within 1 week; crevicular fluid flow was significantly increased at 1 and 3 weeks but mobility did not change significantly. There was progressive loss of the gingival attachment level, which peaked at 10 weeks on experimental teeth; there was no significant loss on control teeth. Probing velocity around experimental teeth exhibited large and significant increases above controls between 1 and 3 weeks, coinciding with the largest weekly losses of the gingival attachment level. Thus probing velocity may be a useful clinical method for the ancillary measurement of early loss of the gingival attachment level. It can also detect biophysical changes in the dento-gingival junction of progressive inflammatory lesions.