Library

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.

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?

Notes: Abstract 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〈0.002) 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. The experimental group demonstrated a significant increase (p〈0.002) in the frequency of plateau formation and a decrease in mean slope between baseline and day 28 (p〈0.02). No significant change was observed in the control until day 56, 28 days after treatment. These data indicate a direct relationship between improved clinical health and increased resistance to probe penetration near the base of the pocket, as reflected by the increased frequency of plateau formation and decreased slope for the terminal segment of the velocity profile curve.

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.

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.

Notes: The growth fraction in the mouse periodontal ligament (PL) was determined by means of continuous labelling for up to 60 days with 3H-Tdr via the drinking water. The method was tested by comparing the labelling indices (LI) of PL cells obtained in this way with those obtained by repeated injections or via osmotic mini-pumps. There was no significant difference between the LI obtained by the 3 different routes. Cell density was assayed in the PL of mice who had received no 3H-Tdr or that were labelled for 60 days with 3H-Tdr. There were no detectable differences in cell density, and we concluded that 3H-Tdr cytocide did not significantly change the growth fraction. The growth fraction of PL cells was 30%, and 25 days of continuous labelling were required to obtain this value. No significant change of LI was observed between 25 and 60 days of labelling. This plateau could not be accounted for by loss of PL cells to bone as osteocytes because very few labelled osteocytes were observed, even after 25 days of continuous labelling. The growth fraction of cells within 10 μm of blood vessels was 40% and was significantly higher than the overall growth fraction. These data suggest that cell division in the PL occurs predominantly in a paravascular location and that some of the progeny of dividing cells in the PL die.

Notes: The mammalian periodontal ligament contains heterogeneous populations of connective tissue cells, the precise function of which is poorly understood. Despite close proximity to bone and the application of high amplitude physical forces, cells in the periodontal ligament (PL) are capable of expressing regulatory factors that maintain PL width during adult life. The study of PL homeostasis and PL cell differentiation requires culture and phenotypic methods for precise characterization of PL cell populations, in particular those cells with an inherently osteogenic program. Currently it is unknown if cells cultured from the PL are phenotypically similar to the parental cells that are present in the tissues. We have compared the phenotype of cells in vivo with cells derived from the PL and expanded in vitro to assess the general validity of in vitro models for the study of phenotypic regulation in vivo. Rat PL cells were isolated by either scraping the root of the extracted first mandibular molars (Group A), or by scraping the alveolar socket following extraction of first mandibular molars (Group B), or by obtaining a mixture of cells after disaggregating a block of tissue consisting of first mandibular molar, PL and the surrounding alveolar bone (Group C). Cultured cells at confluence were fixed and immunostained for α-smooth muscle actin (α-SMA), osteopontin (OPN), alkaline phosphatase (AP), or bone sialoprotein (BSP). For in vivo assessments, frontal sections of rat first mandibular molar were immunostained for α-SMA, OPN, AP and BSP. We examined osteogenic differentiation of cultured PL cell cultures by bone nodule-forming assays. In vivo and at all examined sites, 〉68% of PL cells were immunostained for AP; ∼50% and ∼51% for OPN and α-SMA (p=0.3), respectively, while only ∼8% were positively stained for BSP (p〈0.01). Analysis of cultured PL cells in Groups A, B and C showed 54%, 53% and 56% positive staining for α-SMA respectively; 51%, 56%, 54% for OPN; 66%, 70%, 69% for AP and 2.2%, 1.4% and 2.8% for BSP. The mean percentage of PL cells in situ stained for the different markers was similar to that of cultured PL cells (Group A∼Group B∼Group C in situ for p〉0.2) except for BSP which was 3 to 4 fold higher in vivo(p〈0.01). PL cell cultures treated with dexamethasone showed mineralized tissue formation for all groups (A, B, C), but no mineralized tissue formation was detected in the absence of dexamethasone. As PL cells express quantitatively similar phenotypes in vitro and in vivo, we conclude that the in vitro models used here for assessment of PL cell differentiation appear to be appropriate and are independent of the cell sampling method. Further, dexamethasone-dependent progenitors are present both on the root and bone-related sides of the PL.