R. Lanese

A new experimental model for studying the response of periodontal ligament cells to hydrostatic pressure

An apparatus was developed to apply positive or negative hydrostatic pressure dynamically to periodontal ligament (PDL) cells in vitro. The objective of this investigation was to construct this apparatus and to determine its effects on PDL cells. Human PDL cells were collected from freshly extracted premolars. At the sixth passage, the cells were mechanically stimulated by this apparatus at different magnitudes of continuous positive or negative hydrostatic pressures (PHP or NHP, respectively). The application of PHP between 0.3 and 30 gm/cm2 significantly enhanced prostaglandin E (PGE) production and intracellular cyclic AMP (cAMP) of the cells. In contrast, perturbation by NHP significantly decreased PGE production and intracellular level of cAMP. Proliferation rate increased significantly at 24 and 48 hours due to stimulation of these cells with -30 gm/cm2 of NHP. Challenging these cells with +30 gm/cm2 of PHP significantly decreased the proliferation rate of these cells at 24 and 48 hours. Stimulation by PHP between +30 to +600 gm/cm2 increased cell length and width and appeared to increase surface area attachment to the bottom of the culture dishes. In contrast, NHP (between -30 and -600 gm/cm2) decreased these dimensions and appeared to reduce the surface area of attachment. These results indicate that this type of mechanical perturbation of PDL cells produces physiologic responses and is not detrimental to their vitality.

The interactive effects of mechanical stress and interleukin-1β on prostaglandin E and cyclic AMP production in human periodontal ligament fibroblasts in vitro: Comparison with cloned osteoblastic cells of mouse (MC3T3-E1)

Human periodontal ligament fibroblasts and a cloned osteogenic cell line (MC3T3-E1) were seeded (4 x 10(5) cells) on 60 mm Petriperm dishes, which have a flexible plastic growth surface. Cells were stretched by placing the dish on top of a spheroidal convex template, equilibrated to 37 degrees C. The amount of stretch was varied by changing the curvature of the template and calculated as percentage stretch. Both types of cell responded to mechanical stress by elevated synthesis of PGE and cAMP; the addition of interleukin-1 beta to mechanically stretched cells produced further elevation. Synergism between mechanical stress and interleukin-1 beta was found at certain lengths of incubation. The production of cAMP was secondary and dependent on the newly synthesized PGE, as shown in the presence of indomethacin. The two cell types were also different in terms of the timing of their response to mechanical stress and interleukin-1 beta. In the absence of stimuli, periodontal fibroblasts tended to produce PGE continually over time, whereas the MC3T3-E1 cells did not. However, both cell types had elevated PGE levels in response to the stimuli used in this experiment. Periodontal fibroblasts responded to mechanical stress and interleukin-1 beta with significant elevations of PGE as early as 15 min, whereas the MC3T3-E1 cells required 2 h to produce significant elevations for mechanical stress and 15 min for interleukin-1 beta. These findings indicate that the chemical and mechanical signals on these cells are mediated by surface receptors. Locally produced autocrine or paracrine factors can modify the effect of mechanical stress on periodontal and bone cells via the cAMP pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of cytokines on prostaglandin E and cAMP levels in human periodontal ligament fibroblasts in vitro

The stimulation of PGE synthesis and cAMP production by cytokines have important physiological effects in many target tissues. The effects of interleukin-1 alpha and -1 beta, tumour necrosis factor-alpha and interferon-gamma on PGE and cAMP production by periodontal ligament fibroblasts were studied. Fibroblasts in the 4th-6th passage, grown and maintained in DMEM supplemented with 10% equine serum, were incubated with graded doses of the various cytokines for 0.25, 0.5, 1.2, 4, 24, 48 or 72 h. At the end of each incubation, PGE in the medium and the cellular content of cAMP were evaluated by a combined immunohistochemical microphotometric procedure, and conventional radiometric assays. The fibroblasts responded to all the cytokines with a dose- and time-related increase in the levels of PGE and cAMP. Such increases were inhibited by the inclusion of indomethacin in the medium. The addition of exogenous PGE reversed that inhibition in respect of cAMP production. Immunohistochemical localization showed PGE predominantly in the cytoplasm and cAMP in the nucleus. These findings indicate that: (1) human periodontal ligament fibroblasts respond to these cytokines by increased synthesis of PGE and the production of cAMP; and (2) the cAMP production is secondary to the PGE synthesis. They suggest that these cytokines may regulate the function of these fibroblasts in physiological remodelling of the periodontium, as well as in inflammatory reactions.

Interactive Effects Between Cytokines on PGE Production by Human Periodontal Ligament Fibroblasts in vitro

Mononuclear cell production of cytokines that stimulate fibroblast production of prostaglandin E (PGE) is an important mechanism by which mononuclear cells regulate fibroblast function. The objective of this investigation was to determine the effects of the cytokines interleukin 1β (IL-1β), interleukin la (IL-1α), tumor necrosis factor a (TNF-a), and interferon γ (IFN--y), alone or in paired combinations, on PGE production by near-confluent human periodontal ligament (PDL) fibroblasts in vitro. Premolars extracted in the course of orthodontic treatment were used for this study. Fibroblast cultures, free of epithelial cells, were obtained after the fourth subculture by the use of accurately-timed trypsin treatment. Cells in the fourth to sixth passage, incubated in DMEM supplemented with 10% equine serum, were used for these experiments. Cells (1 x 105) were seeded in 12- x -75-mm tissue culture tubes and incubated with various doses of IL-1β, IL-la, TNF-a, and IFN-γ, alone or in specific combinations, for 15 min, two, 12, 24, and 72 h. PGE concentrations in the media were measured by radio-immunoassay. The results showed that human PDL fibroblasts responded to the administration of cytokines by an elevation in the synthesis of PGE in a dose- and time-related fashion. The increase in PGE production was inhibited by the addition of indomethacin. The interactions between these cytokines varied in degree, depending on the particular combinations of cytokines. In addition, the administration of cytokine combinations was found to be additive, synergistic, subtractive, or suppressive on the production of PGE by PDL fibroblasts, depending on the duration of incubation. These experiments demonstrate the importance of the consideration of the interplay between cytokines produced by mononuclear cells on the mechanisms that regulate the functions of PDL fibroblasts.

A two-month study of the effects of oral irrigation and automatic toothbrush use in an adult orthodontic population with fixed appliances

Forty-seven adult orthodontic patients with fixed orthodontic appliances were divided into three study groups: (1) oral irrigation with automatic toothbrush, (n = 16); (2) oral irrigation with manual toothbrushing, (n = 16); (3) control group with continued normal toothbrushing only, (n = 15). Gingival and plaque indices, bleeding after probing, and gingival sulcus depths were assessed at baseline, 1-month, and 2-month periods. Marked and significant gingival and plaque improvements from baseline were measured in all three study groups. After 1 to 2 months use of the automatic toothbrush and/or the oral irrigation device, there was a significant reduction in plaque when compared with the control group who used only the manual toothbrush (p = 0.026). Also, there was a significant reduction in gingival inflammation (p = 0.045) and evidence for reducing bleeding after probing (p = 0.037). No significant differences were found in probe depths among the three study groups, however, use of both devices reduced the pocket depth significantly from baseline by 0.5 mm (p < 0.0002). For this population of orthodontic patients, significant reductions in plaque, gingival inflammation, and a tendency for reduced bleeding after probing occurred in both groups with the power device. These improvements were most attributable to the effect of the oral irrigation device.

Effects of parathyroid hormone and cytokines on prostaglandin e synthesis and bone resorption by human periodontal ligament fibroblasts

Cultured human periodontal ligament fibroblasts showed synergistic elevations in the synthesis of prostaglandin E and production of cAMP by the administration of parathyroid hormone and cytokines (interleukin 1 alpha, -1 beta, or tumour necrosis factor-alpha). Unstimulated conditioned media derived from these fibroblasts contained bone-resorbing activity. In addition, conditioned media generated by cytokine-or parathyroid hormone-treated fibroblasts showed further increases in bone-resorbing activity. The effects were additive when the hormone was combined with either one of the cytokines in stimulating bone resorption. These findings suggest that the effect of parathyroid hormones and cytokines together on bone resorption can be mediated in part by human periodontal ligament fibroblasts via PGE production and subsequent PGE action on the osteoclasts.

99mTc-medronate uptake in the temporomandibular joints of young rats treated with a mandibular hyperpropulsor.

To correct maxillomandibular sagittal discrepancies in growing children, most functional appliances position the mandible more anteriorly than its habitual relation with the maxilla. The aim of this study was to evaluate the effects of such a procedure on the mineralization of the temporomandibular joints in young growing rats. Temporomandibular joint uptake of a radioactive bone marker, technetium 99m methylene diphosphonate (99mTc-MDP), was measured over time as a hyperpropulsor appliance was being worn intermittently 12 hours/day. As the uptake of 99mTc-MDP decreased in control animals, it increased significantly in the joints of treated rats before returning to the baseline level after 5 weeks of treatment. These results suggest that intermittent anterior positioning of the mandible induces a high rate of bone and cartilage remodeling in the joints of growing rats and that bone-seeking radiopharmaceutical uptake, with 99mTc-MDP, may be a useful technique to monitor joint adaptation to an experimental functional change.