Bone-resorbing activity and prostaglandin E produced by human periodontal ligament cells in vitro.

Human periodontal ligament (PDL) cells were derived from healthy premolars extracted for orthodontic treatment and were utilized for in vitro experiments in passages 4-6. Human PDL cells were seeded in tissue culture tubes and incubated with interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), indomethacin, parathyroid hormone (PTH), or their combinations, for 1 h. The medium was then replaced with serum-free BGJb medium and incubated for 24 h without further additions. Prostaglandin E (PGE) concentrations in the conditioned media (CM) were measured by radioimmunoassay, and bone-resorbing activity was measured using 45Ca-labeled neonatal mouse calvariae. The results of this study indicated that (1) unstimulated cultured PDL cells produced PGE, and PDL CM stimulated bone resorption; (2) cytokine-treated (IL-1 alpha, IL-1 beta, and TNF-alpha) PDL cells had increased production of PGE and bone-resorbing activity compared to unstimulated PDL cells; (3) indomethacin completely inhibited PGE production from unstimulated PDL cells but only partially inhibited bone-resorbing activity, indicating that PDL cells produced nonprostaglandin bone-resorbing factor(s); (4) IFN-gamma did not change PGE or bone-resorbing activity production by cytokine-stimulated PDL cells; and (5) PTH treatment of PDL cells in addition to cytokines (IL-1 alpha, IL-1 beta, and TNF-alpha) had additive effects on the production of bone-resorbing activity and synergistic effects on PGE production compared to cytokine treatment alone.

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 1 beta (IL-1 beta), interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma), 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 10(5)) were seeded in 12- x -75-mm tissue culture tubes and incubated with various doses of IL-1 beta, IL-1 alpha, TNF-alpha, and IFN-gamma, 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.

Involvement of PGE synthesis in the effect of intermittent pressure and interleukin-1 beta on bone resorption.

Human periodontal ligament (PDL) fibroblasts, cultured from extracted healthy premolars, and a cloned osteogenic cell line (MC3T3-E1) were used in this study to determine the effect of intermittent pressure on bone resorption. Cells (1 x 10(5] were incubated with BGJb medium in the presence or absence of the following factors: intermittent negative (-30 g/cm2) or positive (30 g/cm2) hydrostatic pressure and interleukin-1 beta (IL-1 beta, 1 ng/mL), for 24 h. Conditioned media (CM) generated from cultures of either cell types were used for prostaglandin E (PGE) assay, bone resorption assay, and assessment of osteoclast (OC)-like cell formation. Unstimulated PDL fibroblasts or MC3T3-E1 cells produced measurable amounts of PGE and bone-resorbing activity as measured by 45Ca released from mouse calvaria and OC-like cells. IL-1 beta-treated cells showed significantly elevated levels of PGE, bone resorption, and OC-like cell formation, as compared with unstimulated cells. Intermittent positive pressure (IPP) alone stimulated PGE production, but the resultant CM did not stimulate bone resorption or OC-like cell formation when IPP was applied to either cell type. The application of IPP, together with IL-1 beta in CM, caused a slight increase in the number of alpha-like cells, as compared with that of IL-1 beta-treated CM in both cell types. On the other hand, direct application of IPP on mouse bone-marrow cultures significantly increased the number of OC-like cells. This effect was additive in combination with either CM from unstimulated cells or exogenous addition of PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic AMP in dental and periodontal tissues during tooth eruption in kittens.

Cyclic adenosine 3',5'-monophosphate (cAMP) concentrations and cellular distribution were studied in dental and periodontal tissues during tooth eruption in kittens. Although the mean levels of cAMP around developing teeth were similar in all the tissue samples, there were marked differences in cAMP stainability of tissues apical and occlusal to the erupting teeth.

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.

Immunohistochemical assessment of the effect of chemical and mechanical stimuli on cAMP and prostaglandin E levels in human gingival fibroblasts in vitro.

These were evaluated by: (1) a combined immunohistochemical-microphotometric procedure (IH) and (2) conventional radiometric assays. Human gingival fibroblasts were in the sixth passage, grown and maintained in Dulbecco minimal essential medium (DMEM) supplemented with 10 per cent horse serum. For chemical and hormonal stimuli, cells (2 x 10(4] were seeded on tissue-culture chamber/slides, and incubated with graded doses of either parathyroid hormone (PTH) or prostaglandin E2 (PGE2) for assessment of their adenosine-3',5'-monophosphate (cAMP) levels, and with indomethacin or colchicine for their effect on PGE levels. For mechanical stimuli, cells (1 x 10(6] were seeded on culture dishes with a flexible plastic membrane and stretched for 5, 30, 60 or 120 min by placing the membrane over a convex surface and weighting the dish cover. After freeze drying, cells were stained by an immunoperoxidase technique for either cAMP or PGE, using monoclonal antibodies. The staining intensity of fibroblasts was determined at 600 nm wavelength. Per cent light absorbance of 15 cells in each slide was measured and the results tested by analysis of variance. The gingival fibroblasts responded to the drugs and hormones in a dose- and time-related fashion. Stretching significantly increased their synthesis of PGE with concomitant increase in cAMP. The IH results were compared with the radiometric assays to confirm the validity of this technique; both assays were valid for describing the quantitative responses of these cells to the stimuli. In particular, the IH method could localize those intracellular sites which demonstrated chances in relative cAMP and PGE concentrations in response to hormonal stimuli.

The interactive effects of mechanical stress and interleukin-1 beta 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.

The increase in vivo by electric currents of the 99mTc-methylene diphosphonate uptake and cyclic AMP content of the bone of the cat mandible.

Technetium-99-methylene diphosphonate (99mTc-MDP) bone images were obtained from live cats whose mandibles had been stimulated non-invasively and unilaterally with electric current (12 +/- 2 microA) for 1 week. The images were obtained using a gamma camera interfaced to a digital computer for quantitative comparison of the mandible. Radionuclide uptake by the stimulated sides increased by 59 +/- 26 per cent when compared with the contralateral control sites. Immunohistochemical staining of mandibular sections for adenosine 3',5'-monophosphate (cyclic AMP) showed that periosteal osteoblasts opposite the electrodes were intensely stained. Thus 99mTc-MDP scintigraphy is a sensitive non-invasive technique for determining in vivo enhanced bone remodelling activity. The immunohistochemistry indicates that the stimulation of bone cells was limited to surfaces adjacent to both cathode and anode.

Neurotransmitters, cytokines, and the control of alveolar bone remodeling in orthodontics.

This article described research aimed at testing the hypothesis that tissue remodeling during orthodontic tooth movement is modulated, at least in part, by factors derived from the nervous and vascular (immune) systems. Specifically, the neurotransmitters SP and VIP and the cytokines IL-1 alpha and IL-1 beta were localized immunohistochemically in paradental tissues of cat canines that had been treated by the application of an 80 g tipping force for 1 hour to 14 days. Increased staining (concentrations) of these agents were found in areas of PDL tension and compression at different time periods. Moreover, administration of SP and IL-1 beta to human PDL fibroblasts in vitro for 1 to 60 minutes resulted in significant increases in the levels of the intracellular "second messenger" cAMP, as well as of PGE2, a plasma membrane-associated fatty acid believed to serve as a local regulator of bone cell activity. Taken together, these results tend to support the hypothesis that neurotransmitters and cytokines play a regulatory role in orthodontic force-induced alveolar bone remodeling. Consequently, determination of the cytokine synthetic activity by leukocytes of orthodontic patients may inform about their alveolar bone remodeling potential.

On a path to unfolding the biological mechanisms of orthodontic tooth movement.

Orthodontic forces deform the extracellular matrix and activate cells of the paradental tissues, facilitating tooth movement. Discoveries in mechanobiology have illuminated sequential cellular and molecular events, such as signal generation and transduction, cytoskeletal re-organization, gene expression, differentiation, proliferation, synthesis and secretion of specific products, and apoptosis. Orthodontists work in a unique biological environment, wherein applied forces engender remodeling of both mineralized and non-mineralized paradental tissues, including the associated blood vessels and neural elements. This review aims at identifying events that affect the sequence, timing, and significance of factors that determine the nature of the biological response of each paradental tissue to orthodontic force. The results of this literature review emphasize the fact that mechanoresponses and inflammation are both essential for achieving tooth movement clinically. If both are working in concert, orthodontists might be able to accelerate or decelerate tooth movement by adding adjuvant methods, whether physical, chemical, or surgical.

The effect of drugs on orthodontic tooth movement.

OBJECTIVE: Molecules produced in various diseased tissues, or drugs and nutrients consumed regularly by patients, can reach the mechanically stressed paradental tissues through the circulation, and interact with local target cells. The combined effect of mechanical forces and one or more of these agents may be inhibitory, additive or synergistic. The objective of this review is to outline the mechanisms of action and effects of some commonly used drugs on tissue remodeling and orthodontic tooth movement. DESIGN: All the existing published literature on the effects of various drugs that are prescribed by orthodontists, which are consumed by patients for systemic diseases and those that are known to promote and retard the tooth movement process was obtained and subjected to thorough review process. RESULTS: All the drugs reviewed have therapeutic effects, as well as side effects, that may influence the cells targeted by orthodontic forces. Therefore, it is imperative that the orthodontist pays close attention to the drug consumption history of each and every patient, before and during the course of orthodontic treatment. When the use of drugs is revealed, their effects and side effects on tissue systems should be explored, to determine their potential influence on the outcome of mechanotherapy. CONCLUSION: Drug-consumption history must be an integral part of every orthodontic diagnosis and treatment plan.

Tooth movement.

This article reviews the evolution of concepts regarding the biological foundation of force-induced tooth movement. Nineteenth century hypotheses proposed two mechanisms: application of pressure and tension to the periodontal ligament (PDL), and bending of the alveolar bone. Histologic investigations in the early and middle years of the 20th century revealed that both phenomena actually occur concomitantly, and that cells, as well as extracellular components of the PDL and alveolar bone, participate in the response to applied mechanical forces, which ultimately results in remodeling activities. Experiments with isolated cells in culture demonstrated that shape distortion might lead to cellular activation, either by opening plasma membrane ion channels, or by crystallizing cytoskeletal filaments. Mechanical distortion of collagenous matrices, mineralized or non-mineralized, may, on the other hand, evoke the development of bioelectric phenomena (stress-generated potentials and streaming potentials) that are capable of stimulating cells by altering the electric charge on their membrane or their fluid envelope. In intact animals, mechanical perturbations on the order of about 1 min/d are apparently sufficient to cause profound osteogenic responses, perhaps due to matrix proteoglycan-related "strain memory". Enzymatically isolated human PDL cells respond biochemically to mechanical and chemical signals. The latter include endocrines, autocrines, and paracrines. Histochemical and immunohistochemical studies showed that during the early places of tooth movement, PDL fluids are shifted, and cells and matrix are distorted. Vasoactive neurotransmitters are released from periodontal nerve terminals, causing leukocytes to migrate out of adjacent capillaries. Cytokines and growth factors are secreted by these cells, stimulating PDL cells and alveolar bone lining cells to remodel their related matrices. This remodeling activity facilitates movement of teeth into areas in which bone had been resorbed. This emerging information suggests that in the living mammal, many cell types are involved in the biological response to applied mechanical stress to teeth, and thereby to bone. Essentially, cells of the nervous, immune, and endocrine systems become involved in the activation and response of PDL and alveolar bone cells to applied stresses. This fact implies that research in the area of the biological response to force application to teeth should be sufficiently broad to include explorations of possible associations between physical, cellular, and molecular phenomena. The goals of this investigative field should continue to expound on fundamental principles, particularly on extrapolating new findings to the clinical environment, where millions of patients are subjected annually to applications of mechanical forces to their teeth for long periods of time in an effort to improve their position in the oral cavity.(ABSTRACT TRUNCATED AT 400 WORDS)