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Summary Background In long-term studies of orthodontic, orthognathic, and prosthodontic treatments, control subjects are needed for comparison. Objectives To study the craniofacial (skeletal, soft tissue, and dental) changes that occur in untreated subjects with normal occlusion between 13 and 62 years of age. Materials/Methods Thirty subjects with a Class I normal occlusion and harmonious facial profile were studied. X-ray examinations were performed at 13 (T1), 16 (T2), 31 (T3), and 62 (T4) years of age, and data were obtained from cephalograms. In total, 53 angular and linear parameters were measured using superimposition-based and conventional cephalometric methods to describe the craniofacial changes. Results The jaws showed significant anterior growth from T1 to T2, and significant retrognathism from T3 to T4. The anterior face height and jaw dimensions increased significantly until T3. Significant posterior rotation of the mandible and opening of the vertical jaw relation, in addition to significant retroclination of the incisors and straightening of the facial profile, were found from T3 to T4. Limitations Given the small sample size at T4, it was not possible to analyse the gender dimension. Conclusions/Implications Craniofacial changes continue up to the sixth decade of life. These changes are consistent, albeit to a lesser extent, with the adolescent growth patterns for most of the studied parameters, with the exceptions of incisor inclination, sagittal jaw position, vertical jaw relation and inclination, and posterior face height.

Summary Background The long-term effects on dentofacial morphology of interceptive treatment with premolar extractions, in the absence of subsequent orthodontic treatment, have not been fully explored. Objective The aim was to investigate the effects of premolar extractions (without subsequent orthodontic treatment) on the dentoskeletal and soft tissue profile of patients aged between 12 and 62 years with Class I malocclusion with severe crowding, as compared to untreated controls. Materials and methods The Extraction group (N = 30 with Class I crowding malocclusion) had their first premolars removed in early adolescence without subsequent orthodontic treatment. The Control group included 30 untreated subjects with Class I normal occlusion. Cephalograms were taken at 12 (T1), 15 (T2), 30 (T3), and 62 (T4) years of age. A superimposition-based cephalometric method was used to assess the dentoskeletal and soft tissue changes. Results There were no significant differences between the Extraction and Control groups in terms of skeletal sagittal relation, incisor inclination, and protrusion, or most of the soft tissue parameters throughout the observation period. However, significant differences were observed between the groups with respect to the vertical relations in T2–T3, such that the Extraction group showed more-pronounced decreases in the ML/NSL, ML/NL, and Gonial angles and more-pronounced increases in facial heights. Conclusions and implications Treatment for subjects with Class I malocclusion with severe crowding by the extraction of four premolars, without subsequent orthodontic treatment, does not affect the long-term dentoskeletal and soft tissue profile, as compared to an untreated Control group. The degree of crowding, rather than changes in dentofacial morphology, is crucial in deciding on extraction therapy.

The aim of this study was to evaluate if delayed dental development is a cause of postponed care for patients with impacted maxillary canine (IMC).This case-control study was based on 403,355 children and adolescents in Region Västra Götaland, Sweden. The subjects, who were in the age range of 9-16 years during the period of 2011-2013, underwent surgical exposure or removal of a maxillary canine. Demirjian's dental age assessment was carried out on panoramic radiographs.In total, 1028 patients, 514 with IMC and 514 age- and gender-matched controls, were enrolled. The patients with IMC exhibited a dental development delay of 0.2 years compared to the control group. In the impaction sub-groups, the female patients, patients in the chronological age group of 12-13 years, and patients with palatally positioned IMC had a significantly lower dental age than their paired-control subjects.Overall, the difference in dental age between patients with or without IMC is significant but small, and as such is likely of minor clinical relevance. Therefore, the timing of preventive care and treatment for patients with IMC should be the same as that for patients with normally erupting canines.

Objective: Descriptions of the craniofacial morphology in Saethre-Chotzen syndrome (SCS) are primarily based on case reports or visual assessments of affected families. The aim of this study was to compare cephalometric measurements of the craniofacial skeleton in a cohort of individuals with SCS and age- and sex-matched individuals without craniofacial anomalies. Design: Retrospective case series. Patients: Eight girls and 4 boys with SCS (age range, 7.0-19.2 years). Methods: Cephalometric measurements were performed using lateral and frontal cephalograms. Results: Most of the individuals with Saethre-Chotzen syndrome exhibited lower values for SNA, SNB, s-n and s-ar, while their NSL/NL, NSL/ML, NL/ML, and n-s-ba values were higher than the respective mean reference values for healthy individuals. In comparison with age- and sex-matched individuals without craniofacial anomalies, the individuals with SCS showed higher values for the maxillary and mandibular angular measurements, as well as for the menton midline angle. Conclusions: This sample of 12 unrelated individuals with SCS is the largest collected to date for cephalometric measurements. We found that the syndrome is associated with bimaxillary retrognathism, posterior maxillary and mandibular inclination, neutral sagittal relation as well as a tendency toward an open vertical skeletal relation, a short and flattened skull base, and facial asymmetry, as compared to individuals without the syndrome.

Summary Background In dentistry, epidemiological studies are important for establishing high-quality care for the individual patient as well as for socio-economic reasons. Objective The aim of this epidemiological study was to evaluate the prevalence of impacted maxillary canines in a geographical region in which interceptive treatment is implemented systematically. Furthermore, the aim was to study the age and gender of the patients, and the location and surgical technique used for the impacted maxillary canine. Method The study was based on 54 716 adolescents in the Region Västra Götaland, Sweden who were born in the period 1996–98. All patients in the three cohorts who had impacted maxillary canines treated with surgical exposure or surgical removal were identified in the dental record system used in the region. Results The prevalence of impacted maxillary canines when interceptive treatment was systematically implemented was 1.1% (N = 601). Overall, the cohorts of patients with impacted canines comprised 65% girls and 35% boys. Most of the canines were palatally impacted and the most common surgical technique was closed exposure. Conclusion The prevalence of impacted maxillary canines in a geographical area in which interceptive treatment is systematically implemented is lower than that reported previously. The distributions of impacted canines with respect to gender and location are in accordance with those reported previously in similar studies.

To assess the craniofacial changes related to growth and/or to orthodontic and orthognathic treatments, it is necessary to superimpose serial radiographs on stable structures. However, conventional superimposition provides only a graphical illustration of these changes. To increase the precision of growth and treatment evaluations, it is desirable to quantitate these craniofacial changes. The aims of this study were to (1) evaluate a superimposition-based cephalometric method to process numerical data for craniofacial growth changes and (2) identify a valid, reliable, and feasible method for superimposition. Forty pairs of cephalograms were analyzed at T1 and T2 (mean age 9.9 and 15.0 years, respectively). The superimposition-based cephalometric method involved relating the sagittal and vertical measurements on the T2 radiographs to the nasion and sella landmarks on the T1 radiographs. Validity and reliability were evaluated for three superimposition methods: the sella-nasion (SN), the tuberculum sella-wing (TW), and Björk’s structural. Superimposition-based cephalometrics can be used to quantify craniofacial changes digitally. The numerical data from the superimposition-based cephalometrics reflected a graphical illustration of superimposition and differed significantly from the data acquired through conventional cephalometrics. Superimposition using the TW method is recommended as it is valid, reliable, and feasible.

Funder: Swedish Dental Society, Numerous studies have reported on the positive effects of silicon (Si) on bone metabolism, particularly on the stimulatory effects of Si on osteoblast cells and on bone formation. Inhibitory effects of Si on osteoclast formation and bone resorption have also been demonstrated in vitro and are suggested to be mediated indirectly via stromal and osteoblast cells. Direct effects of Si on osteoclasts have been less studied and mostly using soluble Si, but no characterisation of the Si treatment solutions are provided. The aims of the present study were to (a) further investigate the direct inhibitory effects of Si on osteoclastogenesis in RANKL-stimulated RAW264.7 cells, (b) determine at what stage during osteoclastogenesis Si acts upon, and (c) determine if these effects can be attributed to the biologically relevant soluble orthosilicic acid specie. Our results demonstrate that silicon, at 50 μg/ml (or 1.8 mM), does not affect cell viability but directly inhibits the formation of TRAP+ multinucleated cells and the expression of osteoclast phenotypic genes in RAW264.7 cells. The inhibitory effect of Si was clearly associated with the early stages (first 24 hr) of osteoclastogenesis. Moreover, these effects can be attributed to the soluble orthosilicic acid specie.

Silicon is a trace element found mainly in plant-based food and proposed to be beneficial for bone health. Urinary excretion of Si has been shown to be a surrogate measure of its uptake in the gastrointestinal tract. The objective of this study was to describe and compare the levels of urinary Si excretion, and consequently Si uptake, in Swedish men, non-pregnant women, and pregnant women. No formal assessment of dietary Si intake was carried out in this study. This cross-sectional study included 89 men, 42 non-pregnant women, and 60 pregnant women. The subjects collected urine over a 24-h period and the samples were assayed for total Si using inductively coupled plasma optical emission spectrometry. The excretion levels of creatinine were used to validate the completeness of the urine sample collections. The mean 24-h urinary excretions of Si were 7.8 mg for the cohort of young men, 7.6 mg for the cohort of non-pregnant women, and 12.4 mg for the cohort of pregnant women. Creatinine excretion was similar between pregnant and non-pregnant women (10.4 vs. 10.8 mmol/day) and significantly higher in men (15.4 mmol/day). The pregnant women excreted significantly higher levels of Si than the young men and non-pregnant women, respectively (p < 0.05). The higher urinary excretion of Si by pregnant women compared with men and non-pregnant women is a novel finding possibly caused by temporary physiological changes during pregnancy such as increased gastrointestinal uptake of Si, altered bone metabolism, and increased renal excretion of Si.

Osteoporosis is a major cause of age-related fractures. Healing complications in osteoporotic patients are often associated with increased mortality and morbidity. Stimulation of the implant-adjacent bone could be beneficial in terms of the surgical outcome. Over the past decade, numerous investigations have implicated insulin in normal bone growth, and recent studies have described the advantages of administering insulin locally to increase bone formation. Therefore, we hypothesized that insulin-coated titanium implants would increase bone formation in osteoporotic animals. The aim of this study was to evaluate the effects of insulin delivered from an implant surface on bone-related gene expression and bone formation in osteoporotic rats. Characterizations of the surfaces of insulin-coated and control implants were performed using ellipsometry and interferometry. Forty ovariectomized and four healthy Sprague Dawley rats were used and implants were inserted in the tibias. The systemic effect of insulin was assessed by measuring the blood glucose levels and total body weight. The animals were sacrificed either 1 day or 3 weeks postimplantation. Implant-adherent cells were analyzed by quantitative real-time PCR, and the bone adjacent to the implants was examined by microcomputed tomography and histomorphometry. The insulin-coated implants had no systemic effects. The insulin-coated samples demonstrated significantly lower expression of the gene for interleukin 1β (p = 0.019) at 1 day, and significantly exhibited more periosteal callus (p = 0.029) at 3 weeks. Locally delivered insulin has potential for promoting bone formation and it exerts potentially anti-inflammatory effects in osteoporotic rats. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2472-2480, 2018.

Animal studies suggest that the dental follicle (DF) plays a major role in tooth eruption. However, the role of the DF during tooth impaction and related root resorptions in adjacent teeth is not clear. The hypothesis for the present study is that expression of regulatory factors involved in the bone remodelling process necessary for tooth eruption may differ between dental follicles from teeth with different clinical situations. We have analysed the gene expression profiles in the DF obtained from impacted canines, with (N = 3) or without (N = 5) signs of root resorption, and from control teeth (normal erupting teeth, mesiodens) (N = 3). DF from 11 patients (mean age: 13 years) obtains at the time of surgical exposure of the tooth. Due to the surgical time point, all teeth were in a late developmental stage. Gene expression related to osteoblast activation/bone formation, osteoclast recruitment and activation was analysed by RTqPCR. Genes related to bone formation (RUNX2, OSX, ALP, OCN, CX43) were highly expressed in all the samples, but osteoclast recruitment/activation markers (OPG, RANKL, MCP-1, CSF-1) were negligible. No apparent patterns or significant differences in gene expression were found between impacted canines, with or without signs of root resorption, or when compared to control teeth. Our results suggest the DF regulation of osteoclastic activity is limited in the late pre-emergent stage of tooth development, irrespective if the tooth is normally erupting or impacted. We suggest that the follicle may have an important regulatory function for alveolar bone formation in the final eruption process and CX43-gap junction communication could be an important signalling pathway.

Surface characterization of insulin-coated Ti6Al4V medical implants, after incubation in α-minimum essential medium (α-MEM), was done by X-ray photoelectron spectroscopy (XPS), in order to analyze ...

Objective: To compare the reliability of digital occlusal contacts quantification and the validity of digital occlusal contacts quantification with traditional methods used for occlusal contact determination. Subjects and Methods: Thirty participants, all of whom were students at the Sahlgrenska Academy, University of Gothenburg in Gothenburg, Sweden, were included in the study. Three different methods were used to evaluate occlusal contacts: (I) a digital examination of the patients’ casts, using the Ortho 3D Models (O3DM) software and measuring the total occlusal contact area in square millimeters (DE), (II) an examination involving the measurement of the total number of occlusal contacts on stone casts mounted in an articulator (AE), and (III) a clinical examination with the measurement of the total number of occlusal contacts with 8 μm-thick articulating foil (CE). Results: The repeated digital measurements (same casts scanned multiple times) showed a significant correlation of 0.85 (p &lt, 0.01), which shows a diagnostic consistency. Furthermore, there was a significant correlation between the results obtained with the DE method and the AE of 0.41 (p &lt, 0.05), and between those acquired with the AE method and the CE of 0.37 (p &lt, 0.05). However, no significant correlation was found between the DE method and the CE method with a correlation coefficient of 0.10 (p &gt, 0.05). Conclusions: Digital casts can be used for quantification of the total occlusal contact area (in mm2) owing to the high reliability of repeated measurements and the strong validity of the method compared to traditionally employed stone cast measurements.

Local administration of insulin from a titanium surface has been demonstrated to increase bone formation in non-diabetic rats. The authors hypothesized that insulin was released from the titanium surface and with preserved biological activity after the release. Thus, in the present in vitro study, human recombinant insulin was immobilized onto titanium discs, and the insulin release kinetics was evaluated using Electro-chemiluminescence immunoassay. Neutral Red uptake assay and mineralization assay were used to evaluate the biological effects of the released insulin on human osteoblast-like MG-63 cells. The results confirmed that insulin was released from titanium surfaces during a six-week period. Etching the disc prior to insulin coating, thickening of the insulin coating and incubation of the discs in serum-enriched cell culture medium increased the release. However, longer storage time decreased the release of insulin. Furthermore, the released insulin had retained its biological activity, as demonstrated by the significant increase in cell number and a stimulated mineralization process, upon exposure to released insulin. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1847-1854, 2017.

Introduction The introduction of digital cast models is inevitable in the otherwise digitized everyday life of orthodontics. The introduction of this new technology, however, is not straightforward, and selecting an appropriate system can be difficult. The aim of the study was to compare 4 orthodontic digital software systems regarding service, features, and usability. Methods Information regarding service offered by the companies was obtained from questionnaires and Web sites. The features of each software system were collected by exploring the user manuals and the software programs. Replicas of pretreatment casts were sent to Cadent (OrthoCAD; Cadent, Carlstadt, NJ), OthoLab (O3DM; OrthoLab, Poznan, Poland), OrthoProof (DigiModel; OrthoProof, Nieuwegein, The Netherlands), and 3Shape (OrthoAnalyzer; 3Shape, Copenhagen, Denmark). The usability of the programs was assessed by experts in interaction design and usability using the "enhanced cognitive walkthrough" method: 4 tasks were defined and performed by a group of domain experts while they were observed by usability experts. Results The services provided by the companies were similar. Regarding the features, all 4 systems were able to perform basic measurements; however, not all provided the peer assessment rating index or the American Board of Orthodontics analysis, simulation of the treatment with braces, or digital articulation of the casts. All systems demonstrated weaknesses in usability. However, OrthoCAD and 03DM were considered to be easier to learn for first-time users. Conclusions In general, the usability of these programs was poor and needs to be further developed. Hands-on training supervised by the program experts is recommended for beginners.

OBJECTIVE: To evaluate the effects of rapid maxillary expansion (RME) on nocturnal enuresis (NE) related to the nasal airway, nasal breathing, and plasma osmolality (as an indicator for antidiuretic hormone). MATERIALS AND METHODS: Nineteen patients with monosymptomatic primary NE, aged 6–15 years, were treated with RME for 10–15 days. To exclude a placebo effect of the RME appliance, seven patients were first treated with a passive appliance. Computed tomography of nasal cavity, rhinomanometric, and plasma osmolality measurements were made 2–3 days before and 2–3 months after the RME period. RME effects on NE were followed for three more years. RESULTS: Two to three months after the expansion there were significant improvements in the breathing function and a decrease in the plasma osmolality. NE decreased significantly in all patients after the RME period, and all patients showed full dryness after 3 years. CONCLUSIONS: This study demonstrates that RME causes complete dryness in all patients, with significant effects on pathophysiological mechanisms related to NE.

Background: Ectopic eruption and/or impaction of maxillary permanent canines is a frequent problem in clinical dentistry. Previous studies aimed to identify potential associated factors and predictors for impacted maxillary canines have only used conventional univariate statistics, which does not allow the analysis of the interaction between and within variables. Multivariate data analysis (MVDA) is a better and more powerful tool for the integration and interpretation of complex datasets. Aim: The aim of this study was to validate previously explored predictors of permanent maxillary canine impaction using MVDA. Subjects and methods: This cohort study included all the patients referred during 2011 to Molndal Hospital, Sweden for surgical exposure of impacted canines ( N = 45). Age- and gender-matched orthodontic patients ( N = 45) with normally erupting canines comprised the control group. The age range for both groups was 11–17 years. The positions of the canine teeth (orthopantograms), the skeletal variables (profile radiographs), and dentoalveolar traits (casts) were evaluated as potential predictive factors for impaction. Results: None of the parameters evaluated with either profile radiography or casts were positively correlated with impacted maxillary canines, with the exception of the location of the already impacted canines, as identified by orthopantogram. Conclusion: No correlation between clinical variables and impaction was found using MVDA. Therefore, these variables could not be used as predictors of canine impaction. Other types of parameters, such as inheritance and molecular factors that regulate the biological mechanisms of the eruption process, need to be further investigated.

To systematically investigate the biological interface of Bio-Oss by analysing dissolution-precipitation behaviour and osteogenic responses using in vitro experimental systems.Different concentrations (1-100 mg/ml) of Bio-Oss were incubated in cell culture medium for 24 h before elemental concentrations for calcium, phosphorus and silicon in the medium were analysed with inductive coupled plasma-optical emission spectroscopy. Radioactive calcium-45 isotope labelling technique was used to study possible precipitation of calcium on the Bio-Oss particle. Biological interface of Bio-Oss was studied in osteogenic experiments using mineralization medium and three different sources of cells (primary mouse bone marrow stromal cells, primary rat calvarial cells and MC3T3-E1 mouse pre-osteoblast cell line). Cells were fixed and stained with Toulidine blue, von Kossa or Alizarin Red staining for confirmation of extracellular matrix mineralization.Elemental analysis of the cell culture medium demonstrated a significant decrease of calcium and phosphorus and a dose-dependent release of silicon to the medium after incubation with Bio-Oss. A significant decrease of calcium and phosphorus in the medium occurred even at low concentrations of Bio-Oss. Uptake of calcium on the Bio-Oss particle was confirmed with radioactive calcium-45 isotope labelling technique. In osteogenic experiments with Bio-Oss (1 mg/ml), matrix mineralization around the Bio-Oss particles were demonstrated in all three cell types with von Kossa and Alizarin Red staining.Dissolution-precipitation reactions occur at the surface of Bio-Oss, and osteogenic responses are seen at the biological interface. The concentration of Bio-Oss is a key factor for the experimental in vitro results, and may also have implications for the clinic.

Biomaterials are widely used in clinical practice as bone graft substitutes for treating patients with bone defects. A molecular level understanding of the chemical processes at the interface betwe ...

We examined the effect of calcitonin in cultured mouse calvarial bones after prestimulation with different activators of adenylyl cyclase. Calcitonin (100 ng/ml), added after 48 h of culture, inhibited bone resorption (assessed as release of 45Ca from prelabeled bones cultured for 96-144 h) stimulated with parathyroid hormone (PTH, 10 nM; 0-144 h) or the adenylyl cyclase stimulator forskolin (2 microM; 0-144 h). However, no effect of calcitonin was demonstrated when bone resorption was prestimulated with the adenylyl cyclase stimulator cholera toxin, at and above 1 ng/ml, at any time point studied. In contrast, two other types of inhibitors of bone resorption in vitro, the carbonic anhydrase inhibitor acetazolamide (10 microM) and the aminobisphosphonate AHPrBP (10 microM), significantly inhibited cholera toxin-stimulated bone resorption. No cyclic AMP response to calcitonin was seen after preculture for 48 h with cholera toxin (0.1-100 ng/ml), although bones precultured in basic medium, in the absence or presence of forskolin, were still able to respond to calcitonin with elevation of cyclic AMP. Binding studies with [125I]calcitonin demonstrated that the preculture with cholera toxin did not affect the binding of calcitonin to the receptor. In summary, our data show that cholera toxin pretreatment makes calvarial bones insensitive to calcitonin-induced inhibition of bone resorption as a result of an interaction with cholera toxin at the level of calcitonin receptor-linked signal transduction. We suggest that the interaction, distal to the calcitonin receptor, is caused by the irreversible activation of Gs produced by cholera toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of this work was to investigate the release of inorganic ionic products from specimens of the polyacid-modified composite resin Dyract AP (DAP) and furthermore, to analyze the biological effect of DAP and the medium extract in human periodontal ligament (PDL) cells and mouse bone marrow cell (BMC) cultures.Ion release from DAP specimens immersed in cell culture medium was analyzed with inductively coupled plasma optical emission spectroscopy (ICP-OES). Cells were cultured with either DAP specimens or with DAP media extract and effects on cell proliferation, osteoblastic gene expression and mineralization capacity were analyzed with direct-contact tests, neutral red (NR) uptake, quantitative real-time PCR and a bone nodule formation assay.ICP-OES analysis of DAP extract demonstrated a significant increase in fluoride, strontium and silica. PDL cells demonstrated normal growth pattern in the direct-contact tests with the material. DAP extracts produced a dose-dependent stimulation of cell proliferation and concomitant inhibition of osteoblast specific markers and nodule formation.The compomer may have possible bioactive properties due to ions leaching out from the filler component.

Introduction Fixed retainers are widely used after orthodontic treatment, sometimes for extended periods, despite insufficient knowledge of their possible long-term adverse effects on the periodontium. The aim of this study was to evaluate whether bonded orthodontic retainers have an adverse long-term effect on the marginal bone levels of the mandibular front teeth. Methods The study included 62 consecutive patients in 3 groups: (1) patients who underwent orthodontic treatment and wore a fixed retainer for 10 years, (2) patients who underwent orthodontic treatment but did not have a fixed retainer, and (3) untreated controls. The marginal bone levels were measured by cone-beam computed tomography 10 years after treatment. Additionally, multivariate data analysis was used to analyze possible correlations between the marginal bone levels at 10 years and the variables obtained from the study casts and profile radiographs. Results The results demonstrated a significantly lower marginal bone level on the buccal side of the mandibular front teeth in the orthodontically treated patients compared with the orthodontically untreated group. There was no difference in the marginal bone levels between the retainer group and the no-retainer group. Multivariate analysis indicated that a low marginal bone level was correlated with a basal open vertical relationship, posterior rotation of the mandible, pretreatment of the incisor protrusion, and extraction therapy. Conclusions Within the limits of this research design, the long-term retention phase in general does not seem to cause any adverse effects on the marginal bone levels after 10 years.

Receptor activator of NF-kappaB ligand (RANKL) is crucial in osteoclastogenesis but signaling events involved in osteoclast differentiation are far from complete and other signals may play a role in osteoclastogenesis. A more direct pathway for cellular crosstalk is provided by gap junction intercellular channel, which allows adjacent cells to exchange second messengers, ions, and cellular metabolites. Here we have investigated the role of gap junction communication in osteoclastogenesis in mouse bone marrow cultures. Immunoreactive sites for the gap junction protein connexin 43 (Cx43) were detected in the marrow stromal cells and in mature osteoclasts. Carbenoxolone (CBX) functionally blocked gap junction communication as demonstrated by a scrape loading Lucifer Yellow dye transfer technique. CBX caused a dose-dependent inhibition (significant > or = 90 microM) of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells formed in 7- to 8-day marrow cultures stimulated by parathyroid hormone (PTH; 10 nM) or forskolin (FSK; 1 microM). Furthermore, CBX (100 microM) significantly inhibited prostaglandin E2 (PGE2; 10 microM) and 1,25(OH)2-vitamin D3 stimulated osteoclast differentiation in the mouse bone marrow cultures. Consequently, quantitative real-time polymerase chain reaction (PCR) analysis demonstrated that CBX downregulated the expression of osteoclast phenotypic markers, but without having any significant effects on RANK, RANKL, and osteoprotegerin (OPG) mRNA expression. However, the results demonstrated that CBX significantly inhibits RANKL-stimulated (100 ng/ml) osteoclastogenesis in the mouse bone marrow cultures. Taken together, our results suggests that gap junctional diffusion of messenger molecules interacts with signaling pathways downstream RANKL in osteoclast differentiation. Further studies are required to define the precise mechanisms and molecular targets involved.

PAR-2 is expressed by osteoblasts and activated by proteases present during inflammation. PAR-2 activation inhibited osteoclast differentiation induced by hormones and cytokines in mouse bone marrow cultures and may protect bone from uncontrolled resorption.Protease-activated receptor-2 (PAR-2), which is expressed by osteoblasts, is activated specifically by a small number of proteases, including mast cell tryptase and factor Xa. PAR-2 is also activated by a peptide (RAP) that corresponds to the "tethered ligand" created by cleavage of the receptor's extracellular domain. The effect of activating PAR-2 on osteoclast differentiation was investigated.Mouse bone marrow cultures have been used to investigate the effect of PAR-2 activation on osteoclast differentiation induced by parathyroid hormone (PTH), 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], and interleukin-11 (IL-11). Expression of PAR-2 by mouse bone marrow, mouse bone marrow stromal cell-enriched cultures, and the RAW264.7 osteoclastogenic cell line was demonstrated by RT-PCR.RAP was shown to inhibit osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11. Semiquantitative RT-PCR was used to investigate expression of mediators of osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11 in mouse bone marrow cultures and primary calvarial osteoblast cultures treated simultaneously with RAP. In bone marrow and osteoblast cultures treated with PTH, 1,25(OH)2D3, or IL-11, RAP inhibited expression of RANKL and significantly suppressed the ratio of RANKL:osteoprotegerin expression. Activation of PAR-2 led to reduced expression of prostaglandin G/H synthase-2 in bone marrow cultures treated with PTH, 1,25(OH)2D3, or IL-11. RAP inhibited PTH- or 1,25(OH)2D3-induced expression of IL-6 in bone marrow cultures. RAP had no effect on osteoclast differentiation in RANKL-treated RAW264.7 cells.These observations indicate that PAR-2 activation inhibits osteoclast differentiation by acting on cells of the osteoblast lineage to modulate multiple mediators of the effects of PTH, 1,25(OH)2D3, and IL-11. Therefore, the role of PAR-2 in bone may be to protect it from uncontrolled resorption by limiting levels of osteoclast differentiation.

Several studies have demonstrated that connexin 43 (Cx43) mediates signals important for osteoblast function and osteogenesis. The role of gap junctional communication in bone resorption is less clear. We have investigated the expression of Cx43 mRNA in osteoclasts and bone resorption cultures and furthermore, the functional importance of gap junctional communication in bone resorption. RT-PCR analysis demonstrated Cx43 mRNA expression in mouse bone marrow cultures and in osteoclasts microisolated from the marrow cultures. Cx43 mRNA was also expressed in bone resorption cultures with osteoclasts and osteoblasts/stromal cells incubated for 48h on devitalized bone slices. An up-regulation of Cx43 mRNA was detected in parathyroid (PTH)-stimulated (0.1 nM) bone resorption. Two inhibitors of gap junction communication, 18alpha-glycyrrhetinic acid (30 microM) and oleamide (100 microM), significantly inhibited PTH- and 1,25-(OH)(2)D(3)-stimulated osteoclastic pit formation. In conclusion, our data indicate a functional role for gap junction communication in bone resorption.

Osteoclasts are hematopoietic in origin and formed by proliferation, differentiation and fusion of osteoclast progenitor cells. However, the signal transducing mechanisms involved in generation of osteoclasts are not clear. We have used two well-known adenylate cyclase stimulators to examine the effect of cyclic AMP (cAMP) on the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in cultured mouse calvarial bones and in mouse bone marrow cultures. The effects of forskolin and cholera toxin were compared with those of parathyroid hormone (PTH) and 1,25(OH)2vitaminD3 (1,25(OH)2D3). PTH, as well as forskolin and cholera toxin, increased the number of osteoclast profiles/mm bone in 24-h and 120-h cultures of mouse calvarial bones. In mouse bone marrow cultures, 1,25(OH)2D3 or PTH stimulated formation of TRAP-positive multinucleated cells. Moreover, forskolin or cholera toxin produced dose-dependent stimulation of these cells at a range of concentrations correlating with their effect on cAMP production. The osteoclastic phenotype of the TRAP-positive cells was demonstrated by autoradiography of 125I-labelled calcitonin binding and by the bone-resorbing activity of the cells. The sustained presence (0-9 d) of forskolin or PTH was required to obtain maximal formation of osteoclasts. However, the presence of 1,25(OH)2D3 was required only for the last 3 d of culture for maximal osteoclast formation. We conclude that PTH may stimulate osteoclast generation using the adenylate cyclase cAMP system as a signal transduction mechanism.

Prostaglandins are involved in force-induced orthodontic tooth movement. Bradykinin (BK) and thrombin are known to cause a significant time- and concentration-dependent burst of prostanoid biosynthesis in cultured human periodontal-ligament (PDL) cells. The aim now was to investigate interactive effects between interleukin 1 alpha, -beta (IL-1 alpha, -1 beta), tumour necrosis factor-alpha,-beta (TNF-alpha, -beta) and BK or thrombin on prostaglandin biosynthesis in human PDL cells. IL-1 alpha and -1 beta produced time- and concentration-dependent stimulation of prostanoid biosynthesis [prostaglandin (PG)E2 and 6-keto-PGF1alpha]. Synergistic stimulation of prostanoid biosynthesis was demonstrated when BK or thrombin were added together with IL-1 alpha or -1 beta. BK and IL-1 beta both significantly stimulated the release of [3H]arachidonic acid. No synergistic effect on [3H]arachidonic acid release was seen when BK and IL-1 beta were added simultaneously. These data suggest that the synergistic effect of BK and IL-1 beta on prostanoid biosynthesis is not due to interactions at the receptor level nor to enhanced release of arachidonic acid, but may be due to increased activity of cyclo-oxygenase. Also, TNF-alpha and -beta produced a concentration-dependent stimulation of PGE2 formation in cultured human PDL cells. Synergistic effects of BK and thrombin were demonstrated when PGE2 production was stimulated in combination with TNF-beta. In addition, a synergistic effect on the PGE2 response to IL-1 alpha or -1 beta was demonstrated when added in combination with TNF-alpha. These experiments demonstrate synergistic interactions between BK, thrombin, IL-1 and TNF on prostaglandin biosynthesis in cultured human PDL cells. The findings suggest that inflammatory mediators may act in concert in stimulating prostanoid production in response to pro-inflammatory stimuli. As an inflammatory reaction is seen in the periodontal ligament when teeth are orthodontically treated, this synergistic interaction may be of importance in force-induced tooth movement.

The thyroid hormones, thyroxine (T4) and triiodothyronine (T3), were found to enhance both neonatal mouse calvarial bone resorption and pit formation on bovine slices by isolated rat osteoclasts. Dosage-dependent release of 45Ca from mouse calvarial bones was observed after 120 hr of culture with 10(-6)-10(-8) MT4 and 10(-6)-10(-10) M T3. Maximum treatment/control ratios of 45Ca release were recorded for 10(-7) M T4 and 10(-8) MT3. Inhibition of 45Ca release stimulated by 10(-8) M T3 was observed in the presence of 30 nM salmon calcitonin at 48 hr and 120 hr of culture with no indication of "escape" by T3-treated bones. In contrast, stimulation of 45Ca release from mouse calvarial bones by 10(-7) MT4 and 10(-8) MT3 was not inhibited by 10(-6) M indomethacin. Formation of PGE2 and PGI2 (evaluated by measuring 6-keto-PGF1alpha) by mouse calvariae was also not increased by 10(-8) MT3 after 120 hr of culture. Furthermore, no increases in cAMP formation were observed in calvarial bone cultures after either 10 min or 24 hr of exposure to 10(-8) MT3. However, significant inhibition of 45Ca release stimulated by 10(-8) M T3 was found at 120 hr in the presence of 10(-3) M hydroxyurea. When isolated rat osteoclasts were cultured in the presence of 10(-7) MT3, a 1.4-fold stimulation of pit number was observed. Pit formation was not affected by addition of 10(-6) M indomethacin to either the control or T3-treated cultures. These data suggest that the stimulation of bone resorption in neonatal mouse calvariae and activation of isolated rat osteoclasts by the thyroid hormones is not related to either prostaglandin or cAMP formation. In mouse calvariae, the effect on bone resorption of the thyroid hormones is dependent on increased cellular replication, perhaps of osteoclast precursors, or other bone cells involved in the resorptive process.

Background: The role of orthodontic retainers in maintaining stability after orthodontic treatment, and side effects associated with orthodontic retainers have not been well established. Objective: To investigate whether fixed retainers improve stability after orthodontic treatment, or increase the risk of side effects on the teeth and periodontium in comparison with removable retainers, no retainer, or fiberotomy. Methods: A systematic literature search was conducted in PubMed, Embase, the Cochrane Library, HTAdatabases of NHS Centre for Reviews and Dissemination, Swedish Council on Health Technology Assessment (SBU), Norwegian Knowledge Centre for the Health Services (NOKC), Danish Health and Medicines Authority, and reference lists. Data extraction was verified by at least two authors. The quality of evidence was rated. Meta-analysis was not suitable. Results: Two systematic reviews (SR), two randomized controlled trials (RCT), four non-randomized controlled studies (CT), and five case series were included. The SRs were well reported, but addressed issues that differed from the addressed question, or did not add to the identified primary studies. Both RCTs and CTs had study limitations, and problems with directness and/or precision. None of the studies compared fixed retainers to fiberotomy. Conclusion: According to the literature there is only low quality of evidence, that treatment stability may be improved by a fixed retainer after orthodontic treatment in comparison with a removable retainer, or no retainer (GRADE ⊕⊕⃝⃝). Furthermore, there is very low quality of evidence, whether periodontal outcomes, dental caries prevalence, or presence of calculus differ between the various types of retainer regimens (GRADE ⊕⃝⃝⃝).

Porphyromonas gingivalis strongly correlates with periodontitis, but the underlying mechanisms causing dentoalveolar bone resorption are not fully understood. As contradictory effects of P. gingivalis on osteoclastogenesis have been reported, this study investigates the effect of P. gingivalis extract on osteoclast formation. Osteoclast formation in mouse bone marrow (MBM) cell cultures and RAW 264.7 cells was stimulated by nuclear factor-κB ligand (RANKL) or parathyroid hormone (PTH). Cells were cultured with and without P. gingivalis surface-associated material and phenotypic characteristics were examined using microscopy, flow cytometry, and RT-PCR. P. gingivalis significantly decreased osteoclast formation and the expression of osteoclast phenotypic markers in PTH-stimulated MBM cultures. Additionally, P. gingivalis inhibited expression of osteoclast differentiation factors and stimulated expression of the mouse macrophage marker F4/80. The presence of P. gingivalis in RANKL-stimulated MBM cultures and RAW 264.7 cells inhibited osteoclastogenesis. Interestingly, a transient exposure with P. gingivalis before PTH stimulation increased osteoclastogenesis in MBM cultures. Flow cytometric analyses of cells transiently exposed to P. gingivalis demonstrated an increased proportion of potential osteoclast precursor cells. We conclude that a transient exposure of MBM cultures to P. gingivalis increases the number of osteoclast precursors and osteoclast formation, whereas a prolonged exposure completely abolishes osteoclastogenesis.

Helodermin and helospectin are peptides structurally similar to vasoactive intestinal polypeptide (VIP) which were recently isolated from the salivary gland venom of the lizard Heloderma suspectum. Pituitary adenylate cyclaseactivating polypeptide (PACAP) has been isolated from ovine hypothalamus and also shows sequence homology to VIP. A helodermin-like peptide has been detected by combined immunohistochemical and immunochemical techniques in the thyroid C-cells. In the present study, lizard helodermin was found to cause a time- and dose-dependent stimulation of cyclic AMP (cAMP) formation in neonatal mouse calvarial bones. Also, helospectin I, PACAP 27, and the C-terminally extended PACAP 38 stimulated cAMP accumulation in the mouse calvariae. The cAMP rise in response to helodermin was comparable to that induced by VIP, both in terms of potency and magnitude of the response. Helodermin, helospectin I, PACAP 27, and PACAP 38, at concentrations of 1 μmol/liter, stimulated cAMP accumulation in enzymatically isolated mouse calvarial bone cells. A significant response to all peptides was observed in both early and late released bone cells isolated from the calvariae, with low and high alkaline phosphatase activity, respectively. Helodermin and VIP stimulated cAMP accumulation in the cloned mouse calvarial osteoblastic cell line MC3T3-E1, in rat (UMR 106-01), and human (Saos-2) osteoblastic osteosarcoma cell lines, but not in the rat osteosarcoma cell line ROS 17/2.8. The effect of helodermin was synergistically and dose-dependently enhanced by forskolin (0.1 and 1 μmol/liter). These data show that bone cells, including osteoblasts, respond to several peptides of the VIP family, including helodermin, helospectin I, PACAP 27, and PACAP 38. Whether the responses are mediated via one or several receptor populations remains to be established. The finding that VIP and helodermin, at maximally effective concentrations, did not cause additive effect on cAMP formation in intact mouse calvariae suggests that these two agonists may use a common receptor.

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The protein kinase C-(PKC) activating phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 nmol/l) and phorbol 12, 13-dibutyrate (PDBU; 100 nmol/l) enhanced basal cyclin AMP accumulation in cultured neonatal mouse calvaria. The cyclic AMP response to parathyroid hormone (PTH; 10 nmol/l) and the adenylate cyclase activators forskolin (1–3 μmol/l) and choleratoxin (0.1 μmg/ml) was potentiated in a more than additive manner by TPA and PDBU. In contrast, phorbol 13-monoacetate (phorb-13; 100 nmol/l), a related compound but inactive on PKC, had no effect on basal or stimulated cyclic AMP accumulation. In the presence of indomethacin (1μmol/l), TPA and PDBU had no effect on cyclic AMP accumulation in calvarial bones per se, but were still able to cause a significant enhancement of the response to PTH, forskolin and choleratoxin. PTH-, forskolin- and choleratoxin-stimulated cyclic AMP accumulation in rat osteosarcoma cells UMR 106-01 was synergistically potentiated by TPA and PDBU, but not by phorb.-13. These data indicate that PKC enhances cyclic AMP formation and that the level of interaction may be at, or distal to, adenylate cyclase.

Calcitonin is a well known inhibitor of osteoclastic bone resorption, both in vivo and in vitro. However, it is also known that calcitonin has only a transient inhibitory effect on bone resorption. The mechanism for this so-called "escape from inhibition" phenomenon is not clear. In the present study, the inhibitory effect of calcitonin on phorbol ester-induced bone resorption was examined in cultured neonatal mouse calvaria. Bone resorption was assessed as the release of radioactivity from bones prelabelled in vivo with 45Ca. Two protein kinase C-activating phorbol esters, phorbol-12-myristate-13-acetate and phorbol-12,13-dibutyrate, both stimulated 45Ca release in 120-h cultures at a concentration of 10 nmol/l. Calcitonin (30 nmol/l) inhibited phorbol esterstimulated bone resorption without any "escape from inhibition". This was in contrast to the transient inhibitory effect of calcitonin on bone resorption stimulated by parathyroid hormone (10 nmol/l), prostaglandin E2 (2 μmol/l), and bradykinin (1 μmol/l). Our results suggest that activation of protein kinase C produces a sustained inhibitory effect of calcitonin on bone resorption.

The effects of cystatin C and other cysteine proteinase inhibitors on osteoclast formation and differentiation have been investigated. Cystatin C decreased osteoclast formation stimulated by parathyroid hormone (PTH), 1,25(OH)2-vitamin D3 or interleukin-6 (IL-6) (in the presence of its soluble receptor) as assessed by the number of tartrate-resistant acid phosphatase (TRAP+) multinucleated cells in mouse bone marrow cultures. The inhibitory effect was associated with decreased mRNA expression for the calcitonin receptor as well as decreased number of specific binding sites for 125I-calcitonin, and without any effect on the mRNA expression of receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL). Similarly, the cysteine proteinase inhibitors leupeptin, E-64 and benzyloxycarbonyl-Phe-Ala-diazomethane (Z-FA-CHN2) decreased PTH-stimulated formation of TRAP+ multinucleated cells and binding of 125I-calcitonin. A peptidyl derivative synthesized to mimic part of the proteinase-binding site of cystatin C (benzyloxycarbonyl-Arg-Leu-Val-Gly-diazomethane, or Z-RLVG-CHN2) also decreased PTH-stimulated osteoclast formation. In a 9-day culture, addition of cystatin C during the last 5 days was sufficient to cause substantial inhibition of osteoclast formation. Cystatin C-induced decrease of osteoclast formation was associated with enhanced number of F4/80-positive macrophages and increased mRNA expression of the macrophage receptor c-fms in the bone marrow culture. Osteoclast formation in mouse bone marrow cultures as well as in mouse spleen cell cultures, stimulated by macrophage colony-stimulating factor (M-CSF) and RANKL was also decreased by different cysteine proteinase inhibitors. In addition, cystatin C inhibited M-CSF/RANKL induction of calcitonin receptor mRNA in spleen cell cultures. The inhibitory effect by cystatin C in spleen cells was associated with decreased mRNA expression of RANK and the transcription factor NFAT2. It is concluded that cysteine proteinase inhibitors decrease formation of osteoclasts by interfering at a late stage of pre-osteoclast differentiation.

Skeletal tissue contains a network of nerve fibers expressing several neuropeptides, including vasoactive intestinal peptide (VIP) and the related peptide pituitary adenylate cyclase activating peptide (PACAP). These peptides have been demonstrated to regulate osteoclast formation and osteoclast activity. Using atomic force microscopy and by analysing changes of the intracellular calcium concentrations, we have recently demonstrated that multinucleated rat osteoclasts have cell membrane binding sites recognising VIP and PACAP. In the present study, we have further studied the expression of VIP receptor subtypes in mouse bone marrow cultures and isolated osteoclasts. A micromanipulation technique was used to isolate pure populations of osteoclasts formed in PTH-stimulated mouse bone marrow cultures. By reverse transcriptase polymerase chain reaction (RT-PCR), we studied the expression of mRNA for VIP-1, VIP-2, and PACAP receptors. The purity of the microisolated osteoclasts was determined by studying the expression of specific mRNA associated with the phenotypic trait of osteoclasts or osteoblasts/stromal cells. In this study, we show that mouse osteoclasts express VIP-1 and PACAP, but not VIP-2, receptor mRNA.

The presence of a network of peptidergic nerve fibers in the skeleton, expressing several neuropeptides including vasoactive intestinal peptide (VIP), has been demonstrated. This observation, together with our findings in vitro showing that VIP can regulate the activities of osteoblasts and osteoclasts as well as the recruitment of osteoclasts, has suggested the existence of a neuro-osteogenic interplay in bone metabolism. In the present study, the effects of VIP and pituitary adenylate cyclase-activating polypeptide (PACAP), two members of the VIP/secretin/glucagon superfamily, on osteoclast formation and mRNA expression of three key regulatory proteins involved in osteoclast formation have been investigated. VIP, PACAP-27, and PACAP-38, at concentrations of 10 −6 M, all significantly inhibited formation of tartrate-resistant acid phosphatase-positive multinuclear cells (TRAP + MNC) in mouse bone marrow cultures stimulated by 1,25(OH) 2 -vitamin D3 (D3; 10 −8 M). By using semiquantitative RT-PCR, it was found that D3 upregulated the mRNA expressions of receptor activator of NF-κB ligand (RANKL) and receptor activator of NF-κB (RANK), whereas the expression of osteoprotegerin (OPG) was downregulated in mouse bone marrow cultures stimulated by D3 for 7 days. Both VIP and PACAP-38 decreased the stimulatory effects of D3 on RANKL and RANK expression, whereas the inhibitory effect of D3 on OPG expression was reversed by VIP and PACAP-38. These observations indicate that the inhibitory effects of VIP and PACAP on osteoclast recruitment are due to regulation of the expression of key proteins involved in later stages of osteoclast differentiation.

Previous reports have demonstrated the importance of prostaglandin formation in orthodontic tooth movement, but the mechanisms involved in the activation of prostaglandin biosynthesis in the periodontal ligament (PDL) are still unclear. There is, however, evidence that inflammatory reactions appear after application of orthodontic forces. In the present study, the effect of bradykinin (BK) and thrombin, two inflammatory mediators, on prostanoid biosynthesis in human PDL-cells was investigated. BK and thrombin caused a time-dependent burst of prostaglandin E2 (PGE2) formation (maximal effect after 2-5 min). The stimulatory actions of BK and thrombin on PGE2 biosynthesis were dose-dependent; seen in PDL-cells isolated from four different patients and abolished by the non-steroidal anti-inflammatory drug indomethacin. BK and thrombin also dose-dependently stimulated the biosynthesis of PGI2. BK agonists, with affinity to the B2 subtype of BK receptors, caused a significant increase of PGE2 biosynthesis in human PDL-cells. In contrast, BK agonists with affinity to BK-B1 receptors did not cause a burst of PGE2 biosynthesis. BK and BK-B2 receptor agonists as well as thrombin, but not BK-B1 receptor agonists, also significantly increased [3H] release in human PDL-cells prelabelled with [3H]-arachidonic acid, indicating that BK and thrombin stimulate prostanoid biosynthesis, at least partly, due to activation of phospholipase A2. These data show that BK via BK-B2 receptors, as well as thrombin, have the capacity to stimulate arachidonic acid release and subsequent prostanoid biosynthesis in human PDL-cells and thus may be implicated in the tissue reactions involved in orthodontic tooth movement.

It has been reported that osteoclastic function is regulated by calcium-induced alterations in cytoplasmic free calcium ([Ca2+]i), possibly through a specific receptor. We have investigated whether osteoclasts, isolated from neonatal rat long bones, possess the divalent cation-receptor that has been demonstrated on parathyroid cells. Studies with fura-2 loaded adherent single cells showed that an increase in extracellular Ca2+ ([Ca2+]e) from 0.5 mM to 10 mM resulted in an increase in [Ca2+]i in isolated rat osteoclasts, from a basal value of 94.7 +/- 16.2 to 150.6 +/- 22.4 nM (means +/- SEM; n = 14). The shape and time course of the [Ca2+]i increase varied considerably from cell to cell. Less than half of the cells responded with a rapid transient increase whereas the rest responded with a slow increase that reached a plateau within 1-2 minutes. When [Ca2+]e was changed back to 0.5 mM, a slow decrease in [Ca2+]i was monitored. Immunohistochemical staining with two different monoclonal antibodies, recognizing the putative Ca2+ receptor on parathyroid cells, did not indicate any staining on freshly isolated rat osteoclasts. Thus, our data demonstrate that an increase in [Ca2+]e causes an elevation of [Ca2+]i in osteoclasts. This increase is not mediated via the putative cation-receptor found on parathyroid cells.

Calcitonin (CT) is a well-known inhibitor of osteoclastic bone resorption both in vivo and in vitro. The effect is mediated by activation of adenylate cyclase and subsequent increased levels of cyclic AMP (cAMP). We report here that CT-induced (30 nmol/liter) accumulation of cAMP in cultured neonatal mouse calvaria is enhanced two-fold by 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 nmol/liter) and phorbol 12,13-dibutyrate (PDBU; 100 nmol/liter), two protein kinase C (PKC)-activating phorbol esters, whereas phorbol 13-monoacetate (phorb-13; 100 nmol/liter), a related compound that does not activate PKC, has no effect. The ability of TPA and PDBU to enhance CT-stimulated cAMP accumulation was obtained also in the presence of indomethacin (1 mumol/liter). Kinetic studies revealed that TPA enhanced the cAMP response to CT at all the time points at which CT had a significant effect per se and that TPA did not alter the time-course of the cAMP response to CT. Treatment with pertussis toxin (100 ng/ml) enhanced cAMP response to parathyroid hormone (10 nmol/liter) and prostaglandin E2, but not to CT. From these data it is concluded that PKC, but not pertussis toxin-sensitive guanyl nucleotide-binding proteins (G-proteins), can interact with and modify the signal transducing system for CT in osteoclasts.

Bone resorption in a modified bone culture system, based on incubation of small fragments from neonatal mouse calvarial bones, has been studied. Four bone fragments were dissected out from each mouse calvaria and were thereafter cultured in CMRL 1066 medium in plastic multiwell dishes. Bone resorption was assessed by 45Ca release from prelabeled bones. The rate of bone resorption in response to parathyroid hormone (PTH) was less in the anterior part of the calvaria compared to the posterior part. After removing the anterior region, four parietal bone fragments that showed identical basal and PTH-stimulated release of 45Ca could be dissected out from each mouse. Excretion of lactate dehydrogenase and beta-glucuronidase was the same in bones cultured submerged or on grids. Uptake of [3H]thymidine in bones cultured submerged was 54% of [3H]thymidine uptake in bones cultured on grids. Dose-response curves, established by using parietal bone fragments, showed that the sensitivity and the magnitude of the increase in 45Ca release seen after stimulation with PTH, prostaglandin E2, and 1 alpha-hydroxyvitamin D3 were the same for bones cultured submerged or on grids. The 45Ca release in response to stimulation with PTH, prostaglandin E2, and 1 alpha-OHD3 was the same in calvarial fragments cultured submerged and those previously obtained with calvarial halves cultured on grids. Thus, even though the rate of DNA synthesis was slower in bones cultured submerged, the rate and the magnitude of resorption were the same in bones cultured on grids or submerged. These data show that it is possible to perform studies on bone resorption with small fragments of neonatal mouse parietal bones.

The effects of mouse recombinantγ-interferon (γ-IFN) and indomethacin on bone resorption stimulated by bradykinin, Lys-bradykinin, Met-Lys-bradykinin, des-Arg9-bradykinin and prostaglandin E2 (PGE2) have been studied using cultures of neonatal calvarial bones and analyzing the release of45Ca from prelabelled bones as a paramenter of bone resorption. In addition, the effects ofγ-IFN and indomethacin on formation of PGE2 in bone cultures stimulated by bradykinin was analyzed. Indomethacin (1 μmol/l) totally abolished bradykinin (1 μmol/l) induced45Ca release. The inhibitory effect of indomethacin could be fully reversed by addition of PGE2 (1 μmol/l).γ-IFN (1000 U/ml) almost totally inhibited45Ca release stimulated by bradykinin (1 μmol/l), but the inhibitory effect could only be partially overcome by PGE2.γ-IFN and indomethacin also inhibited the stimulatory effects of Lys-bradykinin, Met-Lys-bradykinin and des-Arg9-bradykinin (1 μmol/l) on45Ca release. The stimulatory effects of PGE2 (1 μmol/l) on radioactive calcium mobilization was partially inhibited byγ-IFN (1000 U/ml), whereas indomethacin (1 μmol/l) was without effect. The inhibitory effect ofγ-IFN on45Ca release stimulated by bradykinin and PGE2 was dose-dependent with threshold for action at 3–30 U/ml. Comparative dose-response curves showed thatγ-IFN was most potent as inhibitor of bradykinin induced45Ca release. Bradykinin (1 μmol/l) significantly stimulated PGE2 formation by a mechanism that was completely inhibited by indomethacin (1 μmol/l).γ-IFN (1000 U/ml) partially inhibited the stimulatory effect of bradykinin on PGE2 formation. These data show that i)γ-IFN is a potent inhibitor of bone resorption induced by bradykinin and bradykinin analogues and ii) that the mechanism of action can be mainly explained by an inhibition of kinin induced prostaglandin biosynthesis. The results, however, also show thatγ-IFN can inhibit bone resorption by mechanisms unrelated to prostaglandin formation.

The adenylate cyclase activator forskolin (1–10 μmol L ) inhibited 45 Ca release from parathyroid hormone (PTH; 10 nmol L ) stimulated prelabeled neonatal mouse calvaria in short term culture (24 h). This effect of forskolin was potentiated by rolipram, Ro 20-1724, and isobutyl-methylxanthine, three structurally different inhibitors of cyclic AMP phosphodiesterase. Forskolin (10 μmol L ) and calcitonin (30 mU/mL) inhibited the mobilization of stable calcium and inorganic phosphate as well as the release of the lysomal enzymes beta-glucuronidase and beta-N-acetylglucosaminidase from PTH-stimulated unlabeled bones. Osteoclasts in PTH-stimulated calvaria showed active ruffled borders with numerous membrane infoldings. Treatment of PTH-stimulated bones with forskolin and calcitonin resulted in a rapid (2 h) loss of the active ruffled border. In addition, forskolin and calcitonin induced similar changes with respect to the number and size distribution of cytoplasmic vesicles in PTH-activated osteoclasts. After 24 h, all signs of osteoclast inactivation were still prominent, whereas after 48 h of treatment with forskolin or calcitonin, the reappearance of a ruffled border on a number of osteoclasts signaled an escape from the inhibitory action of both calcitonin or forskolin. These data indicate that forskolin inhibits bone resorption by a cyclic AMP dependent mechanism and that the effect of forskolin and calcitonin on bone resorption and osteoclast morphology are comparable. These observations lend further support to the view that cyclic AMP may be an intracellular mediator of the inhibitory action of calcitonin on multinucleated osteoclasts.

The effect of prostaglandin E2 (PGE2) on the kinetic of bone resorptionin vitro was assessed by following the release of minerals and degradation of matrix in cultured mouse calvarial bones. PGE2 (1 and 3 μmol/liter) caused an initial inhibition of the release of45Ca, stable calcium, and inorganic phosphate from unstimulated calvarial bones. The effect was transient and after 24 and 48 hours the release of45Ca, stable calcium, and inorganic phosphate from PGE2-treated bones was enhanced. 0.3 μmol/liter of PGE2 stimulated the release of45Ca after 24 hours, but at this concentration no initial inhibition was observed. The initial inhibitory effect of PGE2 (1 μmol/liter) could be further increased by three structurally different inhibitors of cyclic AMP breakdown. PGE2 (1 μmol/liter) caused not only an initial inhibition of mineral release but also an initial inhibition of matrix degradation, as assessed by the release of3H from [3H]-proline labeled bones. In addition, PGE2 (3 μmol/liter), in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine, caused a rapid (6 hours) inhibition of the release of the lysosomal enzymes β-glucuronidase and β-N-acetyl-glucosaminidase, without affecting the release of the cytosolic enzyme lactate dehydrogenase. Similar specific initial inhibition of lysosomal enzyme release was also seen in the presence of calcitonin and dibutyryl cyclic AMP, but not in the presence of parathyroid hormone (PTH). Neither PGE2 nor the phosphodiesterase inhibitors rolipram and Ro 20.1724, could inhibit the initial stages of PTH-induced45Ca release. Nor did PGE2 inhibit the stimulation of radioactive calcium mobilization induced by 1α(OH)-vitamin D3. Enhancement of cyclic AMP formation by forskolin, choleratoxin, and dibutyryl cyclic AMP in unstimulated bones resulted in an initial reduction of45Ca release (6 hours) followed by a delayed stimulation (96 hours). These data indicate that PGE2 has the capacity to reduce the activity of unstimulated, preexisting osteoclasts by a cyclic AMP-dependent process.

Earlier we have shown that bone resorption is impaired in cultured calvaria from beige mice (an animal equivalent of the Chediak-Higashi syndrome in man). In the present study we have compared the concentrations of cyclic AMP and cyclic GMP in calvarial bones from beige mice with the nucleotide concentrations in bones from corresponding normal mice. In six independent experiments the basal concentrations of cyclic AMP was significantly (P less than 0.01) higher in bones from beige mice (on an average 50% augmented). The ratio of cyclic AMP/cyclic GMP was 2.43 times higher (P less than 0.01) in bones from beige mice. After stimulation with the phosphodiesterase inhibitor isobutylmethylxanthine and prostaglandin E2 no significant differences of cyclic AMP concentrations between beige and control mice could be registered. The response to adenosine was significantly higher (P less than 0.005) in bones from beige mice (4.3 +/- 0.4-fold of basal cyclic AMP concentrations, mean +/- SE) compared to control mice (1.9 +/- 0.4-fold of basal, mean +/- SE). The increased basal concentration of cyclic AMP in calvaria from beige mice may be due to increased sensitivity to some agonists, such as adenosine, rather than simply being a function of cell mass.

We have utilized the adenylate cyclase stimulator, cholera toxin, as a tool to test the role of cyclic AMP as a mediator of the effects on bone resorption by the calcium-regulating hormones, parathyroid hormone (PTH) and calcitonin. The effects on bone resorption were studied in an organ culture system using calvarial bones from newborn mice. Cyclic AMP response was assayed in calvarial bone explants and isolated osteoblasts from neonatal mouse calvaria. Cholera toxin caused a dose-dependent cAMP response in calvarial bones, seen at and above approx. 1–3 ng/ml and calculated half-maximal stimulation (EC 50 ) at 18 ng/ml. The stimulatory effect of cholera toxin could be potentiated by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX, 0.2 mmol/l). Cyclic AMP accumulation in the bones was maximal after 4–6 h, and thereafter declined. However, activation of the adenylate cyclase was irreversible and the total amount (bone + medium) of cAMP produced, in the presence of IBMX (0.2 mmol/l), increased with time, for at least 48 h. In osteoblast-like cells cholera toxin (1 μg/ml) stimulated the cellular levels of cAMP with a peak after 60–120 min, which could be potentiated with IBMX. The total cAMP accumulation indicated an irreversible response. In short-term bone organ cultures (at most, 24 h) cholera toxin, at and above 3 ng/ml, inhibited the stimulatory effect of PTH (10 nmol/l) on 45 Ca release from prelabelled calvarial bones. The inhibitory effect of cholera toxin (0.1 μg/ml) on 45 Ca release was significant after 6 h and the calculated IC 50 value at 24 h was 11.2 ng/ml. Cholera toxin (0.1 μg/ml) also inhibited PTH-stimulated (10 nmol/l) release of Ca 2+ , inorganic phosphate (P i ), α-glucuronidase, β - N -acetylglucosaminidase and degradation of organic matrix (release of 3 H from [ 3 H]proline-labelled bones) in 24 h cultures. 45 Ca release from bones stimulated by prostaglandin E 2 (1 μmol/l) and 1α-hydroxyvitamin D 3 (0.1 μmol/l) was also inhibited by cholera toxin (0.3 μg/ml) in 24-h cultures. The inhibitory effect of cholera toxin on bone resorption was transient, and in long-term cultures (120 h) cholera toxin caused a dose-dependent, delayed stimulation of mineral mobilization (Ca 2+ , 45 Ca, P i ), degradation of matrix and release of the lysosomal enzymes β-glucuronidase and β - N -acetylglucosaminidase. The calculated EC 50 value for 45 Ca release in 120 h cultures was 0.8 ng/ml. Cholera toxin-stimulated (0.01 μg/ml) 45 Ca release could be inhibited by calcitonin (0.1 U/ml) but not by indomethacin (1 μmol/I). We conclude that cholera toxin-stimulated cAMP accumulation in bone transiently inhibits bone resorption, indicating that cAMP may be a mediator of the effect of calcitonin but that the nucleotide does not mediate the rapid bone-resorptive effects of PTH. The late bone-resorptive effect of cholera toxin suggest that cAMP may be involved in a delayed action of PTH on bone resorption.

The effect of forskolin on parathyroid hormone (PTH) stimulated bone resorption, as assessed in vitro by the release of 45Ca from prelabelled neonatal mouse calvarial bones, and cyclic AMP formation in mouse calvarial bones and osteoblast-like cells was investigated. Much higher concentrations (100–300-times) of PTH were required to stimulate cyclic AMP accumulation than to stimulate mineral mobilization in murine calvarial bones. PTH, in the absence of phosphodiesterase inhibitor, stimulated cyclic AMP formation in mouse calvarial bones at and above concentrations of 3–10 nmol/l with EC50 at 10–15 nmol/l. In the presence of forskolin ( 1 or 10 μmol/l) the minimal concentration required to obtain a cyclic AMP response to PTH was decreased by a factor of 30–100 and the EC50 value was decreased to 1–2 nmol/l. Similar results were seen in osteoblast-cnriched cells. In addition, the magnitude of the PTH-induced cyclic AMP response was substantially potentiated by forskolin, both in calvarial bones and in isolated osteoblasts. Forskolin, in the absence of PTH, stimulated cyclic AMP levels in mouse calvaria at and above 1 μmol/l. In the presence of PTH, the response to forskolin was potentiated over the whole dose-response curve with apparent EC50 value at 1–2 μmol/l of forskolin. Forskolin (1 μmol/l) did not affect the magnitude of the 45Ca release response to PTH in 24 or 48 h cultures. In 96 h cultures, forskolin, in an additive manner, potentiated the effect of PTH on calcium mobilization. These results show that forskolin, in mouse calvarial bones and in isolated osteoblasts, in addition to directly stimulating cyclic AMP, can enhance receptor-mediated activation of adenylate cyclase. The finding that forskolin did not synergistically potentiate PTH-induced bone resorption suggests that there is no simple relationship between PTH-induccd cyclic AMP formation and stimulation of bone resorption.

The effect of bradykinin on prostaglandin production in mouse calvarial bones and in isolated osteoblasts has been examined. Bradykinin (1 mumol/l) stimulated prostaglandin formation in neonatal mouse calvarial bones incubated for 30 min. In isolated osteoblast-like cells from neonatal mice calvarial bones and in a cloned mouse calvarial osteoblastic cell lineage (MC3T3-E1) bradykinin stimulated the production of prostaglandin E2 (PGE2) and 6-keto-prostaglandin F1 alpha (the stable breakdown product of prostacyclin). The stimulation of PGE2 production occurred rapidly (30 s) and reached its maximum after 5-10 min. The stimulatory effect of bradykinin on PGE2 production in isolated osteoblast-like cells and in MC3T3-E1 cells was dose dependent with apparent half maximal stimulation seen at 10 and 3 nmol/l, respectively. Bradykinin-induced prostaglandin production was totally reversible after withdrawal of the agonist. Pretreatment with bradykinin (1 mumol/l) resulted in desensitization to a subsequent challenge with bradykinin (1 mumol/l), while pretreatment with bradykinin had no effect upon arachidonic acid (30 mumol/l) induced prostaglandin formation. Bradykinin-induced production of PGE2 was abolished by several structurally unrelated, competitive and non-competitive inhibitors of arachidonic acid metabolism as well as by corticosteroids. The mouse calvarial osteoblast-like cells also showed a PGE2 and 6-keto-PGF1 alpha response to thrombin, but not to parathyroid hormone (PTH), calcitonin and 1 alpha(OH)D3. The formation of cyclic AMP in mouse calvarial osteoblasts was enhanced by PTH, bradykinin, thrombin and arachidonic acid but not by calcitonin and 1 alpha(OH)D3. The cyclic AMP response to bradykinin, thrombin and arachidonic acid, but not that to PTH, was abolished by indomethacin. The degree of confluency of the cell cultures greatly influenced the amount of prostaglandins being produced. At higher cell density the amount of prostanoids synthesized per cell was substantially decreased in untreated control cultures as well as in bradykinin- and arachidonic acid-treated cells. These data suggest that osteoblasts are equipped with receptors for bradykinin coupled to prostaglandin production.