Systemic treatment with resveratrol and/or curcumin reduces the progression of experimental periodontitis in rats.

BACKGROUND AND OBJECTIVE: Periodontitis is a chronic inflammatory disease of periodontal tissues that leads to the destruction of bone and other connective tissues. Resveratrol and curcumin are plant-derived substances with biological properties that may have immunomodulatory properties. This study investigated the effect of continuous administration of resveratrol and curcumin and the association of resveratrol and curcumin on the progression of experimental periodontitis in rats. MATERIAL AND METHODS: Forty Wistar rats were assigned randomly to the following groups: group 1, experimental periodontitis + placebo (PL) (n = 10); group 2, experimental periodontitis + resveratrol (RSV) (n = 10); group 3, experimental periodontitis + curcumin (C) (n = 10); and group 4, experimental periodontitis + resveratrol + curcumin (COMBI) (n = 10). Periodontitis was induced in rats by tying a silk suture, as a ligature, around one of the first molars. Daily administration of the placebo solution, 10 mg/kg of resveratrol, 100 mg/kg of curcumin or 10 mg/kg of resveratrol plus 100 mg/kg of curcumin was carried out from day 0 to day 30. At the end of the relevant experimental periods, rats were killed and the specimens obtained were processed for morphometric analysis of bone loss. Gingival tissues surrounding the first molar were collected for quantification of interleukin (IL)-1ß, IL-4, interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) using a Luminex/MAGPIX assay. RESULTS: Intergroup comparisons of the morphometric outcomes revealed higher bone-loss values in the PL group (p < 0.05) when compared with RSV, C and COMBI groups. There was no difference in bone-loss values among RSV, C and COMBI groups (p > 0.05). The immunoenzymatic assay of the gingival tissue showed a lower concentration of IL-1ß in the COMBI group in comparison with the PL group (p < 0.05). Higher values of IL-4 were demonstrated in groups RSV, C and COMBI in comparison with the PL group (p < 0.05). Only RSV caused a reduction in the levels of IFN-γ (p < 0.05). There was no difference in the concentration of TNF-α amongst the four groups (p > 0.05). CONCLUSION: Resveratrol and curcumin are capable of reducing alveolar bone loss in an animal model of periodontitis. This occurred when these agents were added singly or in combination with one another, but there did not appear to be either synergistic or additive effects.

Cytokines during periodontal wound healing: potential application for new therapeutic approach.

Regeneration of periodontal tissues is one of the main goals of periodontal therapy. However, current treatment, including surgical approach, use of membrane to allow maturation of all periodontal tissues, or use of enamel matrix derivatives, presents limitations in their indications and outcomes leading to the development of new tissue engineering strategies. Several cytokines are considered as key molecules during periodontal destruction process. However, their role during each phase of periodontal wound healing remains unclear. Control and modulation of the inflammatory response and especially, release of cytokines or activation/inhibition in a time- and spatial-controlled manner may be a potential perspective for periodontal tissue engineering. The aim of this review was to summarize the specific role of several cytokines during periodontal wound healing and the potential therapeutic interest of inflammatory modulation for periodontal regeneration especially related to the expression sequence of cytokines.

Quantifying oral inflammatory load: oral neutrophil counts in periodontal health and disease.

BACKGROUND AND OBJECTIVE: Neutrophils are the primary white blood cells that are recruited to fight the initial phases of microbial infections. While healthy norms have been determined for circulating blood neutrophil counts in order to identify patients with suspected systemic infections, the levels of oral neutrophils (oPMNs) in oral health and in the presence of periodontal diseases have not been described. It is important to address this deficiency in our knowledge as neutrophils are the primary immune cell present in the crevicular fluid and oral environment and previous work has suggested that they may be good indicators of overall oral inflammation and periodontal disease severity. The objective of this study was to measure oPMN counts obtained in a standardized oral rinse from healthy patients and from those with chronic periodontal disease in order to determine if oPMN levels have clinical relevance as markers of periodontal inflammation. A parallel goal of this investigation was to introduce the concept of 'oral inflammatory load', which constitutes the inflammatory burden experienced by the body as a consequence of oral inflammatory disease. MATERIAL AND METHODS: Periodontal examinations of patients with a healthy periodontium and chronic periodontal disease were performed (n = 124). Two standardized consecutive saline rinses of 30 s each were collected before patient examination and instrumentation. Neutrophils were quantified in the rinse samples and correlated with the clinical parameters and periodontal diagnosis. RESULTS: Average oPMN counts were determined for healthy patients and for those with mild, moderate and severe chronic periodontal diseases. A statistically significant correlation was found between oPMN counts and deep periodontal probing, sites with bleeding on probing and overall severity of periodontal disease. CONCLUSIONS: oPMN counts obtained through a 30-s oral rinse are a good marker of oral inflammatory load and correlate with measures of periodontal disease severity.

Impact of matrix metalloproteinases on inhibition of mineralization by fetuin.

BACKGROUND AND OBJECTIVE: Human subjects affected by inflammatory diseases, such as periodontitis, may be at increased risk for the development of cardiovascular diseases and calcification of atheromas; however, the potential mechanisms have not been defined. Alpha-2-Heremans Schmid glycoprotein (fetuin A) is an abundant serum glycoprotein of ~49 kDa that inhibits ectopic arterial calcification. We examined whether matrix metalloproteinases (MMPs), which are increased in inflammatory diseases, including periodontitis, bind and degrade fetuin and alter its ability to inhibit calcification in vitro. MATERIAL AND METHODS: Binding and cleavage of fetuin by MMPs were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-silico analyses and mass spectrometry. The effects of intact and MMP-degraded human fetuin on mineralization were measured in a cell-free assay. RESULTS: From in-silico analyses and literature review, we found that only MMP-3 (stromelysin) and MMP-7 (matrilysin) were predicted to cleave human fetuin at levels that were physiologically relevant. In-vitro assays showed that MMP-7, and, to a lesser extent, MMP-3, degraded human fetuin in a time- and dose-dependent manner. Fetuin peptides generated by MMP-7 cleavage were identified and sequenced by mass spectrometry; novel cleavage sites were found. Hydroxyapatite mineralization in vitro was strongly inhibited by fetuin (> 1 µm), as was MMP-3-cleaved fetuin, while MMP-7-cleaved fetuin was threefold less effective in blocking mineralization. CONCLUSION: MMP-7 and, to a lesser extent, MMP-3, affect the ability of fetuin to inhibit the formation of hydroxyapatite in vitro. These data suggest that the MMPs increased in inflammatory diseases, such as periodontitis, could affect regulation of mineralization and potentially enhance the risk of calcified atheroma formation.

Periodontal diseases and stress: a brief review.

Periodontal diseases are common chronic inflammatory diseases caused by pathogenic microorganisms colonising the subgingival area and inducing local and systemic elevations of pro-inflammatory cytokines resulting in tissue destruction. Apparition and evolution of periodontal diseases are influenced by many local or systemic risk factors. Psychological stress has been suggested as one of them and may negatively influence the outcome of periodontal treatment. However, mechanisms explaining the possible relationship between stress and increased susceptibility to periodontal disease remain poorly understood. Several stress markers are found in blood and saliva of patients with periodontal diseases and influence the development of periodontal diseases by several mechanisms including modifications of the inflammatory response and changes in the composition of the dental biofilm. The aim of this review is to provide an insight into the relationship between psychological stress and periodontal diseases.

Periodontal Inflammation Primes the Systemic Innate Immune Response.

The presence of periodontal diseases (PDs) often strongly correlates with other severe chronic inflammatory conditions, including cardiovascular disease, diabetes, and arthritis. However, the mechanisms through which these diseases interact are unclear. In PD, tissue and bone destruction in the mouth is driven by elevated recruitment of polymorphonuclear neutrophils (PMNs), which are primed and recruited from the circulation to sites of inflammation. We predicted that systemic effects on PMN mobilization or priming could account for the interaction between PD and other inflammatory conditions. We tested this using a mouse model of ligature-induced PD and found elevated PMN counts specifically in bone marrow, supporting a systemic effect of periodontal tissue inflammation on PMN production. In contrast, mice with induced peritonitis had elevated PMN counts in the blood, peritoneum, and colon. These elevated counts were further significantly increased when acute peritonitis was induced after ligature-induced PD in mice, revealing a synergistic effect of multiple inflammatory events on PMN levels. Flow cytometric analysis of CD marker expression revealed enhanced priming of PMNs from mice with both PD and peritonitis compared to mice with peritonitis alone. Thus, systemic factors associated with PD produce hyperinflammatory PMN responses during a secondary infection. To analyze this systemic effect in humans, we induced gingival inflammation in volunteers and also found significantly increased activation of blood PMNs in response to ex vivo stimulation, which reverted to normal following resolution of gingivitis. Together, these results demonstrate that periodontal tissue inflammation has systemic effects that predispose toward an exacerbated innate immune response. This indicates that peripheral PMNs can respond synergistically to simultaneous and remote inflammatory triggers and therefore contribute to the interaction between PD and other inflammatory conditions. This suggests larger implications of PD beyond oral health and reveals potential new approaches for treating systemic inflammatory diseases that interact with PD.

The Biology of Social Adversity Applied to Oral Health.

Biological embodiment is a concept derived from Engel's biopsychosocial model to health, theorized as the process by which adverse social exposures trigger neuroendocrine and immune responses, leading to disease and/or increased disease susceptibility. This critical review discusses the biopsychosocial model as applied to oral health and its relevance to oral health policy while deciphering some of the pathobiological processes underlying social adversity. In periodontal disease, for example, such processes can occur via the activation of the hypothalamic-pituitary-adrenal axis and the consequent release of the chronic stress hormone cortisol. The latter contributes to a proinflammatory immune state that increases the risk for periodontal inflammation. Recent research shows that cortisol relates to an elevated oral inflammatory load, demonstrated as hyperactive neutrophils that are pivotal to periodontal tissue damage. Consistent with the biopsychosocial model, this relationship is amplified in those of lower income and higher financial stress. Similarly, among children from lower socioeconomic backgrounds, cortisol is linked to a higher cariogenic bacterial load. Such findings implicate the stress pathway as key in the oral pathogenic process, particularly under social/socioeconomic adversity. Collectively, this work emphasizes the importance of addressing social factors in alleviating oral disease burden and reducing the social gaps therein.

Prevalence of periodontal pathogens in subgingival lesions, atherosclerotic plaques and healthy blood vessels: a preliminary study.

BACKGROUND AND OBJECTIVE: Previous studies have reported different periodontal bacteria in atherosclerotic lesions, but their involvement in plaque formation remains unclear. The aim of the present study was to investigate the presence of 20 periodontal bacteria in atherosclerotic samples and healthy blood vessels (used as controls) and to clarify their relationship in regard to clinical and bacteriological periodontal status. MATERIAL AND METHODS: The day before vascular surgery the patients had a thorough periodontal examination and bacteriological samples were taken from periodontally diseased sites. Atheromatous plaques, internal mammary arteries and saphenous veins were harvested during surgery. A DNA-DNA hybridization procedure was used to screen periodontal and vascular samples for the 20 selected bacterial species. RESULTS: Periodontal samples from the severe periodontitis group were found to have a higher prevalence and biomass of bacterial species than the moderate periodontitis group. In vessel samples, the prevalence of the same 20 bacterial species analyzed together was similar in the two groups, except for saphenous veins. CONCLUSION: The presence of periodontal pathogens in atherosclerotic plaques and in apparently healthy vessels appeared to reflect a higher level of bacteremia rather than infection of endothelial cells.

Oral and Blood Neutrophil Activation States during Experimental Gingivitis.

Polymorphonuclear neutrophils (PMNs) are the primary leukocytes present in the healthy and inflamed oral cavity. While unique PMN activation states have been shown to differentiate health and periodontitis, little is known about the changes in PMN activation states that occur during the transition from periodontal health to gingivitis. The objective of this study was to characterize oral and circulatory PMNs during induction and resolution of experimental gingivitis. Healthy volunteers were recruited to undergo experimental gingivitis. Clinical assessment of pocket depths, bleeding on probing, gingival index, and plaque index, as well as flow cytometric analysis of CD (cluster of differentiation) activation markers on blood and oral PMNs, was performed weekly. All clinical parameters increased significantly during the induction period and returned to baseline levels during the resolution phase. During the induction phase, while oral PMN counts increased, oral PMN activation state based on surface expression of CD63, CD11b, CD16, and CD14 was diminished compared to those seen in health and during the resolution phase. PMNs in circulation during onset showed increased activation based on CD55, CD63, CD11b, and CD66a. Using clinical parameters and oral PMN counts assessed at day 21, we noted 2 unique disease patterns where one-third of subjects displayed an exaggerated influx of oral PMNs with severe inflammation compared to the majority of the population who experienced a moderate level of inflammation and PMN influx. This supports the notion that PMN influx and severe inflammatory changes during gingivitis could identify subjects at risk for the development of severe gingival inflammation and progression toward destructive periodontitis. This study demonstrates that oral PMN activation states are reduced in gingivitis and suggest that only in periodontitis do PMNs become hyperactivated and tissue damaging. Knowledge Transfer Statement: Our article creates a paradigm for future studies of the evolution of essential oral and circulatory biomarkers to identify individuals at risk to develop periodontitis at an early stage of periodontal disease, which is reversible upon proper oral hygiene practices and dental treatments.

Porphyromonas gingivalis and its lipopolysaccharide differently modulate epidermal growth factor-dependent signaling in human gingival epithelial cells.

Periodontitis is an inflammatory disease induced by pathogenic bacteria such as Porphyromonas gingivalis. Little is known about epidermal growth factor (EGF) signals in human gingival epithelial cells (HGEC), which are major targets of P. gingivalis, and how the expression of proteins participating in EGF signaling-that is, EGF-receptor (EGFR), suppressor of cytokine signaling-3 (SOCS-3), interferon regulatory factor-1 (IRF-1), and signal transducers and activators of transcription (STAT-3)-are modified. This study aimed to assess the effects of P. gingivalis and its purified lipopolysaccharide (LPS-Pg) on EGF signaling. HGEC were infected for 2 h in a dose-dependent manner with P. gingivalis and with heat-killed P. gingivalis, and activated for 2 and 24 h by 1 µg/mL of purified LPS-Pg. Quantitative reverse transcription polymerase chain reaction and Western blotting were performed to measure mRNA and protein levels for SOCS-3, IRF-1 EGF, EGFR, and STAT-3. The tyrosine-phosphorylation status of STAT-3 was also examined. The results showed that infection of HGEC cells with P. gingivalis, but not with heat-killed P. gingivalis, led to significant reductions in expression levels of mRNAs and proteins for SOCS-3, IRF-1, and EGFR, while LPS-Pg over time significantly increased the expression of these mRNAs and proteins. Tyrosine-phosphorylation of STAT-3 was significantly increased during infection with P. gingivalis and activation by LPS-Pg but not modified during infection with heat-killed P. gingivalis. This study highlights that P. gingivalis and its purified LPS differentially modulated the expression of proteins (SOCS-3, IRF-1, EGFR, and STAT-3) interfering with EGF signaling.

Dexamethasone effects on induction of neoplastic transformation by Fujinami sarcoma virus in an in vitro chick embryo periosteal model for osteosarcoma.

Recently we have developed a model in vitro system for the study of factors regulating the histogenesis of osteosarcoma. In this system, Fujinami sarcoma virus (FSV) induces osteosarcomatous changes such as increased cell proliferation and altered patterns of bone and nonmineralized matrix (osteoid) formation. Such changes can be quantitated at the individual cell level, by computer-assisted morphometry. Here we report on the effects of dexamethasone (DEX) on FSV-induced neoplastic transformation and osteogenesis in chick embryonic periosteum cultures, as reflected by a series of histopathological parameters. Most significantly, it was found that compared to 10(-9) M DEX treated cultures, in 10(-7) M DEX pretreated cultures, the bone/osteoid ratio was increased due to a relative increase in the area of bone and a decrease in the area of osteoid. The number of [3H]thymidine-labeled cells decreased significantly, while the proportion of alkaline phosphatase positive cells increased. Double-label immunohistochemistry (with anti-P140gag-fps) and histochemistry for alkaline phosphatase activity was performed, to demonstrate production of the oncogene-encoded protein, and osteoblastic differentiation, respectively. In an in vitro transformation assay single cells derived from 10(-9) M DEX treated cultures formed a significantly higher number of colonies than those obtained from 10(-7) M DEX pretreated cultures. Taken together, the data indicate that in the chick embryonic periosteum culture system, pretreatment with 10(-7) M DEX inhibits the ability of FSV to induce neoplastic transformation. This effect is probably the result of DEX-induced cell differentiation, prior to infection with FSV.

[Microbial contamination of the implant-abutment connections: Review of the literature]. / Pénétration microbienne dans la connectique pilier-implant : revue de littérature.

Today manufacturing process of dental implant parts allows for a precision of fit between implant and abutment of several microns. This microgap opens and closes under occlusal forces, leading to a pumping effect and to a contamination of the implant from bacteria and oral fluids. This kind of contamination is seen in all systems even if less often with internal connections that offers a better fit. Apart from this junction area, the screw well is another contamination pathway if the filling materials do not guarantee a hermetic sealing. The nature of contamination depends on the surrounding oral flora. When present, contamination leads to a persistent inflammatory reaction nearby the seal. The use of antiseptics or other materials for sealing the microgap decreases the risk of contamination and improves the gingival reaction. However, these solutions are time-limited.

Inhibition of osteoclast differentiation by polycyclic aryl hydrocarbons is dependent on cell density and RANKL concentration.

We investigated the effect of representative polycyclic aryl hydrocarbons (PAHs), benzo[a]pyrene (BaP), and 7,12-dimethylbenz[a]anthracene (DMBA) on osteoclast differentiation and function by using dispersed cancellous bone derived rabbit osteoclasts and the RAW264.7 cells. These cells differentiate into osteoclasts when exposed to receptor activator of NF-kappaB ligand (RANKL). The rabbit osteoclasts were exposed to 10(-6) to 10(-9)M BaP or DMBA and the tartrate-resistant acid phosphatase (TRAP)-positive cells were counted. The effect of PAHs on osteoclast differentiation in dispersed rabbit osteoclast-containing stromal cell populations was cell density dependent, suggesting that the cell density of stromal cells, osteoclast precursors, and/or mature osteoclasts are factors regulating the effect of PAHs. To investigate the direct effect of BaP on osteoclast differentiation, RAW264.7 cells were exposed to 10(-5) to 10(-6) M BaP. Treatment of RAW264.7 cells cultured with 25 ng/ml soluble RANKL and 10(-5)M BaP for 5 days decreased osteoclast differentiation, TRAP activity levels, and resorption of bone-like substrata. The inhibition was prevented by 10(-6) to 10(-7) M resveratrol, an aryl hydrocarbon receptor (AhR) antagonist, and by higher concentrations of RANKL. To investigate the ability of RANKL to reverse BaP-mediated inhibition, gene expression was determined by RT-PCR. Cytochrome P450 1B1 (CYP1B1) mRNA, one of the genes activated by BaP, was present only in the groups exposed to BaP; the levels of CYP1B1 mRNA decreased in the presence of increasing concentrations of RANKL. These results suggest that the inhibitory effects of PAHs on osteoclastogenesis are direct and likely involve interaction of the RANKL and PAH signaling pathways.

The relationship between mouthrinse matrix metalloproteinases (MMP-1, 8, 13) and albumin levels with the degree of oral mucositis in allogeneic stem cell transplant patients.

Our aim was to examine the relationship between mouthrinse matrix metalloproteinases (MMPs) and whole albumin levels (AL) relative to oral mucositis (OM) in allogeneic stem cell transplant (alloSCT) patients. Mouthrinse vertebrate collagenase levels are positively correlated with connective tissue destruction (CTD) in periodontitis and may also be involved in CTD associated with OM. Increases in salivary AL have been noted prior to OM onset and may serve as a predictive tool for OM and as a positive control in this study. A total of 23 alloSCT patients were visited eight times over 4 weeks following the transplant. OM was scored via a previously validated examiner-based ordinal system. Mouthrinse samples were collected and analyzed for MMP-1, 8, 13 (members of the vertebrate collagenase group) and AL. No significant correlation was found for MMP levels relative to OM scores. AL were positively and significantly associated with OM scores (P<0.001). MMP levels may not be an important factor in OM development and severity; however, mouthrinse AL may serve as a more objective measure of OM development and severity.

Regulation of BMP-induced ectopic bone formation by Ahsg.

alpha2-HS-glycoprotein (Ahsg), also known as fetuin is a serum and bone resident glycoprotein, which binds to TGF-beta superfamily members including bone morphogenetic proteins (BMP) and inhibits dexamethasone-induced osteogenesis in bone marrow cultures in vitro. Here we demonstrate that Ahsg reduces cytokine binding to its cognate receptor in HOS osteocyte cells and suppresses intracellular signaling, while in vivo, we test the hypothesis that Ahsg-deficient mice are hyper-responsive to BMP-induced osteogenesis. Human native BMP was implanted into the hindquarter muscles of Ahsg(+/+), Ahsg(+/-) and Ahsg(-/-) mice and 4 weeks later, ossicle formation was analyzed by radiography, bone density scanning (DEXA) and histomorphometry. Alkaline phosphatase (AP) activity was measured in ossicles as a marker for bone cell differentiation, and was significantly higher in Ahsg(-/-) versus Ahsg(+/-) and/or Ahsg(+/+) mice. Ectopic ossicle size in the Ahsg(+/-) mouse was 4-fold greater than that in the wild type (Ahsg(+/+)), and intermediate to that shown in Ahsg(-/-) mouse. Bone mineral density (BMD) was lower in the Ahsg(-/+) and Ahsg(-/-) mice compared to Ahsg(+/+) littermates. The ratio of cortical to cancellous bone was found to be >2-fold higher in Ahsg(-/-) mouse in comparison to the Ahsg(+/+) mice with no significant change in the Ahsg(-/+) mouse. Finally, a significantly higher incidence of satellite ossification; small islands of immature bone, was shown in Ahsg(-/-) mice as compared to Ahsg(+/+) mice. Although Ahsg binds to TGF-beta/BMP and blocks receptor signalling, it may also sequester cytokines in matrix, thereby acting as a reservoir of osteoinductive activity when released. This may explain the non-linear relationship between ectopic bone formation characteristics and Ahsg(+/+), Ahsg(+/-) and Ahsg(-/-) genotypes, although the increase in satellite bone formation might also explain this phenomenon. Our results suggest that Ahsg may be useful for prevention of the heterotopic ossification and the regulation of osteoinductive effects of BMP used with grafts.

Dexamethasone stimulates osteogenesis in chick periosteum in vitro.

Folded periosteal explants derived from 16-day-old chick embryo calvariae differentiate and form bone when cultured for 6 days in chemically defined, hormone-supplemented medium or on plasma clots. We studied the effect of dexamethasone on generation of cells with osteoblastic phenotype in such cultures. Bone cell phenotype was evaluated by determination of alkaline phosphatase (AP) activity. Cellular proliferation was assessed by measuring ornithine decarboxylase (ODC) activity, [3H]thymidine uptake, and radioautography of cultures that had incorporated [3H]thymidine. Cultures were exposed to various medium concentrations of dexamethasone (10(-6) M, 10(-7) M, 10(-8) M) from the outset or from the second or fourth day of culture onwards. In cultures continuously exposed to dexamethasone and maintained in chemically defined media or on plasma clots, dexamethasone increased AP activity measured at day 6. This effect was maximal at 10(-7) M dexamethasone. Cultures exposed to dexamethasone after day 2 in culture also showed increased AP activity, but only in the cultures maintained on plasma clots. There was no stimulation of AP activity when dexamethasone was added at day 4 of culture with either medium, thus suggesting that the effect of glucocorticoids depends on the stage of differentiation of the cultures. In addition to AP stimulation, dexamethasone also stimulated ODC activity. Since ODC activity has been associated with mesenchymal cell proliferation, this suggested that dexamethasone stimulated the proliferation of similar cells in the cultured periostea. Measuring [3H]thymidine uptake in and performing autoradiography of control cultures and cultures treated with dexamethasone confirmed that stimulation of proliferation did occur and located this proliferation within the cell layer adjacent to the bone surface. These results demonstrate that dexamethasone stimulates in vitro osteogenesis, and that this effect appears to be mediated through stimulation of progenitor cell proliferation. In addition, our data indicate that factors in the clot medium modulate the responsiveness of the precursor cell population.

Tamoxifen attenuates the effects of exogenous glucocorticoid on bone formation and growth in piglets.

Tamoxifen (Tam) has been shown to inhibit dexamethasone (Dex)-mediated effects on bone formation in vitro. Our objective was to determine whether Tam would block Dex-induced osteopenia and growth inhibition in growing piglets. Four-day-old male Yorkshire piglets were adapted to a liquid formula diet (400 ml/kg x day) and randomized to one of four groups (n = 5/group): Dex (0.5 mg/kg x day), Tam (1 mg/kg x day), Dex plus Tam, or placebo control (vehicle only). Both drugs were administered by orogastric gavage twice daily for 12 days. At baseline and at the end of treatment, whole body bone mineral density (BMD) was determined by dual energy x-ray absorptiometry (Hologic QDR1000W). Plasma osteocalcin and PTH were measured on days 0 and 12, and urinary N-telopeptide was measured on day 12. Changes in axial length and daily weight were also measured. Delta whole body BMD was 29% lower (P < 0.05) in Dex alone treated piglets than in controls (0.033 vs. 0.047 g/cm2, respectively), whereas the maximum change in BMD in Dex plus Tam group (0.046 g/cm2) was similar to that in controls. Concurrent Tam administration reduced the Dex-induced deficit in weight gain by 56% (P < 0.05) and the deficit in axial length gain by 72% (P < 0.01). In Dex alone treated piglets, PTH was significantly elevated (7-fold), whereas osteocalcin and N-telopeptide were significantly reduced compared with control values. These effects were prevented by Tam. These data suggest that the suppression of growth and other changes in parameters of bone metabolism induced by glucocorticoids in vivo can be attenuated by Tam.

Tamoxifen attenuates glucocorticoid actions on bone formation in vitro.

Tamoxifen is a synthetic estrogen analog which may regulate osteogenesis in vivo by virtue of its antiglucocorticoid properties. We have examined tamoxifen regulation of glucocorticoid-induced osteogenesis in two different in vitro bone systems: the chicken periosteal osteogenesis model (CPO) and rat bone marrow stromal cells (RBMC). Hormone uptake studies were conducted with the osteosarcoma cell line, ROS 17/2.8. In the CPO model, alkaline phosphatase (AP) activity and collagen synthesis were stimulated by the glucocorticoid dexamethasone (Dex; 0.1 microM). These Dex-mediated effects were inhibited by increasing concentrations of tamoxifen (10-100 microM). Similarly, in the RBMC model, Dex-dependent (0.01 microM Dex) mineralized tissue formation and AP activity were blocked by tamoxifen (0.1 microM). Although tamoxifen inhibited Dex-mediated increases of AP activity in ROS 17/2.8 cells, it did not inhibit uptake of 3H-Dex or of 3H-estrogen. Northern analyses showed that tamoxifen did not affect messenger RNAs (mRNAs) for AP. Tamoxifen did seem to reduce mRNA for collagen type I, but not bone sialoprotein, osteopontin, and osteocalcin. Dex-induced increases for all proteins mRNAs in the RBMC model were not reduced by tamoxifen. Similarly, tamoxifen had no effects on cellular proliferation. We conclude that tamoxifen has no direct effect on gene expression of bone-related proteins of osteoblastic cells. Further, in the ROS 17/2.8 cell line, the antiglucocorticoid properties of tamoxifen do not appear to be mediated through either Dex or estrogen receptors.

Expression of bone sialoprotein and osteopontin in breast cancer bone metastases.

Bone sialoprotein (BSP) and osteopontin (OPN) are prominent, mineral-associated proteins in the extracellular matrix of bone that have been implicated in the metastatic activity of cancer cells. The expression of BSP, which is normally restricted to mineralizing tissues, has been observed in cancers with a high propensity for forming bone metastases. To investigate the relationship between BSP expression and the formation of bone metastases we have conducted an initial study of the expression of BSP in 10 intraductal breast carcinoma bone metastases using immunostaining and in situ hybridization, and compared the expression with OPN. The metastases were characterized by the infiltration of tumour cells into bone with extensive bone resorption evident. Moderate to strong staining for BSP was observed in all (100%) carcinomas, which also expressed BSP mRNA as determined by in situ hybridization. Variable staining for BSP was also observed in the mineralized bone and expression of BSP mRNA could be observed in osteoblastic cells on the bone surface and in some osteocytes at sites of bone remodelling. Contrary to a previous report, BSP expression could be demonstrated by PCR in three breast cancer cell lines, MCF-7, T47-D and MDA-MB-231. Moreover, in sub-cutaneous tumours formed by MDA-MB-231 breast cancer cells injected into athymic mice, higher immunostaining for BSP was seen in large ulcerating tumours in which mineral deposits were formed. In contrast to BSP, staining for OPN in bone metastases was generally restricted to the interface between tumor cells and bone surface of the carcinomas. While OPN staining was also observed in the cytoplasm of osteoclasts, which showed strong hybridization to a digoxygenin-labelled OPN cRNA probe, expression of OPN was not clearly detectable in the tumour cells. These studies provide the first demonstration of BSP expression by tumour cells in bone metastases and support the concept that BSP may have a role in targeting metastatic cells to bone. Expression of OPN in bone metastases appears to be related to increased bone resorptive activity by osteoclasts.

Effects of bisphosphonates and inorganic pyrophosphate on osteogenesis in vitro.

The bisphosphonates, which are chemically related to pyrophosphate, have been studied extensively both in vivo and in vitro to elucidate their effects on bone tissues and cells. However, because these agents have important effects on bone resorption, the majority of investigations have focused on this area. Few studies regarding direct bisphosphonate effects on bone formation have been carried out in the past and, thus, we chose to use the chick periosteal osteogenesis (CPO) in vitro model system to test the direct effects of pyrophosphate and the bisphosphonates ethane-1-hydroxy-1,1-diphosphate (HEBP) and disodium-1-hydroxy-1-amino-propylidine (APD) on various parameters of osteogenesis in vitro. The data show that the bisphosphonate HEBP inhibits bone mineralization reversibly while APD, at low doses, may actually enhance mineralization of bone. Similarly, pyrophosphate (PPi) will prevent mineralization in CPO cultures. However, CPO cultures can circumvent PPi-mediated blockage of mineralization with longer-term, continuous (10-day) incubation, whereas this does not occur if cultures are incubated continuously with bisphosphonates. Both drugs appear to be able to reverse beta-glycerophosphate-induced changes in alkaline phosphatase activity, but do not appear on their own to regulate the activity of this enzyme. The findings show that in addition to their well-known effects on resorption, bisphosphonates have significant and direct effects on mineralization in bone-forming cultures. Their direct effects on osteoblastic activity and differentiation remain to be determined.