Protocol for a randomized controlled trial to compare bone-loading exercises with risedronate for preventing bone loss in osteopenic postmenopausal women.

BACKGROUND: In the United States, over 34 million American post-menopausal women have low bone mass (osteopenia) which increases their risk of osteoporosis and fractures. Calcium, vitamin D and exercise are recommended for prevention of osteoporosis, and bisphosphonates (BPs) are prescribed in women with osteoporosis. BPs may also be prescribed for women with low bone mass, but are more controversial due to the potential for adverse effects with long-term use. A bone loading exercise program (high-impact weight bearing and resistance training) promotes bone strength by preserving bone mineral density (BMD), improving bone structure, and by promoting bone formation at sites of mechanical stress. METHODS/DESIGN: The sample for this study will be 309 women with low bone mass who are within 5 years post-menopause. Subjects are stratified by exercise history (≥2 high intensity exercise sessions per week; < 2 sessions per week) and randomized to a control or one of two treatment groups: 1) calcium + vitamin D (CaD) alone (Control); 2) a BP plus CaD (Risedronate); or 3) a bone loading exercise program plus CaD (Exercise). After 12 months of treatment, changes in bone structure, BMD, and bone turnover will be compared in the 3 groups. Primary outcomes for the study are bone structure measures (Bone Strength Index [BSI] at the tibia and Hip Structural Analysis [HSA] scores). Secondary outcomes are BMD at the hip and spine and serum biomarkers of bone formation (alkaline phosphase, AlkphaseB) and resorption (Serum N-terminal telopeptide, NTx). Our central hypothesis is that improvements in bone strength will be greater in subjects randomized to the Exercise group compared to subjects in either Control or Risedronate groups. DISCUSSION: Our research aims to decrease the risk of osteoporotic fractures by improving bone strength in women with low bone mass (pre-osteoporotic) during their first 5 years' post-menopause, a time of rapid and significant bone loss. Results of this study could be used in developing a clinical management pathway for women with low bone mass at their peak period of bone loss that would involve lifestyle modifications such as exercises prior to medications such as BPs. TRIAL REGISTRATION: Clinicaltrials.gov NCT02186600 . Initial registration: 7/7/2014.

Combined exposure to big endothelin-1 and mechanical loading in bovine sternal cores promotes osteogenesis.

Increased bone formation resulting from mechanical loading is well documented; however, the interactions of the mechanotransduction pathways are less well understood. Endothelin-1, a ubiquitous autocrine/paracrine signaling molecule promotes osteogenesis in metastatic disease. In the present study, it was hypothesized that exposure to big endothelin-1 (big ET1) and/or mechanical loading would promote osteogenesis in ex vivo trabecular bone cores. In a 2×2 factorial trial of daily mechanical loading (-2000µÎµ, 120cycles daily, "jump" waveform) and big ET1 (25ng/mL), 48 bovine sternal trabecular bone cores were maintained in bioreactor chambers for 23days. The bone cores' response to the treatment stimuli was assessed with percent change in core apparent elastic modulus (ΔEapp), static and dynamic histomorphometry, and prostaglandin E2 (PGE2) secretion. Two-way ANOVA with a post hoc Fisher's LSD test found no significant treatment effects on ΔEapp (p=0.25 and 0.51 for load and big ET1, respectively). The ΔEapp in the "no load + big ET1" (CE, 13±12.2%, p=0.56), "load + no big ET1" (LC, 17±3.9%, p=0.14) and "load + big ET1" (LE, 19±4.2%, p=0.13) treatment groups were not statistically different than the control group (CC, 3.3%±8.6%). Mineralizing surface (MS/BS), mineral apposition (MAR) and bone formation rates (BFR/BS) were significantly greater in LE than CC (p=0.037, 0.0040 and 0.019, respectively). While the histological bone formation markers in LC trended to be greater than CC (p=0.055, 0.11 and 0.074, respectively) there was no difference between CE and CC (p=0.61, 0.50 and 0.72, respectively). Cores in LE and LC had more than 50% greater MS/BS (p=0.037, p=0.055 respectively) and MAR (p=0.0040, p=0.11 respectively) than CC. The BFR/BS was more than two times greater in LE (p=0.019) and LC (p=0.074) than CC. The PGE2 levels were elevated at 8days post-osteotomy in all groups and the treatment groups remained elevated compared to the CC group on days 15, 19 and 23. The data suggest that combined exposure to big ET1 and mechanical loading results in increased osteogenesis as measured in biomechanical, histomorphometric and biochemical responses.

A comparative study of the bone metabolic response to dried plum supplementation and PTH treatment in adult, osteopenic ovariectomized rat.

Dried plum has been reported to have potent effects on bone in osteopenic animal models, but the mechanisms through which bone metabolism is altered in vivo remain unclear. To address this issue, a study comparing the metabolic response of dried plum to the anabolic agent, parathyroid hormone (PTH), was undertaken. Six month-old female Sprague Dawley rats (n=84) were sham-operated (SHAM) or ovariectomized (OVX) and maintained on a control diet for 6wks until osteopenia was confirmed. Treatments were initiated consisting of a control diet (AIN-93M) supplemented with dried plum (0, 5, 15 or 25%; w/w) or a positive control group receiving PTH. At the end of 6wks of treatment, whole body and femoral bone mineral density (BMD) were restored by the two higher doses of dried plum to the level of the SHAM group. Trabecular bone volume and cortical thickness were also improved with these two doses of dried plum. Dried plum suppressed the OVX-induced increase in bone turnover as indicated by systemic biomarkers of bone metabolism, N-terminal procollagen type 1 (P1NP) and deoxypyridinoline (DPD). Dynamic bone histomorphometric analysis of the tibial metaphysis revealed that dried plum restored the OVX-induced increase in cancellous bone formation rate (BFR) and mineralizing surface (MS/BS) to the SHAM group, but some doses of dried plum increased endocortical mineral apposition rate (MAR). As expected, PTH significantly increased endocortical MAR and BFR, periosteal BFR, and trabecular MAR and BFR beyond that of the OVX and maintained the accelerated rate of bone resorption associated with OVX. Dried plum up-regulated bone morphogenetic protein 4 (Bmp4) and insulin-like growth factor 1 (Igf1) while down-regulating nuclear factor T cell activator 1 (Nfatc1). These findings demonstrate that in the adult osteopenic OVX animal, the effects of dried plum differ from that of PTH in that dried plum primarily suppressed bone turnover with the exception of the indices of bone formation at the endocortical surface.

Apparent elastic modulus of ex vivo trabecular bovine bone increases with dynamic loading.

Although it is widely known that bone tissue responds to mechanical stimuli, the underlying biological control is still not completely understood. The purpose of this study was to validate required methods necessary to maintain active osteocytes and minimize bone tissue injury in an ex vivo three-dimensional model that could mimic in vivo cellular function. The response of 22 bovine trabecular bone cores to uniaxial compressive load was investigated by using the ZETOS bone loading and bioreactor system while perfused with culture medium for 21 days. Two groups were formed, the "treatment" group (n = 12) was stimulated with a physiological compressive strain (4000 µÎµ) in the form of a "jump" wave, while the "control" group (n = 10) was loaded only during three measurements for apparent elastic modulus on days 3, 10, and 21. At the end of the experiment, apoptosis and active osteocytes were quantified with histological analysis, and bone formation was identified by means of the calcium-binding dye, calcein. It was demonstrated that the treatment group increased the elastic modulus by 61%, whereas the control group increased by 28% (p<0.05). Of the total osteocytes observed at the end of 21 days, 1.7% (±0.3%) stained positive for apoptosis in the loaded group, whereas 2.7% (±0.4%) stained positive in the control group. Apoptosis in the center of the bone cores of both groups at the end of 21 days was similar to that observed in vivo. Therefore, the three-dimensional model used in this research permitted the investigation of physiological responses to mechanical loads on morphology adaptation of trabecular bone in a controlled defined load and chemical environment.

Smoke-induced signal molecules in bone marrow cells from altered low-density lipoprotein receptor-related protein 5 mice.

Mechanism underlying smoke-induced loss of bone mass is unknown. In this study, we hypothesized that protein signals induced by smoking in bone marrow may be associated with the loss of bone mass. Using a proteomics approach, we identified 38 proteins differentially expressed in bone marrow cells from low-density lipoprotein receptor-related protein 5 (Lrp5) mice exposed to cigarette smoking. Smoking effects on protein expression in bone marrow among three genotypes (Lrp5(+/+), Lrp5(G171V), and Lrp5(-/-)) varied. On the basis of the ratio of protein expression induced by smoking versus nonsmoking, smoke induced protein expression significantly in wild-type mice compared to the other two genotypes (Lrp5(G171V) and Lrp5(-/-)). These proteins include inhibitors of ß-catenin and proteins associated with differentiation of osteoclasts. We observed that S100A8 and S100A9 were overexpressed in human smokers compared to nonsmokers, which confirmed the effect of smoking on the expression of two proteins in Lrp5 mice, suggesting the role of these proteins in bone remodeling. Smoke induced expression of S100A8 and S100A9 in a time-dependent fashion, which was opposite of the changes in the ratio of OPG/RANKL in bone marrow cells, suggesting that the high levels of S100A8 and S100A9 may be associated with smoke-induced bone loss by increasing bone resorption.

N-acetylcysteine increases the frequency of bone marrow pro-B/pre-B cells, but does not reverse cigarette smoking-induced loss of this subset.

BACKGROUND: We previously showed that mice exposed to cigarette smoke for three weeks exhibit loss of bone marrow B cells at the Pro-B-to-pre-B cell transition, but the reason for this is unclear. The antioxidant N-acetylcysteine (NAC), a glutathione precursor, has been used as a chemopreventive agent to reduce adverse effects of cigarette smoke exposure on lung function. Here we determined whether smoke exposure impairs B cell development by inducing cell cycle arrest or apoptosis, and whether NAC treatment prevents smoking-induced loss of developing B cells. METHODOLOGY/PRINCIPAL FINDINGS: Groups of normal mice were either exposed to filtered room air or cigarette smoke with or without concomitant NAC treatment for 5 days/week for three weeks. Bone marrow B cell developmental subsets were enumerated, and sorted pro-B (B220(+)CD43(+)) and pre-B (B220(+)CD43(-)) cell fractions were analyzed for cell cycle status and the percentage of apoptotic cells. We find that, compared to sham controls, smoke-exposed mice have ∼60% fewer pro-B/pre-B cells, regardless of NAC treatment. Interestingly, NAC-treated mice show a 21-38% increase in total bone marrow cellularity and lymphocyte frequency and about a 2-fold increase in the pro-B/pre-B cell subset, compared to sham-treated controls. No significant smoking- or NAC-dependent differences were detected in frequency of apoptotic cells or the percentage cells in the G1, S, or G2 phases of the cycle. CONCLUSIONS/SIGNIFICANCE: The failure of NAC treatment to prevent smoking-induced loss of bone marrow pre-B cells suggests that oxidative stress is not directly responsible for this loss. The unexpected expansion of the pro-B/pre-B cell subset in response to NAC treatment suggests oxidative stress normally contributes to cell loss at this developmental stage, and also reveals a potential side effect of therapeutic administration of NAC to prevent smoking-induced loss of lung function.

Biological constraints that limit compensation of a common skeletal trait variant lead to inequivalence of tibial function among healthy young adults.

Having a better understanding of how complex systems like bone compensate for the natural variation in bone width to establish mechanical function will benefit efforts to identify traits contributing to fracture risk. Using a collection of pQCT images of the tibial diaphysis from 696 young adult women and men, we tested the hypothesis that bone cells cannot surmount the nonlinear relationship between bone width and whole bone stiffness to establish functional equivalence across a healthy population. Intrinsic cellular constraints limited the degree of compensation, leading to functional inequivalence relative to robustness, with slender tibias being as much as two to three times less stiff relative to body size compared with robust tibias. Using Path Analysis, we identified a network of compensatory trait interactions that explained 79% of the variation in whole-bone bending stiffness. Although slender tibias had significantly less cortical area relative to body size compared with robust tibias, it was the limited range in tissue modulus that was largely responsible for the functional inequivalence. Bone cells coordinately modulated mineralization as well as the cortical porosity associated with internal bone multicellular units (BMU)-based remodeling to adjust tissue modulus to compensate for robustness. Although anecdotal evidence suggests that functional inequivalence is tolerated under normal loading conditions, our concern is that the functional deficit of slender tibias may contribute to fracture susceptibility under extreme loading conditions, such as intense exercise during military training or falls in the elderly. Thus, we show the natural variation in bone robustness was associated with predictable functional deficits that were attributable to cellular constraints limiting the amount of compensation permissible in human long bone. Whether these cellular constraints can be circumvented prophylactically to better equilibrate function among individuals remains to be determined.

The synthesis of a multiblock osteotropic polyrotaxane by copper(I)-catalyzed huisgen 1,3-dipolar cycloaddition.

The design and synthesis of a novel bone-targeting polyrotaxane delivery system that utilizes alendronate (ALN) as targeting moiety is presented in this manuscript. For the introduction of ALN, it is first conjugated to α-cyclodextrin (α-CD) and subsequently threaded onto a short poly(ethylene glycol) (PEG) chain, forming a pseudopolyrotaxane. Using click chemistry, this assembly is copolymerized with bulky monomers that bear imaging and/or therapeutic agent(s) to prevent ALN-functionalized α-CD from dethreading. Overall bone affinity of this novel polymer conjugate can be easily controlled by changing the number of ALN-α-CD incorporated. The osteotropicity of the delivery system was also confirmed in vivo.

Cigarette smoke-induced effects on bone marrow B-cell subsets and CD4+:CD8+ T-cell ratios are reversed by smoking cessation: influence of bone mass on immune cell response to and recovery from smoke exposure.

Cigarette smoking adversely affects the immune system, and is a risk factor for developing osteoporosis. How smoking contributes to osteoporosis is unclear, but since lymphocytes help maintain bone homeostasis and lymphocyte depletion results in bone loss, one potential mechanism for how smoke exposure promotes osteoporosis is by reducing bone marrow lymphocytes. Since the risk for developing osteoporosis is reportedly greater in smokers with polymorphisms in LRP5, a gene involved in canonical Wnt signaling that regulates bone metabolism, smoking-induced effects on lymphocytes may be influenced by Lrp5 functionality. To test these possibilities, we examined how the duration and cessation of cigarette smoke exposure affects lymphocyte distribution and function in normal mice and mice predisposed to low or high bone mass due to disruption or mutation of Lrp5. We find that, independent of genotype, mice exposed to cigarette smoke for 3-12 weeks showed a significant reduction in bone marrow B220(+)CD43(-) B cells and splenic transitional T1 B cells, and exhibited a splenic CD4(+):CD8(+) T-cell ratio that was skewed toward CD8(+) T cells. Smoke exposure had little or no effect on other lymphocyte subsets or on lymphocyte function ex vivo. Interestingly, these differences were no longer apparent after 6 weeks without smoke exposure, except in mice with high bone mass where bone marrow B220(+)CD43(-) B cells failed to fully recover. These data provide the first evidence that smoke exposure reduces bone marrow B cells, providing a plausible mechanism for how smoking contributes to osteoporosis.

Effects of glucocorticoid treatment on bone strength.

Glucocorticoids (GCs) are prescribed for the treatment of several diseases, but their long-term use causes osteoporosis. Current research suggests that GCs suppress the canonical Wnt/beta pathway, resulting in decreased expression of critical bone proteins. This study examined how bone structure and strength of high bone mass (HBM) mice and low density lipoprotein receptor-related protein 5 (LRP5) knockout (KO+/-) mice are affected by GC treatment in comparison to wild-type (WT) mice, and if changes were specific to either trabecular or cortical bone. Mice were treated with either prednisone or placebo. The femurs and L4 vertebral bodies were analyzed by micro-CT for structure and mechanically tested to determine strength and apparent material strength properties. Differences in all measured variables corresponding to GC treatment and genotype were tested using two-way ANOVA. GC treatment caused decreased structural strength parameters, weakened apparent material strength properties, and disruption of bone structure in HBM, but not LRP5+/- or WT, mice. Despite treatment-related loss, trabecular bone structure and strength remained elevated as compared to LRP5+/- and WT mice. In HBM femurs, both cortical and trabecular structure, but not strength parameters, were negatively affected by treatment. In HBM vertebral bodies, both structural and strength parameters were negatively affected by treatment.

Induction of osteopontin expression by nicotine and cigarette smoke in the pancreas and pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease with etiological association with cigarette smoking. Nicotine, an important component of cigarettes, exists at high concentrations in the bloodstream of smokers. Osteopontin (OPN) is a secreted phosphoprotein that confers on cancer cells a migratory phenotype and activates signaling pathways that induce cell survival, proliferation, invasion, and metastasis. Here, we investigated the potential molecular basis of nicotine's role in PDA through studying its effect on OPN. Nicotine significantly (p < 0.02) increased OPN mRNA and protein secretion in PDA cells through activation of the OPN gene promoter. The OPN mRNA induction was inhibited by the nicotinic acetylcholine receptor antagonist, mechamylamine. Further, the tyrosine kinase inhibitor genistein inhibited the nicotine-mediated induction of OPN, suggesting that mitogen activated protein kinase signaling mechanism is involved. Nicotine activated the phosphorylation of ERK1/2, but not p38 or c-Jun NH2-terminal MAP kinases. Inhibition of ERK1/2 activation reduced the nicotine-induced OPN synthesis. Rats exposed to cigarette smoke showed a dose-dependent increase in pancreatic OPN that paralleled the rise of pancreatic and plasma nicotine levels. Analysis of cancer tissue from invasive PDA patients, the majority of whom were smokers, showed the presence of significant amounts of OPN in the malignant ducts and the surrounding pancreatic acini. Our data suggest that nicotine may contribute to PDA pathogenesis through upregulation of OPN. They provide the first insight into a nicotine-initiated signal transduction pathway that regulates OPN as a possible tumorigenic mechanism in PDA.

Vitamin D3 distribution and status in the body.

OBJECTIVE: To estimate the amount, type, and tissue distribution of vitamin D in the adult body under typical inputs. METHODS: Review and reanalysis of published measurements and analysis of tissue samples from growing pigs raised in confinement on diets providing about 2000 IU vitamin D/day. Cholecalciferol and 25-hydroxyvitamin D [25(OH)D] concentration measured by HPLC. RESULTS: Mean serum 25(OH)D in all studies combined was 45 nmol/L. At the level of vitamin D repletion represented by this concentration, total body vitamin D would be 14,665 IU for a 70 kg adult woman. 65% of this total was present as native cholecalciferol and 35% as 25(OH)D. Nearly three-quarters of the cholecalciferol was in fat, while 25(OH)D was more evenly distributed throughout the body (20% in muscle, 30% in serum, 35% in fat, and 15% in all other tissues). At the daily vitamin D consumption rates in these animals total body stores provided only a approximately 7-day reserve. CONCLUSIONS: At total intakes on the order of 2000 IU/day, an adult has very little vitamin D reserve, despite intakes 10x the current recommendations. Those recommended inputs need to be increased by at least an order of magnitude. Food tables that fail to take into account 25(OH)D content of various meat products lead to underestimation of dietary vitamin D intake.

Calcium and vitamin d supplementation decreases incidence of stress fractures in female navy recruits.

INTRODUCTION: Stress fractures (SFx) are one of the most common and debilitating overuse injuries seen in military recruits, and they are also problematic for nonmilitary athletic populations. The goal of this randomized double-blind, placebo-controlled study was to determine whether a calcium and vitamin D intervention could reduce the incidence of SFx in female recruits during basic training. MATERIALS AND METHODS: We recruited 5201 female Navy recruit volunteers and randomized them to 2000 mg calcium and 800 IU vitamin D/d or placebo. SFx were ascertained when recruits reported to the Great Lakes clinic with symptoms. All SFx were confirmed with radiography or technetium scan according to the usual Navy protocol. RESULTS: A total of 309 subjects were diagnosed with a SFx resulting in an incidence of 5.9% per 8 wk. Using intention-to-treat analysis by including all enrolled subjects, we found that the calcium and vitamin D group had a 20% lower incidence of SFx than the control group (5.3% versus 6.6%, respectively, p = 0.0026 for Fisher's exact test). The per protocol analysis, including only the 3700 recruits who completed the study, found a 21% lower incidence of fractures in the supplemented versus the control group (6.8% versus 8.6%, respectively, p = 0.02 for Fisher's exact test). CONCLUSIONS: Generalizing the findings to the population of 14,416 women who entered basic training at the Great Lakes during the 24 mo of recruitment, calcium and vitamin D supplementation for the entire cohort would have prevented approximately 187 persons from fracturing. Such a decrease in SFx would be associated with a significant decrease in morbidity and financial costs.

The effect of local simvastatin delivery strategies on mandibular bone formation in vivo.

Systemic simvastatin is known to reduce cholesterol and stimulate modest bone formation, but local surgical placement in polylactic acid domes causes robust bone formation and local swelling. A less invasive and more flexible injection protocol was studied to evaluate the bone-inducing effects compared to surgical implantation. Bone formation rate, short- and long-term bone augmentation histology, and mechanical properties were evaluated to characterize the new bone in a rat bilateral mandible model (test and control sides in same animal). Results demonstrated that multiple (3) injections of 0.5 mg simvastatin effectively reduced soft tissue swelling while preserving bone growth (60% increase of bone width at 24 days) compared to simvastatin dome placement (43% increase at 24 days). Compared to controls, bone formation rate was significantly higher on the simvastatin side, especially in the dome. Three-point bending tests revealed higher maximum force to fracture and stiffness at 24 days with simvastatin injections. Long-term evaluation showed that 55% of maximum new bone formed 24 days post-injection was retained at 90 days.

Osteotropic beta-cyclodextrin for local bone regeneration.

An osteotropic alendronate-beta-cyclodextrin conjugate (ALN-beta-CD) was developed as a bone-targeting delivery system for improved treatment of skeletal diseases. The conjugate shows very strong binding to hydroxyapatite (HA, main component of the skeleton). Its ability in forming molecular inclusion complex with prostaglandin E(1) (PGE(1), a potent bone anabolic agent) was confirmed by phase solubility experiments and differential scanning calorimetry (DSC). In a bilateral rat mandible model, ALN-beta-CD/PGE(1) molecular complex was shown to stimulate strong local bone anabolic reaction. In the control study, ALN-beta-CD itself was also found to be bone anabolic. To investigate this finding, other control groups were studied. The histomorphometry data suggest that ALN-beta-CD itself could generate more new bone at the injection site than its complex with PGE(1). Alendronate (ALN) injection could also cause new bone formation, which locates peripheral to the site of injection. PGE(1), saline or ethanol injections do not have anabolic effect. These findings were also confirmed by micro-CT evaluation of mandibular bones. It is clear that the bone anabolic effect of ALN-beta-CD is independent of mechanical stimuli of the periosteum or ALN injection alone. Further studies are warranted to understand the working mechanism of ALN-beta-CD as a bone anabolic agent.

Cigarette smoke-induced differential expression of the genes involved in exocrine function of the rat pancreas.

OBJECTIVE: Little is known about the molecular and biological aspects of the epidemiological association between smoking and pancreatic pathology, such as chronic pancreatitis and pancreatic cancer. Recently, we reported that tobacco smoke exposure induced morphological alterations in the rat pancreas. Here, we have investigated the alterations in the expression of genes associated with exocrine pancreatic function and cellular differentiation upon exposure to cigarette smoke. METHODS: Female rats were exposed to environmental smoke inhalation for 2 d/wk (70 min/d) for 12 weeks. The expression profiles of trypsinogen, pancreas-specific trypsin inhibitor, cholecystokinin A receptor, cystic fibrosis transmembrane conductance regulator (CFTR), carbonic anhydrase, and Muc1 and Muc4 mucins transcripts were analyzed by RNA slot blot analysis. Muc4 expression was also examined by immunohistochemistry. RESULTS: Our data revealed that the ratio of trypsinogen to that of the protective pancreas-specific trypsin inhibitor was elevated upon cigarette smoke exposure. The expression of carbonic anhydrase and CFTR remained unaltered when inflammatory signs were not detected in histological examinations. On the other hand, when pancreatic inflammation was present, the levels of CFTR and carbonic anhydrase were increased, indicating ductal and/or centroacinar cell involvement. No changes in the expression of Muc1 and Muc4 mucins were observed. CONCLUSIONS: Our data show that cigarette smoke exposure leads to an increased vulnerability to pancreatic self-digestion. Moreover, the concomitant involvement of pancreatic ducts occurs only when focal pancreatic inflammation is present.

Wnt/beta-catenin signaling is a normal physiological response to mechanical loading in bone.

A preliminary expression profiling analysis of osteoblasts derived from tibia explants of the high bone mass LRP5 G171V transgenic mice demonstrated increased expression of canonical Wnt pathway and Wnt/beta-catenin target genes compared with non-transgenic explant derived osteoblasts. Therefore, expression of Wnt/beta-catenin target genes were monitored after in vivo loading of the tibia of LRP5 G171V transgenic mice compared with non-transgenic mice. Loading resulted in the increased expression of Wnt pathway and Wnt/beta-catenin target genes including Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43 in both genotypes; however, there was a further increased in transcriptional response with the LRP5 G171V transgenic mice. Similar increases in the expression of these genes (except cyclin D1) were observed when non-transgenic mice were pharmacologically treated with a canonical Wnt pathway activator, glycogen synthase kinase 3beta inhibitor and then subjected to load. These in vivo results were further corroborated by in vitro mechanical loading experiments in which MC3T3-E1 osteoblastic cells were subjected to 3400 microstrain alone for 5 h, which increased the expression of Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43. Furthermore, when MC3T3-E1 cells were treated with either glycogen synthase kinase 3beta inhibitor or Wnt3A to activate Wnt signaling and then subjected to load, a synergistic up-regulation of these genes was observed compared with vehicle-treated cells. Collectively, the in vivo and in vitro mechanical loading results support that Wnt/beta-catenin signaling is a normal physiological response to load and that activation of the Wnt/beta-catenin pathway enhances the sensitivity of osteoblasts/osteocytes to mechanical loading.

Chronic pancreatic inflammation induced by environmental tobacco smoke inhalation in rats.

OBJECTIVE: Despite a strong epidemiological association between cigarette smoking and pancreatic diseases, such as pancreatic cancer and chronic pancreatitis, the effects of long-term cigarette smoke inhalation on the pancreas have not been clearly determined. In the present study, we investigated the effect of cigarette smoke inhalation on the pancreas. METHODS: Thirty-six female Sprague Dawley rats were exposed to two different doses of environmental tobacco smoke averaging 100 mg or 160 mg/m3 total suspended particulate matter (TSP) per m3 for 70 min twice a day for 12 wk. The animals were sacrificed and examined for the effects of tobacco smoke exposure on pancreatic morphology and function. RESULTS: In 58% (7/12) of the animals, exposure to 160 mg/m3 TSP cigarette smoke induced a chronic pancreatic inflammatory process with fibrosis and scarring of pancreatic acinar structures. Animals with fibrotic alterations showed an induction of pancreatic pro-collagen 1 gene expression, and the infiltration of immune cells was accompanied by the expression of the inflammatory mediators MIP-1alpha, IL-1beta, and TGF-beta in 33% (4/12) of the animals. Acinar cell stress was manifested by a significant up-regulation of pancreatitis-associated protein expression (PAP) in smoke-exposed animals (smoke-exposed 6,932 +/- 1,236 vs control 3,608 +/- 305, p < 0.05). Possibly contributing to the morphological damage to the exocrine pancreas, the inhalation of cigarette smoke induced trypsinogen and chymotrypsinogen gene expression and, furthermore, reduced pancreatic enzyme content. CONCLUSIONS: This study provides experimental evidence of morphological pancreatic damage induced by the inhalation of cigarette smoke, which is likely to be mediated by alterations of acinar cell function.

Local simvastatin effects on mandibular bone growth and inflammation.

BACKGROUND: Simvastatin has been shown to increase bone growth when applied topically to murine bone; however, it causes considerable soft tissue inflammation at high doses (2.2 mg), making future clinical use problematic. This study evaluated the effect of lower simvastatin doses and cyclooxygenase (COX) synthase inhibitors on tissue inflammation and bone growth in rats and gene expression in mice. METHODS: Adult female rats were untreated or treated with a single dose of 0.1, 0.5, 1.0, 1.5, or 2.2 mg simvastatin in methylcellulose gel in a polylactic acid membrane (SIM) on the lateral aspect of the mandible. The contralateral mandible side was implanted with methylcellulose gel/polylactic acid membrane alone (GEL), and five rats in each dose pairing were evaluated histomorphometrically after 3, 7, and 24 days. Subsequent rats were similarly treated with 0.5 mg simvastatin (optimal dose) and daily intraperitoneal injections of COX-2 inhibitor (NS-398; 1 mg/kg x 7 days; N = 16), general COX inhibitor (indomethacin; 1 mg/kg x 7 days; N = 16), or no inhibitor (N = 10) and evaluated histomorphometrically after 7 or 24 days by analysis of variance (ANOVA). Gene arrays were also used to evaluate osteogenic gene expression from 0.5 mg simvastatin in murine calvaria (N = 12). RESULTS: There was a 45% increase in bone area with 0.5 mg simvastatin versus gel control (P <0.001; similar to the 2.2-mg dose), and clinical swelling was reduced compared to the high simvastatin dose (P <0.05). The 0.1-mg simvastatin dose failed to stimulate significant bone growth. NS-398 and indomethacin reduced inflammation and bone growth. Simvastatin significantly upregulated procollagen, fibronectin, and matrix metalloproteinase-13 genes. CONCLUSION: Reducing the simvastatin dose from 2.2 to 0.5 mg reduced inflammation to a more clinically acceptable level without sacrificing bone-growth potential, but COX-associated inflammation appears to be necessary for in vivo bone growth.

Comparative effect of soy protein, soy isoflavones, and 17beta-estradiol on bone metabolism in adult ovariectomized rats.

UNLABELLED: This study provided a comprehensive investigation on the effect of soy protein and soy isoflavones on both calcium and bone metabolism in virgin adult rats. The measurements included bone histology, calcium kinetic modeling, calcium balance, bone densitometry, and whole body densitometry. Results confirmed the bone-preserving effect of estrogen but did not support a bone-sparing role of soy isoflavones. INTRODUCTION: Several animal and short-term human studies have indicated that soy protein isolate enriched with isoflavones may be used as an alternative therapy to estrogen replacement therapy. However, none of the previous studies have investigated this estrogenic effect on both calcium and bone metabolism in animals or humans, which is essential in ascertaining the mode of action of isoflavones. MATERIALS AND METHODS: This study was designed to determine the effects of soy protein versus isoflavones on calcium and bone metabolism in an ovariectomized rat model. Unmated 6-month-old ovariectomized and sham-operated female Sprague-Dawley rats were randomly assigned to nine groups (16 rats/group) and pair-fed soy- or casein-based diets with or without isoflavones for 8 weeks. A reference group was administered estrogen through subcutaneous implants (20-35 pg/liter plasma). Bone densitometry, histomorphometry, and mechanical testing were used to study bone metabolism and quality. Calcium metabolism was studied using calcium tracer balance and kinetics. RESULTS: After ovariectomy, estrogen prevented bone loss in trabecular bone and suppressed formation on both trabecular and cortical bone surfaces. Isoflavones given as enriched soy protein isolate or supplements did not prevent trabecular bone loss. Combining isoflavones with estrogen had no additional benefits over estrogen alone. There were no differences in response to isoflavones caused by protein source. None of the treatments significantly affected either total Ca balance or (45)Ca absorption. However, soy protein showed significant effects on reducing urinary loss of Ca in animals, irrespective of isoflavone level, perhaps because of the lower amount of sulfur-containing amino acids in soy protein. CONCLUSION: Estrogen, but not isoflavones at the levels tested, suppressed bone remodeling in both trabecular and cortical bone after ovariectomy.