Over-expression of TIMP-1 in osteoblasts increases the anabolic response to PTH

Intermittent PTH treatment induces structural changes that affect cancellous bone mass and have led to its indication for the treatment of osteoporosis. PTH is also known to upregulate the expression of matrix metalloproteinases (MMP) in osteoblasts. We wanted to find out whether inhibiting osteoblastic MMPs can affect the anabolic action of PTH in vivo. We had shown previously that mice over-expressing TIMP-1 (tissue inhibitor of MMPs) specifically in osteoblasts display an increase in bone mineral density and bone mass combined with an overall decrease in bone turnover. In the present study, 10-week-old wild-type (WT) and transgenic (TG) mice were treated with PTH at 40 microg/kg/day for 1.5 months. DEXA analysis was performed before and after treatment, and histomorphometric and molecular analysis were carried out at the end of the experiment. Our findings indicate that the transgene boosted the anabolic action of PTH. The femurs of PTH-treated TG mice displayed a greater increase in bone mineral density and trabecular bone volume than treated WT mice. Interestingly, the positive effect of the transgene on the action of PTH resulted from both reduced bone resorption activity and an increase in the bone formation rate. Osteoclastic surfaces that were increased in PTH-treated WT mice remained unchanged in TG mice, suggesting a decrease in osteoclastic differentiation. Histomorphometric data also indicate that PTH administration increased osteoblast activity in TG mice and affected the number of osteoblasts in WT mice. In conclusion, we demonstrate that inhibiting osteoblastic MMPs can potentiate the anabolic effect of PTH by decreasing osteoclast activity and increasing osteoblast activity. Our data also suggest that osteoblastic MMPs have some role in mediating the anabolic effects of PTH in vivo and indicate that inhibitors of MMPs could constitute a new therapy for degenerative diseases.     

Constrained tibial vibration does not produce an anabolic bone response in adult mice

Osteoporosis is characterized by low bone mass and increased fracture risk. High frequency, low-amplitude whole-body vibration (WBV) has been proposed as a treatment for osteoporosis because it can stimulate new bone formation and prevent trabecular bone loss. We developed constrained tibial vibration (CTV) as a method for controlled vibrational loading of the lower leg of a mouse. We first subjected mice to five weeks of daily CTV loading (0.5 G maximum acceleration) with loading parameters chosen to independently investigate the effects of strain magnitude, loading frequency, and cyclic acceleration on the adaptive response to vibration. We hypothesized that mice subjected to the highest magnitude of dynamic strain would have the largest bone formation response. We observed a slight, local benefit of CTV loading on trabecular bone, as BV/TV was 5.2% higher in the loaded vs. non-loaded tibia of mice loaded with the highest bone strain magnitude. However, despite these positive differences, we observed significantly lower measures of trabecular structure in both loaded and non-loaded tibias from CTV loaded mice compared to Sham and Baseline Control animals, indicating a negative systemic effect of CTV on trabecular bone. Based on this evidence, we conducted a follow-up study wherein mice were subjected to CTV or sham loading, and tibias were scanned at the beginning and end of the study period using in vivo microCT. Consistent with the findings of the first study, trabecular BV/TV in both tibias of CTV loaded and Sham mice was, on average, 36% and 31% lower on day 36 than day 0, respectively, compared to 20% lower in Age-Matched Controls over the same time period. Contrary to the first study, there were no differences between loaded and non-loaded tibias in CTV loaded mice, providing no evidence for a local benefit of CTV. In summary, 5 weeks of daily CTV loading of mice was, at best, weakly anabolic for trabecular bone in the proximal tibia, while daily handling and exposure to anesthesia was associated with significant loss of trabecular and cortical bone. We conclude that direct vibrational loading of bone in anesthetized, adult mice is not anabolic.     

Low‐magnitude whole‐body vibration does not enhance the anabolic skeletal effects of intermittent PTH in adult mice

Whole‐body vibration (WBV) is a low‐magnitude mechanical stimulus that may be anabolic for bone, yet we recently found that WBV did not improve bone properties in adult mice. Because intermittent parathyroid hormone (PTH) enhances the anabolic effects of high‐magnitude skeletal loading, we sought to determine the skeletal effects of WBV in combination with PTH. Seven‐month‐old male BALB/c mice were assigned to six groups (n = 13–14/group) based on magnitude of applied acceleration (0 or 0.3 G) and PTH dose (0, 10, or 40 µg/kg/day). Mice were exposed to WBV (0.3 G, 90 Hz, sine wave) or sham loading (0 G) for 15 min/day, 5 days/week for 8 weeks. Vehicle or hPTH (1–34) was administered prior to each WBV session. Whole‐body bone mineral content increased by ～5% from 0 to 8 weeks in the 40 µg/kg PTH group only, independent of WBV loading. Similarly, PTH treatment increased tibial cortical bone volume by ～5% from 0 to 8 weeks, independent of WBV loading. Neither PTH nor WBV stimulated trabecular bone formation. Consistent with the cortical bone effect, tibias from the 40 µg/kg PTH group had significantly greater ultimate force and energy to failure than tibias in the 0 and 10 µg/kg PTH groups, independent of WBV treatment. In summary, 8 weeks of intermittent PTH treatment increased cortical bone volume and strength in adult male BALB/c mice. Daily exposure to low‐magnitude WBV by itself did not improve skeletal properties and did not enhance the PTH effect. No WBV‐PTH synergy was found in this preclinical study. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:465–472, 2011     

Disruption of LRP6 in osteoblasts blunts the bone anabolic activity of PTH

Mutations in low‐density lipoprotein receptor‐related protein 6 (LRP6) are associated with human skeletal disorders. LRP6 is required for parathyroid hormone (PTH)‐stimulated signaling pathways in osteoblasts. We investigated whether LRP6 in osteoblasts directly regulates bone remodeling and mediates the bone anabolic effects of PTH by specifically deleting LRP6 in mature osteoblasts in mice (LRP6 KO). Three‐month‐old LRP6 KO mice had a significant reduction in bone mass in the femora secondary spongiosa relative to their wild‐type littermates, whereas marginal changes were found in femoral tissue of 1‐month‐old LRP6 KO mice. The remodeling area of the 3‐month‐old LRP6 KO mice showed a decreased bone formation rate as detected by Goldner's Trichrome staining and calcein double labeling. Bone histomorphometric and immumohistochemical analysis revealed a reduction in osteoblasts but little change in the numbers of osteoclasts and osteoprogenitors/osteoblast precursors in LRP6 KO mice compared with wild‐type littermates. In addition, the percentage of the apoptotic osteoblasts on the bone surface was higher in LRP6 KO mice compared with wild‐type littermates. Intermittent injection of PTH had no effect on bone mass or osteoblastic bone formation in either trabecular and cortical bone in LRP6 KO mice, whereas all were enhanced in wild‐type littermates. Additionally, the anti‐apoptotic effect of PTH on osteoblasts in LRP6 KO mice was less significant compared with wild‐type mice. Therefore, our findings demonstrate that LRP6 in osteoblasts is essential for osteoblastic differentiation during bone remodeling and the anabolic effects of PTH. © 2013 American Society for Bone and Mineral Research.     

Combined treatment with a beta-blocker and intermittent PTH improves bone mass and microarchitecture in ovariectomized mice

Intermittent administration of parathyroid hormone (PTH) induces bone remodeling and renewed bone modeling, resulting in net bone gain. beta-blockers improve trabecular bone architecture in young ovariectomized mice by preventing the inhibition of bone formation and stimulation of bone resorption induced by the adrenergic system. To test the hypothesis that PTH and beta-blockers may exert synergistic effects on the skeleton, 15-week-old ovariectomized mice were either given oral propranolol (PRO) or left untreated for 8 weeks, adding daily hPTH(1-34) (80 microg/kg/day) or vehicle (VEH) during the last 4 weeks. The skeletal response was evaluated using pDXA, microCT, histomorphometry and biochemical markers. PRO significantly attenuated loss of bone mineral density (BMD) at whole body (WB) (-0.1% in PRO vs. -2.4% in VEH, P < 0.05), but not at spine or femur 4 weeks after OVX. Thereafter, PTH increased BMD at all sites in both PRO- and VEH-treated mice (+6.7% to +14%, P < 0.05 to P < 0.0001 vs. VEH). Over 8 weeks, sequential-combined treatment of PRO and PTH significantly improved BMD over PTH alone at WB (+9.1% vs. +4.4% over baseline, respectively, P < 0.005) and spine (+9% vs. -1.7%, respectively, P < 0.05). These effects were paralleled by a decrease in TRACP5b with PRO (P < 0.05 vs. VEH) and an increase in osteocalcin with PTH, irrespective of PRO (P < 0.0001 vs. VEH). Trabecular bone microarchitecture, such as BV/TV, trabecular number and ConnD, was significantly improved by sequential-combined treatment of PRO and PTH compared to PTH alone. At midshaft femur, both PRO and PTH significantly increased cross-sectional area (CSA), but the effects of the two drugs on CSA and cortical thickness were not additive. Dynamic histomorphometry indicated that bone formation was increased by PTH at both cortical and trabecular surfaces, whereas PRO increased osteoblast number and surface on trabecular surfaces. The combined treatment further improved the extent of mineralization and BFR over PTH alone (P < 0.05) at endocortical surfaces and recapitulated the effects of PTH and PRO alone on trabecular surfaces. These results indicate that beta-adrenergic blockade may partially improve the bone remodeling balance induced by estrogen deficiency. In turn, PRO exerted synergistic effects with intermittent PTH on bone mass and cancellous bone architecture. As such, combined therapy of beta-blockers and PTH may be of interest in the treatment of postmenopausal osteoporosis.     

Bmp2 conditional knockout in osteoblasts and endothelial cells does not impair bone formation after injury or mechanical loading in adult mice

Post-natal osteogenesis after mechanical trauma or stimulus occurs through either endochondral healing, intramembranous healing or lamellar bone formation. Bone morphogenetic protein 2 (BMP2) is up-regulated in each of these osteogenic processes and is expressed by a variety of cells including osteoblasts and vascular cells. It is known that genetic knockout of Bmp2 in all cells or in osteo-chondroprogenitor cells completely abrogates endochondral healing after full fracture. However, the importance of BMP2 from differentiated osteoblasts and endothelial cells is not known. Moreover, the importance of BMP2 in non-endochondral bone formation such as intramembranous healing or lamellar bone formation is not known. Using inducible and tissue-specific Cre-lox mediated targeting of Bmp2 in adult (10–24 week old) mice, we assessed the role of BMP2 expression globally, by osteoblasts, and by vascular endothelial cells in endochondral healing, intramembranous healing and lamellar bone formation. These three osteogenic processes were modeled using full femur fracture, ulnar stress fracture, and ulnar non-damaging cyclic loading, respectively. Our results confirmed the requirement of BMP2 for endochondral fracture healing, as mice in which Bmp2 was knocked out in all cells prior to fracture failed to form a callus. Targeted deletion of Bmp2 in osteoblasts (osterix-expressing) or vascular endothelial cells (vascular endothelial cadherin-expressing) did not impact fracture healing in any way. Regarding non-endochondral bone formation, we found that BMP2 is largely dispensable for intramembranous bone formation after stress fracture and also not required for lamellar bone formation induced by mechanical loading. Taken together our results indicate that osteoblasts and endothelial cells are not a critical source of BMP2 in endochondral fracture healing, and that non-endochondral bone formation in the adult mouse is not as critically dependent on BMP2.     

beta-Arrestin2 regulates the differential response of cortical and trabecular bone to intermittent PTH in female mice.

Cytoplasmic arrestins regulate PTH signaling in vitro. We show that female beta-arrestin2(-/-) mice have decreased bone mass and altered bone architecture. The effects of intermittent PTH administration on bone microarchitecture differed in beta-arrestin2(-/-) and wildtype mice. These data indicate that arrestin-mediated regulation of intracellular signaling contributes to the differential effects of PTH at endosteal and periosteal bone surfaces. INTRODUCTION: The effects of PTH differ at endosteal and periosteal surfaces, suggesting that PTH activity in these compartments may depend on some yet unidentified mechanism(s) of regulation. The action of PTH in bone is mediated primarily by intracellular cAMP, and the cytoplasmic molecule beta-arrestin2 plays a central role in this signaling regulation. Thus, we hypothesized that arrestins would modulate the effects of PTH on bone in vivo. MATERIALS AND METHODS: We used pDXA, muCT, histomorphometry, and serum markers of bone turnover to assess the skeletal response to intermittent PTH (0, 20, 40, or 80 mug/kg/day) in adult female mice null for beta-arrestin2 (beta-arr2(-/-)) and wildtype (WT) littermates (7-11/group). RESULTS AND CONCLUSIONS: beta-arr2(-/-) mice had significantly lower total body BMD, trabecular bone volume fraction (BV/TV), and femoral cross-sectional area compared with WT. In WT females, PTH increased total body BMD, trabecular bone parameters, and cortical thickness, with a trend toward decreased midfemoral medullary area. In beta-arr2(-/-) mice, PTH not only improved total body BMD, trabecular bone architecture, and cortical thickness, but also dose-dependently increased femoral cross-sectional area and medullary area. Histomorphometry showed that PTH-stimulated periosteal bone formation was 2-fold higher in beta-arr2(-/-) compared with WT. Osteocalcin levels were significantly lower in beta-arr2(-/-) mice, but increased dose-dependently with PTH in both beta-arr2(-/-) and WT. In contrast, whereas the resorption marker TRACP5B increased dose-dependently in WT, 20-80 mug/kg/day of PTH was equipotent with regard to stimulation of TRACP5B in beta-arr2(-/-). In summary, beta-arrestin2 plays an important role in bone mass acquisition and remodeling. In estrogen-replete female mice, the ability of intermittent PTH to stimulate periosteal bone apposition and endosteal resorption is inhibited by arrestins. We therefore infer that arrestin-mediated regulation of intracellular signaling contributes to the differential effects of PTH on cancellous and cortical bone.     

Antecedent hindbrain glucoprivation does not impair the counterregulatory response to hypoglycemia

Recurrent hypoglycemia impairs hormonal counterregulatory responses (CRRs) to further bouts of hypoglycemia. The hypothalamus and hindbrain are both critical for sensing hypoglycemia and triggering CRRs. Hypothalamic glucose sensing sites are implicated in the pathogenesis of defective CRRs; however, the contribution of hindbrain glucose sensing has not been elucidated. Using a rat model, we compared the effect of antecedent glucoprivation targeting hindbrain or hypothalamic glucose sensing sites with the effect of antecedent recurrent hypoglycemia on CRR to hypoglycemia induced 24 h later. Recurrent hypoglycemia decreased sympathoadrenal (1,470 +/- 325 vs. 3,811 +/- 540 pg/ml in controls [t = 60 min], P = 0.001) and glucagon secretion (222 +/- 43 vs. 494 +/- 56 pg/ml in controls [t = 60]), P = 0.003) in response to hypoglycemia. Antecedent 5-thio-glucose (5TG) injected into the hindbrain did not impair sympathoadrenal (3,806 +/- 344 pg/ml [t = 60]) or glucagon (513 +/- 56 pg/ml [t = 60]) responses to subsequent hypoglycemia. However, antecedent 5TG delivered into the third ventricle was sufficient to blunt CRRs to hypoglycemia. These results show that hindbrain glucose sensing is not involved in the development of defective CRRs. However, neural substrates surrounding the third ventricle are particularly sensitive to glucoprivic stimulation and may contribute importantly to the development of defective CRRs.     

Bone mass is inversely proportional to Dkk1 levels in mice

The Wnt/beta-catenin signaling pathway has emerged as a key regulator in bone development and bone homeostasis. Loss-of-function mutations in the Wnt co-receptor LRP5 result in osteoporosis and "activating" mutations in LRP5 result in high bone mass. Dickkopf-1 (DKK1) is a secreted Wnt inhibitor that binds LRP5 and LRP6 during embryonic development, therefore it is expected that a decrease in DKK1 will result in an increase in Wnt activity and a high bone mass phenotype. Dkk1-/- knockout mice are embryonic lethal, but mice with hypomorphic Dkk1d (doubleridge) alleles that express low amounts of Dkk1 are viable. In this study we generated an allelic series by crossing Dkk1+/- and Dkk1+/d mice resulting in the following genotypes with decreasing Dkk1 expression levels: +/+, +/d, +/- and d/-. Using muCT imaging we scanned dissected left femora and calvariae from 8-week-old mice (n=60). We analyzed the distal femur to represent trabecular bone and the femur diaphysis for cortical endochondral bone. A region of the parietal bones was used to analyze intramembranous bone of the calvaria. We found that trabecular bone volume is increased in Dkk1 mutant mice in a manner that is inversely proportional to the level of Dkk1 expression. Trabeculae number and thickness were significantly higher in the low Dkk1 expressing genotypes from both female and male mice. Similar results were found in cortical bone with an increase in cortical thickness and cross sectional area of the femur diaphysis that correlated with lower Dkk1 expression. No consistent differences were found in the calvaria measurements. Our results indicate that the progressive Dkk1 reduction increases trabecular and cortical bone mass and that even a 25% reduction in Dkk1 expression could produce significant increases in trabecular bone volume fraction. Thus DKK1 is a negative regulator of normal bone homeostasis in vivo. Our study suggests that manipulation of DKK1 function or expression may have therapeutic significance for the treatment of low bone mass disorders.     

The role of Dkk1 in bone mass regulation: Correlating serum Dkk1 expression with bone mineral density

The Wnt/β‐catenin pathway is a major signaling cascade in bone biology, playing a key role in regulating bone development and remodeling, with aberrations in signaling resulting in disturbances in bone mass. The objectives of our study were to correlate serum Dkk1 expression with bone mineral density (BMD) and assess the potential role of Dkk1 as a serological marker of bone mass. Serum was collected from a cohort of patients (n = 36), 18 patients with a reduced BMD and 18 control patients. Serum Dkk1 expression as quantified by ELISA was correlated with lumbar and femoral t‐ and z‐scores. Serum Dkk1 concentration in the osteoporosis group was significantly higher than control group (941 ± 116 vs. 558 ± 47 pg/ml, p < 0.01). Serum Dkk1 expression was highly correlated with bone mass variables with inverse associations found between serum Dkk1 expression and lumbar t‐score (r = ?0.34, p = 0.00433), lumbar z‐score (r = ?0.22, p = 0.1907), femur t‐score (r = ?0.42, p = 0.0101), and femur z‐score (r = ?0.43, p = 0.0089). Our data further emphasizes the pivotal role played by Wnt/β‐catenin signaling in bone mass regulation. Dkk1, a powerful antagonist of canonical Wnt signaling, may have a role to play as a serological marker for disorders of bone mass, warranting further evaluation. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:414–418, 2011     

Long-term treatment of osteoporotic women with bisphosphonates does not impair the response to subsequently administered intravenous pamidronate

Summary

We addressed the question whether the response of osteoporotic patients to bisphosphonate treatment is reduced with time. Bisphosphonate-treated women with postmenopausal or glucocorticoid-induced osteoporosis showed adequate and consistent changes of bone markers to subsequently administered intravenous pamidronate. Response of osteoporotic patients to bisphosphonates is not impaired during their long-term administration.

Introduction

Inadequate response to bisphosphonate treatment has been described in patients with Paget’s disease of bone but has not been addressed in osteoporosis although treatment failure is a clinically relevant problem.

Methods

Twenty one women with postmenopausal osteoporosis (PMO) aged 68?±?8.2 years and 14 women with glucocorticoid-induced osteoporosis (GIOP) aged 65?±?10 years were treated with tri-monthly intravenous infusions of 45 mg of pamidronate for 1 year. All patients had been previously treated with bisphosphonates (alendronate, risedronate, pamidronate) for a mean period of 6.2 years (range, 1.3–14 years). Blood samples were taken for measurement of the bone resorption marker C-terminal crosslinking telopeptide of type I collagen (CTX-I) on days?1 and 4 and of the bone formation marker procollagen type I N propeptide, (P1NP) on day?1 of every tri-monthly treatment course.

Results

With each treatment course there was a significant decrease in serum CTX-I on day?4 and an increase to baseline values 3 months after each infusion in both PMO (mean values, day?1: 291.33?±?160.78 pg/ml vs. day?4: 131?±?91.7 pg/ml, p?<?0.001) and GIOP (day?1: 219.3?±?114.8 pg/ml vs. day?4: 98.8?±?51.6 pg/ml, p?<?0.001). Serum P1NP remained stable during the whole year of treatment.

Conclusions

Long-term bisphosphonate treatment of women with either PMO or GIOP does not impair the response to subsequently administered intravenous pamidronate suggesting that inadequate response to long-term bisphosphonate treatment is not responsible for treatment failure.     

Impaired marrow osteogenesis is associated with reduced endocortical bone formation but does not impair periosteal bone formation in long bones of SAMP6 mice.

We used the SAMP6 osteoporotic mouse to examine the link between marrow osteogenic potential and in vivo cortical bone formation. SAMP6 marrow supported less in vitro osteogenesis than marrow from SAMR1 controls; SAMP6 mice had a corresponding deficit in endocortical mineralizing surface. This marrow/endocortical defect did not affect the periosteum, where SAMP6 mice had normal to enhanced bone formation. INTRODUCTION: With aging, there may be a reduction in the number or proliferative capacity of bone marrow osteoprogenitors that may contribute to age-related decreases in bone formation. To examine the link between the ability of the marrow to support osteogenesis and age-related changes in bone formation, we measured in vitro and in vivo indices of osteogenesis in a model of osteoporosis, the senescence-accelerated mouse SAMP6. MATERIALS AND METHODS: Femora and tibias from SAMP6 and SAMR1 (control) mice were harvested at 2, 4, 6, and 12 months of age (168 bones total). Bone marrow cells were cultured under osteogenic conditions and stained for alkaline phosphatase (ALP) and alizarin red. Dynamic indices of bone formation were assessed histologically from calcein labels. RESULTS: ALP+ and alizarin red-positive areas were significantly less in cultures from SAMP6 bones versus SAMR1 (p < 0.05), indicating less osteogenic potential. For example, SAMP6 tibial cultures had 21% less ALP+ area and 36% less alizarin red-positive area than SAMR1. Marrow from tibias had 2-fold greater osteogenesis than femoral marrow (p < 0.001). SAMP6 mice had a deficit in endocortical mineralizing surface across all age groups (p < 0.05), but no deficit in mineral apposition rate. Last, despite the marrow and endocortical deficits, SAMP6 mice had normal or slightly increased periosteal bone formation, consistent with their larger bone size. CONCLUSION: SAMP6 bone marrow supports less in vitro osteogenesis than SAMR1, consistent with a lower concentration of marrow osteoprogenitors in SAMP6. SAMP6 mice have less endocortical mineralizing surface than SAMR1 at all ages but no detectable deficit in mineral apposition rate, which suggests a reduction in osteoblast number but normal function. Periosteal bone formation is unimpaired in SAMP6 mice, indicating that the marrow/endocortical defect does not affect the periosteal surface.     

Blockade of beta-adrenergic signaling does not influence the bone mechano-adaptive response in mice

The involvement of the sympathetic nervous system (SNS) in the modulation of bone adaptation to its load-bearing demand remains controversial. This study tested the involvement of SNS in the adaptive response of trabecular and cortical bone to either external loading or disuse. External loading consisted of cyclic strain (40 cycles, peak 1500 microstrain) applied for 7 min, 3 days/week, while disuse was induced by unilateral sciatic neurectomy (SN). C57Bl/J6 mice, female, 9 weeks old, were subjected to loading or disuse for 2 weeks. Half of the loaded and SN mice were injected with the beta-adrenergic antagonist, propranolol (PRO, 20 mug/g) 1 week before the start of loading or disuse and during all the duration of the experiment. MicroCT analysis of the tibiae showed that the applied load induced significant changes on both trabecular architecture and cortical geometry compared to the contralateral controls, indicating increased bone mass. In contrast, disuse markedly reduced trabecular and cortical indexes. However, these adaptive responses were not altered by PRO treatment. We further tested whether the lack of protective effect of PRO against disuse-induced bone loss was due to the very short duration of treatment by blocking SNS signaling for 8 weeks with either PRO (0.5 mg/ml in drinking water) or guanethidine sulfate (GS, 40 mug/g, injected). At the end of fourth week of treatment, mice underwent SN surgery so that disuse was induced for the remaining 4 weeks. Again, neither PRO nor GS treatments altered the disuse-induced bone loss in the neurectomized tibia. In addition, blockade of SNS signaling for either 3 or 8 weeks did not affect the basal trabecular bone architecture in control tibiae and in L4 vertebrae. This study shows that the mechano-adaptive response occurring in trabecular and cortical bone upon loading or disuse is not altered by inactivation of beta-adrenergic signaling. Furthermore, sympathectomy had no effect on trabecular bone at different skeletal sites. This suggests that the osteo-regulatory action of beta-adrenergic signaling is not involved in the bone mechano-adaptive response and must therefore affect other bone regulatory pathways.     

Fetal distress does not affect in utero defecation but does impair the clearance of amniotic fluid

PURPOSE: An experimental study was performed to evaluate the effect of fetal distress on in utero defecation and clearance of amniotic fluid (AF). METHODS: Sixteen pregnant New Zealand white rabbits underwent laparotomy at 25 days' gestation (full term, 31 to 32 days) as group A (n = 8) and B (n = 8). Uteroplacental ischemia was achieved by constriction of the aorta below the renal arteries to cause fetal distress in group B, whereas sham operation was done in group A. In both groups, 0.1 mL of technetium-99m (99mTc)-HIDA containing 1 mCi of radioactivity was injected into the gluteus muscle of each fetus, which had been exposed through the uterus. Beginning 2 hours after injection, a live fetus was killed every 2 hours for 48 hours in both groups. Tissue samples from the reference organs (lung, heart, stomach, kidney, bladder) and liver, meconium in proximal, mid and distal bowels, AF, and maternal blood were taken. The radioactivity of each sample was determined by a gamma counter and the percentage injected dose (uptake) per gram of tissue (%ID/g) was calculated. The total uptake and mean transit time (MTT) showing intestinal transport were calculated using the linear trapezoidal approximation and extrapolation. The peak concentration (Cmax, %ID/g) and time corresponding to the peak (tmax, h) were obtained. RESULTS: (1) Significant difference was noted between the groups with regard to uteroplacental perfusion pressure and blood pH (51.0+/-2.6 mm Hg; pH, 6.9+/-0.1 in group B; 80.1+/-2.7 mm Hg, pH, 7.3+/-0.1 in group A; P < .05). (2) 99mTc-HIDA was predominantly trapped by the liver and excreted into the gastrointestinal tract and AF in both groups. (3) In liver and bowel, shape of the profile was bimodal because of fetal swallowing and similar in both groups, tmax was the same in both groups, Cmax was lower in group B than in group A, the total uptake was smaller in group B than in group A, and intestinal transport time was similar (44.2 hours in group A and 43.0 hours in group B). In amniotic fluid, shape of the profile was sigmoidal in group B and reached a Cmax value of 11.6% ID/g, whereas unimodal profiles were observed in group A with a Cmax value of 12.6% ID/g; radioactivity was eliminated from the AF with a rate constant of 0.48% ID/g h in group A (AUC, 273% ID/g h); whereas accumulation of radioactivity was noted in group B (AUC, 308% ID/g h). (5) In maternal blood, shape of the profile was sigmoidal in group A with a Cmax value of 2.9% ID/g and unimodal in group B (Cmax, 1.6% ID/g), accumulation of radioactivity was noted in group A (AUC, 93% ID/g h), whereas a rapid decline of radioactivity (k, 0.06% ID/g h) was noted in group B (AUC, 47% ID/g h). CONCLUSIONS: Fetal distress did not affect the intestinal transport dynamics and in utero defecation but impaired the clearance of AF and the passage into the maternal circulation, which was shown by the accumulation of radioactivity in AF only in group B and in maternal blood only in group A without any elimination rate. This finding suggests that meconium-stained AF is not related to meconium passage after fetal distress; rather, it reflects impaired clearance of AF, which already has containing meconium caused by physiological in utero defecation.     

Low-magnitude whole body vibration does not affect bone mass but does affect weight in ovariectomized rats

Mechanical loading has stimulating effects on bone architecture, which can potentially be used as a therapy for osteoporosis. We investigated the skeletal changes in the tibia of ovariectomized rats during treatment with whole body vibration (WBV). Different low-magnitude WBV treatment protocols were tested in a pilot experiment using ovariectomized rats with loading schemes of 2 × 8 min/day, 5 days/week (n = 2 rats per protocol). Bone volume and architecture were evaluated during a 10 week follow-up using in-vivo microcomputed tomography scanning. The loading protocol in which a 45 Hz sine wave was applied at 2 Hz with an acceleration of 0.5g showed an anabolic effect on bone and was therefore further analyzed in two groups of animals (n = 6 each group) with WBV starting directly after or 3 weeks after ovariectomy and compared to a control (non-WBV) group at 0, 3, 6 and 10 weeks’ follow-up. In the follow-up experiment the WBV stimulus did not significantly affect trabecular volume fraction or cortical bone volume in any of the treatment groups during the 10 week follow-up. WBV did reduce weight gain that was induced as a consequence of ovariectomy. We could not demonstrate any significant effects of WBV on bone loss as a consequence of ovariectomy in rats; however, the weight gain that normally results after ovariectomy was partly prevented. Treatment with WBV was not able to prevent bone loss during induced osteoporosis.     

Targeted expression of calcitonin gene-related peptide to osteoblasts increases bone density in mice.

The neuropeptide calcitonin gene-related peptide (CGRP) is concentrated in fine sensory nerve endings innervating all tissues, including bone. CGRP inhibits osteoclasts, stimulates insulin-like growth factor I and inhibits tumor necrosis factor alpha production by osteoblasts in vitro. To investigate the role of CGRP in bone in vivo, mice were engineered to express CGRP in osteoblasts by placing the human CGRP gene under the control of the rat osteocalcin promoter (Ost-CGRP tg+ mice). Calvaria cultures from transgene positive (tg+), but not tg- mice, produced bioactive CGRP. Trabecular bone density and bone volume, determined by peripheral quantitative computed tomography and bone histomorphometry, respectively, were higher in tg+ than tg- littermates. This increase in bone volume was associated with an increased bone formation rate. Trabecular bone density decreased in tg+ mice as a result of ovariectomy, but remained higher than in sham tg- mice. Targeting CGRP to osteoblasts appears to favor the establishment of a higher trabecular bone mass in mice.     

Selective deletion of the membrane-bound colony stimulating factor 1 isoform leads to high bone mass but does not protect against estrogen-deficiency bone loss

To better define the biologic function of membrane-bound CSF1 (mCSF1) in vivo, we have generated mCSF1 knockout (k/o) mice. Spinal bone density (BMD) was 15.9% higher in k/o mice compared to wild-type (wt) controls (P < 0.01) and total BMD was increased by 6.8% (P < 0.05). A higher mean femur BMD was also observed but did not reach statistical significance (6.9% P = NS). The osteoclastogenic potential of bone marrow isolated from mCSF1 k/o mice was reduced compared to wt marrow. There were no defects in osteoblast number or function suggesting that the basis for the high bone mass phenotype was reduced resorption. In addition to a skeletal phenotype, k/o mice had significantly elevated serum triglyceride levels (123 ± 7 vs. 88 ± 3.2 mg/dl; k/o vs. wt, P < 0.001), while serum cholesterol levels were similar (122 ± 6 vs. 116 ± 6 mg/dl; k/o vs. wt, P = NS). One month after surgery, 5-month-old k/o and wt female mice experienced the same degree of bone loss following ovariectomy (OVX). OVX induced a significant fourfold increase in the expression of the soluble CSF1 isoform (sCSF1) in the bones of wt mice while expression of mCSF1 was unchanged. These findings indicate that mCSF1 is essential for normal bone remodeling since, in its absence, BMD is increased. Membrane-bound CSF1 does not appear to be required for estrogen-deficiency bone loss while in contrast; our data suggest that sCSF1 could play a key role in this pathologic process. The reasons why mCSF1 k/o mice have hypertriglyceridemia are currently under study.