Risedronate does not reduce mechanical loading-related increases in cortical and trabecular bone mass in mice

To establish whether the combination of anti-resorptive therapy with mechanical loading has a negative, additive or synergistic effect on bone structure, we assessed the separate and combined effects of risedronate and non-invasive dynamic loading on trabecular and cortical bone. Seventeen-week-old female C57BL/6 mice were given daily subcutaneous injections of vehicle (n=20) or risedronate at a dose of 0.15, 1.5, 15 or 150 μg/kg/day (n=10 in each) for 17 days. From the fourth day of treatment, the right tibiae were subjected to a single period of axial loading (40 cycles/day) for three alternate days per week for two weeks. The left tibiae were used as internal controls. Trabecular and cortical sites in the tibiae were analyzed by high-resolution micro-computed tomography and imaging of fluorochrome labels. In the non-loaded tibiae, treatment with the higher doses of risedronate at 15 or 150 μg/kg/day resulted in higher trabecular bone volume and trabecular number than in vehicle-treated controls, whereas such treatment was associated with no differences in cortical bone volume at any dose. In the loaded tibiae, loading induced increases in trabecular and cortical bone volume compared with contra-lateral controls primarily through increased trabecular thickness and periosteal expansion, respectively, independently of risedronate treatment. In conclusion, the response to mechanical loading in both trabecular and cortical bone in mice is therefore not impaired by short-term treatment with risedronate, even over a 1000-fold dose range. In considering the optimization of treatments for osteoporosis, it is reassuring that anti-resorptive therapy and mechanical loading can exert independent beneficial effects.     

Reduction of pathological and behavioral deficits following spinal cord contusion injury with the selective cyclooxygenase-2 inhibitor NS-398

Spinal cord injury (SCI) results in loss of locomotor function and development of abnormal chronic pain syndromes (mechanical allodynia, thermal hyperalgesia). Following injury, secondary mechanisms including release of excitatory amino acids, inflammation and lipid peroxidation damage neural cells through release of cytotoxic free radicals. We hypothesized that selective inhibition of cyclooxygenase-2 (COX-2), an inducible inflammatory mediator, would decrease tissue damage and subsequently reduce locomotor deficits and development of chronic central pain syndromes after injury. Fifteen minutes prior to receiving T13 spinal segment spinal cord contusion injury, 200-225-g male Sprague-Dawley rats received either vehicle (0.5 ml 1:1 v/v DMSO/saline, i.p., n = 20) or the selective COX-2 inhibitor NS-398 (5 mg/kg in DMSO/saline v/v, i.p., n = 20). Locomotor function via the BBB scale, and nociceptive behaviors measured by paw withdrawals to von Frey filaments and radiant heat stimuli were tested for 4 weeks postinjury. Histological examination and volumetric analysis of spinal cord tissue were performed concomitantly. Spinally contused animals receiving NS-398 demonstrated significantly (p < 0.05) reduced locomotor alteration and reductions in both fore- and hindlimb mechanical allodynia and thermal hyperalgesia when compared to vehicle controls. Histological examination of spinal segments at the lesion segment demonstrated reduced lesion extent and increased viable tissue when compared to vehicle controls. Prostaglandin E2 levels were significantly lowered in NS-398-treated but not vehicle-treated animals 12 h after injury. These results support the role of COX-2 in reducing pathological and behavioral deficits after spinal cord injury.     

Moderate weight gain does not influence bone metabolism in skeletally mature female rats

Bone mass is correlated with body weight during growth. However, it is unclear how bone mass is influenced by weight gain following skeletal maturity. The purpose of this study was to determine the effects of weight maintenance and two rates of weight gain on bone metabolism using skeletally mature female rats. Eight-month-old female rats were fed one of 3 diets for 13 weeks: Lieber–DeCarli liquid diet ad lib (control diet), the same diet with caloric restriction to maintain initial body weight (calorie-restricted diet), and the same diet fed ad lib with the exception that appetite was enhanced (calorie-increased diet) by replacing a small quantity of maltose–dextran isolcalorically with ethanol (0.5% caloric intake). Compared to baseline, rats fed the calorie-restricted, control, and calorie-increased diets changed in weight by ?1 ± 2% (mean ± SE), 10 ± 3%, and 21 ± 2%, respectively. Weight gain was associated with a significant increase in serum leptin, a putative regulator of bone formation. In contrast, significant differences in tibial bone mineral content and density were not detected among treatments groups following dietary intervention or between treatment groups and the baseline group. Similarly, indices of cancellous bone architecture (area, trabecular number, thickness, and separation) and bone turnover (mineralizing perimeter, mineral apposition rate, and bone formation rate) did not differ among groups following dietary intervention. Our findings suggest that neither weight gain nor increased serum leptin levels, over the range evaluated, influence bone metabolism in skeletally mature female rats.     

Diclofenac and NS-398, a selective cyclooxygenase-2 inhibitor, decrease agonist-induced contractions of the pig isolated ureter

Non-steroidal anti-inflammatory drugs (NSAIDs) are currently considered a first-line treatment of renal colic. Their action has been ascribed to the inhibition of renal prostaglandin synthesis, which decreases renal blood flow and diuresis, and consequently lowers the pressure in the renal pelvis and ureter. However, the effects of NSAIDs on induced contractions of ureteral smooth muscle have received little attention. Also, there is a lack of clinically relevant spasmolytic drugs for the ureter. Therefore, we studied the influence of the non-selective cyclooxygenase (COX) inhibitor diclofenac, a NSAID drug customarily used in the treatment of renal colic, and of NS-398, a selective COX-2 inhibitor, on induced contractions of the pig ureter. Serotonin (0.1–30 μM), norepinephrine (0.1–30 μM) and neurokinin A (0.03–10 μM) induced reproducible concentration-dependent contractions, which were inhibited by diclofenac and NS-398 (10–300 μM) in a concentration-dependent manner. The sensitivity of neurokinin A-induced contractions to diclofenac was 3–4 times greater than that of the amines. Depending on the concentration, inhibition ranged between 25 and 96% of the initially induced contractile activity. In the presence of inhibitors, supramaximal concentrations of agonists were unable to trigger recuperation of the initially induced contractions. Prostaglandin F_2α did not reverse the effect of diclofenac on agonist-induced contractions. Removal of diclofenac or NS-398 from the organ baths showed that the inhibition was totally reversible. Thus, the non-selective COX inhibitor diclofenac and the selective COX-2 inhibitor NS-398 are almost equipotent in reducing agonist-induced contractions in the isolated porcine ureter. Although the clinical relevance of this spasmolytic effect remains to be demonstrated, the data suggest that patients suffering from renal colic may benefit not only from the anti-diuretic and analgesic effects of diclofenac, but also from its potential spasmolytic properties. Moreover, selective COX-2 inhibitors may have clinical potential, as they may cause fewer side effects. Received: 21 February 2000 / Accepted: 27 July 2000     

NS-398 upregulates constitutive cyclooxygenase-2 expression in the M-1 cortical collecting duct cell line.

The cortical collecting duct (CCD) is a major site of intrarenal prostaglandin E2 (PGE2) synthesis. This study examines the expression and regulation of the prostaglandin synthesizing enzymes cyclooxygenase-1 (COX-1) and -2 in the CCD. By indirect immunofluorescence using isoform-specific antibodies, COX-1 and -2 immunoreactivity was localized to all cell types of the murine M-1 CCD cell line. By immunohistochemistry, both COX-1 and COX-2 were localized to intercalated cells of the CCD on paraffin-embedded mouse kidney sections. When COX enzyme activity was measured in the M-1 cells, both indomethacin (COX-1 and -2 inhibitor) and the specific COX-2 inhibitor NS-398 effectively blocked PGE2 synthesis. These results demonstrate that COX-2 is the major contributor to the pool of PGE2 synthesized by the CCD. By Western blot analysis, COX-2 expression was significantly upregulated by incubation with either indomethacin or NS-398. These drugs did not affect COX-1 protein expression. Evaluation of COX-2 mRNA expression by Northern blot analysis after NS-398 treatment demonstrated that the COX-2 protein upregulation occurred independently of any change in COX-2 mRNA expression. These studies have for the first time localized COX-2 to the CCD and provided evidence that the intercalated cells of the CCD express both COX-1 and COX-2. The results also demonstrate that constitutively expressed COX-2 is the major COX isoform contributing to PGE2 synthesis by the M-1 CCD cell line. Inhibition of COX-2 activity in the M-1 cell line results in an upregulation of COX-2 protein expression.     

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.     

Diminished response to in vivo mechanical loading in trabecular and not cortical bone in adulthood of female C57Bl/6 mice coincides with a reduction in deformation to load

Bone loss occurs during adulthood in both women and men and affects trabecular bone more than cortical bone. The mechanism responsible for trabecular bone loss during adulthood remains unexplained, but may be due at least in part to a reduced mechanoresponsiveness. We hypothesized that trabecular and cortical bone would respond anabolically to loading and that the bone response to mechanical loading would be reduced and the onset delayed in adult compared to postpubescent mice. We evaluated the longitudinal adaptive response of trabecular and cortical bone in postpubescent, young (10 week old) and adult (26 week old) female C57Bl/6J mice to axial tibial compression using in vivo microCT (days 0, 5, 10, and 15) and dynamic histomorphometry (day 15). Loading elicited an anabolic response in both trabecular and cortical bone in young and adult mice. As hypothesized, trabecular bone in adult mice exhibited a reduced and delayed response to loading compared to the young mice, apparent in trabecular bone volume fraction and architecture after 10 days. No difference in mechanoresponsiveness of the cortical bone was observed between young and adult mice. Finite element analysis showed that load-induced strain was reduced with age. Our results suggest that trabecular bone loss that occurs in adulthood may in part be due to a reduced mechanoresponsiveness in this tissue and/or a reduction in the induced tissue deformation which occurs during habitual loading. Therapeutic approaches that address the mechanoresponsiveness of the bone tissue may be a promising and alternate strategy to maintain trabecular bone mass during aging.     

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.     

Short-term low-strain vibration enhances chemo-transport yet does not stimulate osteogenic gene expression or cortical bone formation in adult mice

Development of low-magnitude mechanical stimulation (LMMS) based treatment strategies for a variety of orthopaedic issues requires better understanding of mechano-transduction and bone adaptation. Our overall goal was to study the tissue and molecular level changes in cortical bone in response to low-strain vibration (LSV: 70 Hz, 0.5 g, 300 με) and compare these to changes in response to a known anabolic stimulus: high-strain compression (HSC: rest inserted loading, 1000 με). Adult (6-7 months) C57BL/6 mice were used for the study and non-invasive axial compression of the tibia was used as a loading model. We first studied bone adaptation at the tibial mid-diaphysis, using dynamic histomorphometry, in response to daily loading of 15 min LSV or 60 cycles HSC for 5 consecutive days. We found that bone formation rate and mineral apposition rate were significantly increased in response to HSC but not LSV. The second aim was to compare chemo-transport in response to 5 min of LSV versus 5 min (30 cycles) of HSC. Chemo-transport increased significantly in response to both loading stimuli, particularly in the medial and the lateral quadrants of the cross section. Finally, we evaluated the expression of genes related to mechano-responsiveness, osteoblast differentiation, and matrix mineralization in tibias subjected to 15 min LSV or 60 cycles HSC for 1 day (4-h time point) or 4 consecutive days (4-day time point). The expression level of most of the genes remained unchanged in response to LSV at both time points. In contrast, the expression level of all the genes changed significantly in response to HSC at the 4-h time point. We conclude that short-term, low-strain vibration results in increased chemo-transport, yet does not stimulate an increase in mechano-responsive or osteogenic gene expression, and cortical bone formation in tibias of adult mice.     

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.     

The RANKL inhibitor OPG-Fc increases cortical and trabecular bone mass in young gonad-intact cynomolgus monkeys

Summary Weekly treatment of gonad-intact cynomolgus monkeys (for up to 6 months) with the RANKL inhibitor OPG-Fc reduced bone turnover markers and increased volumetric cortical and trabecular BMD and BMC at radial and tibial metaphyses. OPG-Fc was well tolerated in this study without evidence of change in measured toxicologic parameters vs. control. Introduction RANKL is the primary mediator of osteoclast formation, function, and survival. The catabolic effects of RANKL are inhibited by OPG, a soluble decoy receptor for RANKL. We investigated the safety and pharmacology of OPG-Fc in gonad-intact cynomolgus monkeys. Methods Males and females were treated weekly with vehicle (n = 5/sex) or OPG-Fc (15 mg/kg) by s.c. (n = 5/sex) or i.v. (n = 3/sex) injection for 6 months. Results Routine toxicologic investigations, hematologic parameters, body and organ weights, and ophthalmologic and electrocardiographic findings were not affected by OPG-Fc treatment. Because s.c. and i.v. dosing of OPG-Fc caused similar effects, these groups were combined for analyses. The following endpoints were significantly different in males and/or females treated with OPG-Fc relative to sex-matched vehicle controls after 6 months (p < 0.05). Biochemical markers of bone turnover (urine N-telopeptide and serum osteocalcin) were significantly decreased with OPG-Fc treatment. Cortical and trabecular volumetric BMD and BMC, cortical thickness, and cross-sectional moment of inertia were significantly increased by OPG-Fc treatment at the proximal tibia and distal radius metaphyses. Increases in cortical thickness were associated with significantly greater periosteal circumference. Conclusions OPG-Fc increased cortical and trabecular BMD and BMC in young gonad-intact cynomolgus monkeys.     

Mechanotransduction in bone does not require a functional cyclooxygenase-2 (COX-2) gene.

COX-2 is a key enzyme involved in the response of bone to loading. However, using mice with a null mutation of the COX-2 gene, we found that a functional COX-2 gene is not required for mechanotransduction. This paradoxical finding may have resulted, in part, from mechanically induced COX-1 activity. INTRODUCTION: Cyclooxygenase-2 (COX-2) is an important mediator in the response of bone to mechanical loading, with pharmacological inhibition of COX-2 effectively eliminating or reducing mechanically induced bone formation. In this study, we further investigated the role of COX-2 in skeletal mechanotransduction using a genetic approach. The aim was to compare the skeletal responsiveness of COX-2 homozygous mutant (COX-2(-/-)) and wildtype control (COX-2(+/+)) mice to investigate whether a functional COX-2 gene is necessary for mechanotransduction. MATERIALS AND METHODS: Adult female COX-2(+/+) and COX-2(-/-) mice on a C57BL/6x129/ola background were studied using the ulna axial loading model. The response to 2 days of loading for 120 cycles/day at 2 Hz was measured histomorphometrically. Phenotypic characterization of the femurs in these mice was also performed. In a separate group of animals, the expression of the remaining COX isozyme, COX-1, was assessed using real-time RT-PCR 4 h after one bout of 120 loading cycles. RESULTS: Null mutation of the COX-2 gene resulted in a consistent femoral phenotype of reduced bone mass, altered architecture, and inferior mechanical properties. Many of these differences were nullified after adjustment for body weight. Nevertheless, body weight-corrected values showed a consistent trend of reduced mechanical properties in COX-2(-/-) mice. Genotype did not influence the response to mechanical loading, with no histomorphometric differences being found between COX-2(+/+) and COX-2(-/-) mice. Real-time RT-PCR showed COX-2(-/-) mice to express significantly greater COX-1 expression in loaded ulnas than in loaded ulnas in COX-2(+/+) mice. There were no differences in COX-1 expression in nonloaded ulnas. CONCLUSIONS: A functional COX-2 gene was not found to be required for skeletal mechanotransduction. This is in contrast to previous pharmacological studies showing that COX-2 is critical to the response of bone to loading. Investigating a potential reason for the absence of a genotype difference in this study, we found that mice with a null mutation in the COX-2 gene possess inductive skeletal COX-1 expression.     

Selective cyclooxygenase-2 inhibitor prevents reduction of trabecular bone mass in collagen-induced arthritic mice in association with suppression of RANKL/OPG ratio and IL-6 mRNA expression in synovial tissues but not in bone marrow cells

We performed this study to clarify whether celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, prevents trabecular bone mass reduction by suppressing arthritis-related increase of bone resorption, and to discriminate differences in actions on bone among celecoxib, SC-58560 (a selective COX-1 inhibitor), and indomethacin. Eight-week-old DBA/1J male mice were divided into six groups as follows. Control untreated (Normal) and collagen-induced arthritic (CIA) mice were compared with four treatment groups: celecoxib was orally administered to CIA mice at doses of 0 (Vehicle), 16 (COX2L), and 75 (COX2H) mg/kg, in addition to two groups of mice treated with SC-58560 (COX1) or indomethacin (IND). Histomorphometry showed a significant decrease in tibial trabecular bone volume in arthritic mice, which was corrected by COX2H. The increased osteoclast surface and number in the Vehicle group were suppressed by COX2L, COX2H, and IND. The decreased bone formation rate in Vehicle was elevated by COX2H without statistical significance. A high ratio of mRNA expression of receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) in Vehicle synovial tissue was suppressed by COX2L and COX2H. The increased expression of interleukin (IL)-6 mRNA in Vehicle was suppressed by COX2L, COX2H, and IND, although no difference in this expression was observed in bone marrow cells among all groups. In conclusion, in CIA mice, celecoxib suppresses arthritis-related increase in bone resorption at low and high doses and prevents trabecular bone mass reduction at high doses in association with suppression of osteoclast development in bone marrow through inhibition of RANKL/OPG ratio and IL-6 mRNA expression in inflammatory synovial tissue.     

SGLT2 inhibitor therapy improves blood glucose but does not prevent diabetic bone disease in diabetic DBA/2J male mice

Persons with type 1 and type 2 diabetes have increased fracture risk, attributed to deficits in the microarchitecture and strength of diabetic bone, thought to be mediated, in part, by the consequences of chronic hyperglycemia. Therefore, to examine the effects of a glucose-lowering SGLT2 inhibitor on blood glucose (BG) and bone homeostasis in a model of diabetic bone disease, male DBA/2J mice with or without streptozotocin (STZ)-induced hyperglycemia were fed chow containing the SGLT2 inhibitor, canagliflozin (CANA), or chow without drug, for 10 weeks of therapy. Thereafter, serum bone biomarkers were measured, fracture resistance of cortical bone was assessed by μCT analysis and a three-point bending test of the femur, and vertebral bone strength was determined by compression testing. In the femur metaphysis and L6 vertebra, long-term diabetes (DM) induced deficits in trabecular bone microarchitecture. In the femur diaphysis, a decrease in cortical bone area, cortical thickness and minimal moment of inertia occurred in DM (p < 0.0001, for all) while cortical porosity was increased (p < 0.0001). These DM changes were associated with reduced fracture resistance (decreased material strength and toughness; decreased structural strength and rigidity; p < 0.001 for all). Significant increases in PTH (p < 0.0001), RatLAPs (p = 0.0002), and urine calcium concentration (p < 0.0001) were also seen in DM. Canagliflozin treatment improved BG in DM mice by ~ 35%, but did not improve microarchitectural parameters. Instead, in canagliflozin-treated diabetic mice, a further increase in RatLAPs was evident, possibly suggesting a drug-related intensification of bone resorption. Additionally, detrimental metaphyseal changes were noted in canagliflozin-treated control mice. Hence, diabetic bone disease was not favorably affected by canagliflozin treatment, perhaps due to insufficient glycemic improvement. Instead, in control mice, long-term exposure to SGLT2 inhibition was associated with adverse effects on the trabecular compartment of bone.     

Mechanical loading enhances the anabolic effects of intermittent parathyroid hormone (1-34) on trabecular and cortical bone in mice

The separate and combined effects of intermittent parathyroid hormone (iPTH) (1-34) and mechanical loading were assessed at trabecular and cortical sites of mouse long bones. Female C57BL/6 mice from 13 to 19 weeks of age were given daily injections of vehicle or PTH (1-34) at low (20 microg/kg/day), medium (40 microg/kg/day) or high (80 microg/kg/day) dose. For three alternate days per week during the last two weeks of this treatment, the tibiae and ulnae on one side were subjected to a single period of non-invasive, dynamic axial loading (40 cycles at 10 Hz with 10-second intervals between each cycle). Two levels of peak load were used; one sufficient to engender an osteogenic response, and the other insufficient to do so. The whole tibiae and ulnae were analyzed post-mortem by micro-computed tomography with a resolution of 5 microm. Treatment with iPTH (1-34) modified bone structure in a dose- and time-dependent manner, which was particularly evident in the trabecular region of the proximal tibia. In the tibia, loading at a level sufficient by itself to stimulate osteogenesis produced an osteogenic response in the low-dose iPTH (1-34)-treated trabecular bone and in the proximal and middle cortical bone treated with all doses of iPTH (1-34). In the ulna, loading at a level that did not by itself stimulate osteogenesis was osteogenic at the distal site when combined with high-dose iPTH (1-34). At both levels of loading, there were synergistic effects in cortical bone volume of the proximal tibia and distal ulna between loading and high-dose iPTH (1-34). Images of fluorescently labelled bones confirmed that such synergism resulted from increases in both endosteal and periosteal bone formation. No woven bone was induced by iPTH (1-34) or either level of loading alone, whereas the combination of iPTH (1-34) and the "sufficient" level of loading stimulated woven bone formation on endosteal and periosteal surfaces of the proximal cortex in the tibiae. Together, these data suggest that in female C57BL/6 mice, under some but not all circumstances, mechanical loading exerts an osteogenic response with iPTH (1-34) in trabecular and cortical bone.     

Incorporation of sodium fluoride into cortical bone does not impair the mechanical properties of the appendicular skeleton in rats

Summary Clinical studies on the use of sodium fluoride (NaF) in osteoporotic patients have demonstrated increased spinal bone mass without a reduction in vertebral fracture incidence, and a trend towards reduced appendicular bone mass with an increase in peripheral fracture incidence. As previous reports have suggested that NaF becomes incorporated into bone's crystal structure, possibly affecting bone strength, we sought to examine the relationship among bone fluoride content, bone mass, and skeletal fragility. Twenty-one-day-old female Sprague-Dawley rats were treated with four different doses of NaF. The tibiae were subjected to histomorphometric and biochemical analyses, and the femora were tested in torsion for the properties of strength, stiffness, energy storage capacity, and angular deformation. The results showed that over 50% of the skeleton in these rats was turned over in the presence of NaF. The four different doses resulted in a linear increase in bone F concentration and suggested excellent absorption and incorporation of this drug. No changes in histomorphometric indices of bone formation or turnover were found. Despite the large fraction of bone formed during NaF treatment, and the linear increase in bone fluoride content in relation to dose, there were no changes observed in any of the mechanical properties. These results suggest that, even extensive incorporation of fluoride into bone, in the absence of an effect on bone mass or remodeling, does not significantly alter its capacity to withstand mechanical loads.     

The mode of school transportation in pre-pubertal children does not influence the accrual of bone mineral or the gain in bone size - two year prospective data from the paediatric osteoporosis preventive (POP) study

Background  

Walking and cycling to school are one source of regular physical activity. The aim of this two years observational study in pre-pubertal children was to evaluate if walking and cycling to school was associated with higher total amount of physical activity and larger gain in bone mineral content (BMC) and bone width than when going by car or bus.     

选择性COX-2抑制剂NS398对前列腺癌PC-3细胞增殖与凋亡的影响

目的观察环氧化酶-2(COX-2)抑制剂NS398对前列腺癌细胞PC-3增殖和凋亡的影响。方法采用四甲基偶氮唑蓝法(MTT法)检测不同浓度和不同时间NS398对PC-3细胞增殖的影响;RT-PCR法检测不同浓度NS398作用PC-3细胞24h后COX-2 mRNA的表达;酶联免疫测定法(ELISA)检测不同浓度NS398作用PC-3细胞24h后PGE2释放水平;流式细胞仪检测不同浓度NS398作用PC-3细胞24h后细胞凋亡情况。结果NS398可以抑制PC-3细胞的增殖,呈时间和剂量依赖性;RT-PCR和ELISA法检测结果显示,随着NS398浓度增高,PC-3细胞COX-2 mRNA表达和PGE2释放水平呈下调趋势;细胞凋亡检测结果显示100、200μmol/LNS398对PC-3细胞具有诱导凋亡的作用。结论NS398可能通过COX-2依赖性途径抑制前列腺癌PC-3细胞增殖,促进肿瘤细胞凋亡。