Bone

Editorial Board

2009-12-01

IBMS Elected Board of Directors 2009 - 2010

2009-12-01

IBMS 2009 Corporate Partners

2009-12-01

IBMS Membership Application

2009-12-01

Increased calcium content and inhomogeneity of mineralization render bone toughness in osteoporosis: Mineralization, morphology and biomechanics of human single trabeculae

2009-09-03

Abstract: The differentiation and degree of the effects of mineral content and/or morphology on bone quality remain, to a large extent, unanswered due to several microarchitectural particularities in spatial measuring fields (e.g., force transfer, trajectories, microcalli). Therefore, as the smallest basic component of cancellous bone, we focused on single trabeculae to investigate the effects of mineralization and structure, both independently and in superposition. Transiliac Bordier bone cores and T12 vertebrae were obtained from 20 females at autopsy for specimen preparation, enabling radiographical analyses, histomorphometry, Bone Mineral Density Distribution (BMDD) analyses, and trabecular singularization to be performed. Evaluated contact X-rays and histomorphometric limits from cases with osteoporotic vertebral fractures generated two subdivisions, osteoporotic (n=12, Ø 78 years) and non-osteoporotic (n=8, Ø 49 years) cases, based on fracture appearance and bone volume (BV/TV). Measurements of trabecular number (Tb.N.), trabecular separation (Tb.Sp.), trabecular thickness (Tb.Th.), trabecular bone pattern factor (TBPf) and eroded surface (ES/BS) were carried out to provide detailed structural properties of the investigated groups. The mechanical properties of 400 rod-like single vertebral trabeculae, assessed by three-point bending, were matched with mineral properties as quantified by BMDD analyses of cross-sectioned rod-like and plate-like trabeculae, both in superposition and independently. Non-osteoporotic iliac crests and vertebrae displayed linear dependency on structure parameters, whereas osteoporotic compartments proved to be non-correlated with bone structure. Independent of trabecular thickness, osteoporotic rod-like trabeculae showed decreases in Young's modulus, fracture load, yield strength, ultimate stress, work to failure and bending stiffness, along with significantly increased mean calcium content and calcium width. Non-osteoporotic trabeculae showed biomechanically beneficial properties due to a homogeneous mineralization configuration, whereas osteoporotic trabeculae predominantly demonstrated various mineralized bone packets, eroded surfaces, highly mineralized cement lines and microcracks. The Young's moduli of single trabeculae exhibited significantly negative linear correlations with trabecular thickness. Because of increased, but inhomogeneously distributed, calcium content, osteoporotic trabeculae may be subject to shear stresses that render bone fragile beyond structure impairment due to cracks and lacunae.

A randomised, double-blinded, placebo-controlled, trial to determine the individual response in bone turnover markers to lasofoxifene therapy

A. Rogers, S.J. Glover, R. Eastell — 2009-09-01

Abstract: Lasofoxifene is a novel selective estrogen receptor modulator that is being developed for the treatment of postmenopausal osteoporosis. Bone mineral density (BMD) measured by dual-energy X-ray absorptiometry (DXA) is currently used to diagnose osteoporosis. BMD response to therapy, however, is often not apparent until at least one year following start of treatment. Biochemical markers of bone turnover may provide an early indication of BMD response in individual patients.The aims of the study were: 1) to determine the variability in bone turnover markers (BTM) to estimate a value for least significant change (LSC); 2) to determine the number of subjects with a response to lasofoxifene greater than LSC; 3) to determine the number of subjects whose bone turnover is decreased to the lower half of the reference range and 4) to evaluate the use of bone turnover markers to predict the change in bone density in response to lasofoxifene.Fifty-one postmenopausal osteopenic women, ages 55 to 77 (mean 63.7) years, were recruited with 44 women completing the 2 year follow up. Participants received either lasofoxifene (0.25 mg/d) or placebo, in a 1:1 ratio. Duplicate measurements of BTM (bone alkaline phosphatase (bone ALP), N-terminal propeptide of type I collagen (PINP), serum β crosslinked C-telopeptides of type I collagen (sβ-CTX), urinary crosslinked N-telopeptides of type I collagen (U-NTX)) were made at baseline and 6 months with single measurements at 4, 8 and 12 weeks. Duplicate measurements of BMD at the lumbar spine (LS), total hip (TH) and distal forearm (DF) were made by DXA at baseline, one and two years in all subjects.Almost all women (92 to 96%), treated with lasofoxifene, had a reduction in serum-based bone turnover markers greater than LSC, and 52 to 80% had serum-based bone turnover markers in the lower half of the reference range, by six months of lasofoxifene therapy. The change in mean LSBMD from baseline, was significantly greater in the lasofoxifene group compared to placebo at 1 and 2 years (+2.5% and +3.4%, respectively, P<0.0001). Change in PINP and U-NTX at 6 months correlated inversely with change in LS and TH BMD at one and two years.The use of lasofoxifene therapy leads to significant decreases in bone turnover by 4 weeks of lasofoxifene therapy as a group, with a decrease in BTM greater than LSC occurring in almost all women taking lasofoxifene by 6 months. By this time, in over half of women taking lasofoxifene, BTM reached the lower half of the reference range. Our results suggest that bone turnover markers are useful for monitoring response to lasofoxifene. Changes occur early and relate to the BMD response.

Rapid and robust response of biochemical markers of bone formation to teriparatide therapy

2009-09-07

Abstract: Teriparatide, a parathyroid hormone analogue, is a potent anabolic treatment for postmenopausal osteoporosis. Studies have shown that teriparatide induces large increases in biochemical markers of bone formation after 1 month of therapy followed by a delayed increase in bone resorption markers.The aims of this study were to (1) describe changes in bone turnover markers during 28 days of treatment with teriparatide; (2) identify the earliest time point by which most subjects showed a biochemical response to teriparatide; (3) identify potential biomarkers of positive bone response; (4) describe changes in bone turnover markers 4 weeks after stopping teriparatide.We recruited 15 osteopenic postmenopausal women, ages 55–69 (mean 62) years. All received 20 μg teriparatide subcutaneously for 28 days. Serum levels of the bone formation markers type I collagen N-terminal propeptide (PINP), type I collagen C-terminal propeptide (PICP), osteocalcin (OC), bone alkaline phosphatase (bone ALP), and the bone resorption markers crosslinked C-telopeptide of type I collagen (Sβ-CTX), crosslinked N-telopeptide of type I collagen (S-NTX) and tartrate-resistant acid phosphatase type 5b (TRACP5b) were measured on 11 occasions: three times before dosing (baseline) and on days 3, 7, 10, 14, 19, 24 and 28 and at day 56 (i.e., 28 days after stopping teriparatide ).During the first 2 days of teriparatide treatment, PINP levels increased rapidly, by 8.2% (90% confidence interval (CI) 6.9%, 9.5%) and continued to increase until the end of treatment to 110.8%. PICP and OC showed a similar, but less pronounced, pattern. All three markers increased by at least 75% at day 28. A small, transient decrease in bone resorption markers occurred over the same period. Following cessation of treatment, concentrations of bone formation markers decreased to within 20% of baseline values by day 56.In conclusion, the bone formation markers PINP, PICP and OC show a rapid and robust increase in response to teriparatide, which is noticeable during the first week of therapy. PINP is the most responsive marker. These findings have important implications for monitoring patients treated with teriparatide and may also inform the design of studies of new anabolic agents for osteoporosis.

Long-term treatment of postmenopausal osteoporosis with strontium ranelate: Results at 8 years

2009-09-02

Abstract: Objectives: Strontium ranelate 2 g/day has proven efficacy against vertebral and nonvertebral fracture over 5 years in postmenopausal osteoporosis, though many women require longer-term treatment. This article describes the efficacy, safety, and tolerability of this agent over 8 years.Methods: Postmenopausal osteoporotic women having participated in the 5-year efficacy trials SOTI and TROPOS were invited to enter a 3-year open-label extension study. The results presented here focus on patients who received strontium ranelate for 8 years.Results: At the extension baseline, the population treated for 8 years (n=879; 79.1±5.6 years) had femoral neck T-score of −2.61±0.71. The cumulative incidences of new vertebral and nonvertebral fractures (13.7% and 12.0%, respectively) over years 6 to 8 were non-statistically different from the cumulative incidences in the first 3 years of the original studies (11.5% and 9.6%). Lumbar spine, femoral neck, and total hip bone mineral density (BMD) increased throughout the 8-year period. Annual relative change in BMD was significant at every visit, except the 8-year visit for femoral neck and total hip BMD. Strontium ranelate was safe and well tolerated over 8 years.Conclusions: Long-term treatment with strontium ranelate 2 g/day in postmenopausal osteoporotic women leads to continued increases in BMD at all sites. The data also provide some evidence for a sustained antifracture efficacy.

Sex-specific compromised bone healing in female rats might be associated with a decrease in mesenchymal stem cell quantity

2009-09-01

Abstract: Introduction: The clinically known importance of patient sex as a major risk factor for compromised bone healing is poorly reflected in animal models. Consequently, the underlying cellular mechanisms remain elusive. Because mesenchymal stem cells (MSCs) are postulated to regulate tissue regeneration and give rise to essential differentiated cell types, they may contribute to sex-specific differences in bone healing outcomes.Methods: We investigated sex-specific variations in bone healing and associated differences in MSC populations. A 1.5 mm osteotomy gap in the femora of 8 male and 8 female 12-month-old Sprague–Dawley rats was stabilized by an external fixator. Healing was analyzed in terms of biomechanical testing, bridging and callus size over time (radiography at 2, 4, and 6 weeks after surgery), and callus volume and geometry by μCT at final follow-up. MSCs were obtained from bone marrow samples of an age-matched group of 12 animals (6 per gender) and analyzed for numbers of colony-forming units (CFUs) and their capacity to differentiate and proliferate. The proportion of senescent cells was determined by β-galactosidase staining.Results: Sex-specific differences were indicated by a compromised mechanical competence of the callus in females compared with males (maximum torque at failure, p=0.028). Throughout the follow-up, the cross-sectional area of callus relative to bone was reduced in females (p≤0.01), and the bridging of callus was delayed (p2weeks=0.041). μCT revealed a reduced callus size (p=0.003), mineralization (p=0.003) and polar moment of inertia (p=0.003) in female animals. The female bone marrow contained significantly fewer MSCs, represented by low CFU numbers in both femora and tibiae (pfemur=0.017, ptibia=0.010). Functional characteristics of male and female MSCs were similar.Conclusion: Biomechanically compromised and radiographically delayed bone formation were distinctive in female rats. These differences were concomitant with a reduced number of MSCs, which may be causative for the suboptimal bone healing.

Reduced physical activity corresponds with greater bone loss at the trabecular than the cortical bone sites in men

Taru Tervo, Peter Nordström, Martin Neovius, Anna Nordström — 2009-08-28

Abstract: Previous research has been inconclusive as to whether high peak bone mineral density (BMD, g/cm2) resulting from previous physical activity is retained with reduced activity later in life. The aim of this 12-year longitudinal study was to investigate the association between BMD loss and reduced physical activity (h/wk) at trabecular and cortical bone sites in men. Three groups with a mean age of 17 years at baseline were investigated: i) 51 athletes who discontinued their active careers during the follow-up period (former athletes), ii) 16 athletes who were active throughout the follow-up period (active athletes), and iii) 25 controls. BMD loss at the hip, spine, and pelvis (mainly trabecular bone) was compared to BMD loss at femur, humerus, and legs (mainly cortical bone) during a 12-year follow-up period. Across the total follow-up period in the total cohort, reduced physical activity was more strongly associated with changes at trabecular BMD sites, i.e. hip, spine, and pelvis (B=0.008–0.005 g/cm2 per weekly hour physical activity (h), p<0.001), than at cortical bone sites, i.e. humerus, legs (B=0.002–0.003 g/cm2/h, p<0.05), and femur (p>0.05). At the final follow-up, former athletes showed higher BMD than controls only at the cortical bone sites of the humerus, legs, and femur (difference 0.05–0.10 g/cm2, p<0.05). In conclusion, this study indicates that predominantly trabecular bone is lost with reduced physical activity levels in young men. Benefits were still evident at the more cortical sites eight years after the discontinuation of an active sports career.

A 246-km continuous running race causes significant changes in bone metabolism

2009-09-09

Abstract: Background: Regular physical exercise exerts a favorable effect on the skeleton. However, excessive physical exercise may have detrimental effects. A low bone mineral density (BMD) has been registered in highly trained runners. The aim of the present study was to evaluate potential effects of the Spartathlon, an annual ultramarathon race of 246 km, on bone metabolism.Methods: Venous blood samples were taken before and within 15 min after the end of the race as well as three days after the start of the race. The following variables of bone metabolism were studied: osteocalcin (Oc), cross-linked-C-telopeptide of type I collagen (CTX), osteoprotegerin (OPG), and its ligand, receptor activator of nuclear factor κB ligand (RANKL).Results: Blood samples were taken from 18 runners (16 men and 2 women) at the three time points. The median time taken by the runners to complete the race was 32 h and 52 min. Serum levels of CTX were significantly increased immediately after the race as well as three days after the start of the race compared with the time prior to the race. Oc was transiently suppressed after the race. Serum levels of RANKL and OPG were increased three days after the start of the race compared to the time before the start of the race.Conclusions: This study showed that an ultra-distance run of nearly 250 km induced changes in RANK/RANKL/OPG interaction, which suggests a transient uncoupling of bone metabolism, increased bone resorption, and suppressed bone formation.

Direct healthcare costs of hip, vertebral, and non-hip, non-vertebral fractures

Nianwen Shi, Kathleen Foley, Gregory Lenhart, Enkhe Badamgarav — 2009-09-07

Abstract: Limited data exist regarding the cost of non-hip, non-vertebral (NHNV) fractures. Although NHNV fractures may be less expensive than hip and vertebral fractures, they have a higher incidence rate. The objective of this study was to quantify first-year healthcare costs of hip, vertebral, and NHNV fractures. This was a claims-based retrospective analysis using a case-control design among patients with commercial insurance and Medicare employer-based supplemental coverage. Patients were ≥50 years old with a closed hip, vertebral, or NHNV fracture between 7/1/2001 and 12/31/2004, and continuous enrollment 6 months prior to and 12 months after the index fracture. Adjusted mean first-year healthcare costs associated with these fractures were determined. Six cohorts were identified. Patients 50–64 years: NHNV (n=27,424), vertebral (n=3386) and hip (n=2423); patients ≥65 years: NHNV (n=40,960), vertebral (n=11,751) and hip (n=21,504). The ratio of NHNV to hip fractures was 11:1 in the 50–64 cohort and 2:1 in the ≥65 cohort. Adjusted mean first-year costs associated with hip, vertebral, and NHNV fractures were $26,545, $14,977, and $9183 for the 50–64 age cohort, and $15,196, $6701, and $6106 for patients ≥65 years. After taking prevalence rate into account, the proportion of the total fracture costs accounted for by NHNV, hip, and vertebral fractures were 66%, 21% and 13% for the 50–64 age cohort, and 36%, 52% and 12% for the ≥65 age cohort. Limitations included the exclusion of the uninsured and those covered by Medicaid or military-based insurance programs. The results of this study demonstrate that osteoporotic fractures are associated with significant costs. Although NHNV fractures have a lower per-patient cost than hip or vertebral fractures, their total first-year cost is greater for those 50–64 because of their higher prevalence.

Vitamin D receptor gene polymorphisms modulate the skeletal response to vitamin D supplementation in healthy girls

2009-09-01

Abstract: Objectives: Vitamin D receptor (VDR) gene plays an important role in bone mass regulation. We have previously shown a beneficial effect of vitamin D supplementation on bone mass in girls. This study investigated whether the musculo-skeletal response to Vitamin D was modulated by polymorphisms in VDR gene.Design: Randomized placebo-controlled trial.Methods: 179 girls (10–17 years), were randomly assigned to placebo or Vitamin D3 for one year. VDR genotypes were determined in 167 girls using BsmI, TaqI and ApaI restriction enzymes. Bone mass at the spine, hip, forearm and total body, and lean mass were measured by DXA at baseline and at one year.Results: After one year, VDR gene polymorphisms using Bsm1 and TaqI restriction enzymes were associated with percent changes in bone area, BMC and BMD at multiple skeletal sites in the Vitamin D3 group but not in the placebo group. The least increments were observed in the BB and tt genotypes. No similar effect was observed with ApaI enzyme. This relationship between VDR genotypes and changes in BMD and BMC remained significant after adjustment for puberty, changes in lean mass, height and bone area.Conclusion: VDR gene polymorphisms influence the skeletal response to vitamin D supplementation in healthy adolescent girls.

HSD11B1 polymorphisms predicted bone mineral density and fracture risk in postmenopausal women without a clinically apparent hypercortisolemia

2009-09-01

Abstract: Introduction: Endogenous glucocorticoid (GC) may participate in bone physiology, even in subjects with no glucocorticoid excess. 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) is a primary regulator catalyzing the reduction of inactive cortisone to active cortisol. To elucidate genetic relevance of HSD11B1 variants to vertebral fracture and osteoporosis, we investigated the potential involvement of six HSD11B1 SNPs in postmenopausal women.Methods: All exons, their boundaries and the promoter region (approximately 1.5 kb) were directly sequenced in 24 individuals. Six polymorphisms were selected and genotyped in all study participants (n=1329). BMD was measured using dual-energy X-ray absorptiometry.Results: HSD11B1 +16374C>T and +27447G>C were associated with reduced vertebral fracture risk (p=0.016 and 0.032, respectively). Two of these (LD block2) in intron 5 (rs1000283 and rs932335) were significantly associated with bone mineral density (BMD) at the femoral neck (p=0.00005 and 0.0002, respectively). Specifically, HSD11B1 +16374C>T and +27447G>C polymorphisms were associated with higher BMD values of the femoral neck in multiple comparison (p=0.0002 and 0.0004, respectively) and Bonferroni corrected significance level (97% power). Consistent with these results, HSD11B1-ht21 and -ht22 comprising both SNPs also showed the evidence of association with BMD values of the femoral neck (pdomiant=0.0002 and precessive=0.00005, respectively).Conclusion: Our results provide preliminary evidence supporting an association of HSD11B1 with osteoporosis in postmenopausal women. Also, these findings demonstrate that +16374C>T polymorphism may be useful genetic markers for bone metabolism.

Beam hardening artifacts in micro-computed tomography scanning can be reduced by X-ray beam filtration and the resulting images can be used to accurately measure BMD

2009-09-01

Abstract: Bone mineral density (BMD) measurements are critical in many research studies investigating skeletal integrity. For pre-clinical research, micro-computed tomography (μCT) has become an essential tool in these studies. However, the ability to measure the BMD directly from μCT images can be biased by artifacts, such as beam hardening, in the image. This three-part study was designed to understand how the image acquisition process can affect the resulting BMD measurements and to verify that the BMD measurements are accurate. In the first part of this study, the effect of beam hardening-induced cupping artifacts on BMD measurements was examined. In the second part of this study, the number of bones in the X-ray path and the sampling process during scanning was examined. In the third part of this study, μCT-based BMD measurements were compared with ash weights to verify the accuracy of the measurements. The results indicate that beam hardening artifacts of up to 32.6% can occur in sample sizes of interest in studies investigating mineralized tissue and affect mineral density measurements. Beam filtration can be used to minimize these artifacts. The results also indicate that, for murine femora, the scan setup can impact densitometry measurements for both cortical and trabecular bone and morphologic measurements of trabecular bone. Last, when a scan setup that minimized all of these artifacts was used, the μCT-based measurements correlated well with ash weight measurements (R2=0.983 when air was excluded), indicating that μCT can be an accurate tool for murine bone densitometry.

Hydroxyapatite particles maintain peri-implant bone mantle during osseointegration in osteoporotic bone

2009-09-02

Abstract: In osteoporotic bones, resorption exceeds formation during the remodelling phase of bone turnover. As a consequence, decreased bone volume and bone contact result in the peri-implant region. This may subsequently lead to loss of fixation. In this study we investigated whether the presence of nonresorbable, osteoconductive hydroxyapatite (HA) particles could help maintain a denser and more functional peri-implant bone structure. Titanium screws were implanted into the proximal tibial metaphysis of four months old, ovariectomized Wistar rats (n=60). In the right tibia, the drill hole was first filled with HA particles, while the left tibia served as a control without HA particles. Histological analysis demonstrated that during the remodelling phase the amount of newly formed bone was significantly higher on the HA over the control side. Micro-CT analysis corroborated the significant changes over time as well as differences in peri-implant bone volume density between treatment and control group. Mechanical tests demonstrated that the pull-out force was greater with HA particles. These results indicate that HA particles are able to induce and maintain for a longer time a denser peri-implant bone mantle in osteoporotic bone, which may have important implications in the prevention of implant migration and cut-outs.

Mechanical stress induces Interleukin-11 expression to stimulate osteoblast differentiation

2009-09-07

Abstract: Molecular mechanism of mechanical stress-induced bone formation remains unclear. We demonstrate that mechanical unloading suppresses and reloading enhances Interleukin (IL)-11 gene expression in the hindlimb of mice in vivo. Mechanical stress to osteoblasts by fluid shear stress (FSS) in vitro rapidly and transiently enhances fosB gene transcription, stimulates binding of ΔFosB/JunD complex to activator protein (AP)-1 site of the IL-11 gene promoter, and enhances IL-11 gene transcription. Anti-IL-11 antibody blocks mechanical stress-induced enhancement of osteoblastogenesis and suppression of adipogenesis, suggesting the requirement of IL-11 for the stimulation of osteoblast differentiation by mechanical stress. Down-regulation of ΔFosB/JunD by small interfering RNA (siRNA) suppresses and overexpression of ΔFosB/JunD enhances IL-11 gene promoter activity. Consistent with our previous observations that up-regulation of ΔFosB depends upon activation of cyclic AMP response element-binding protein (CREB) via Ca2+-dependent activation of extracellular signal-regulated kinase (ERK) to phosphorylate CREB, mechanical stress-induced activation of IL-11 gene transcription is dependent upon Ca2+-ERK pathway. Present results also demonstrated that FSS to osteoblasts enhances canonical Wnt signaling in vitro, and that mechanical unloading induces and reloading suppresses the expression of a canonical Wnt signal inhibitor, dickkopf2 (Dkk2), in vivo. In addition, IL-11 siRNA enhances Dkk2 expression suppressed by FSS, and osteoblasts from IL-11 transgenic mice show reduced Dkk2 mRNA expression than those from wild-type mice. These observations are consistent with the notion that mechanical stress stimulates IL-11 gene transcription via an enhanced ΔFosB/JunD binding to the IL-11 gene promoter, and that increased IL-11 enhances canonical Wnt signal at least in part via a reduction in Dkk2 expression to stimulate osteoblast differentiation.

The role of Akt1 in terminal stages of endochondral bone formation: Angiogenesis and ossification

2009-09-01

Abstract: Longitudinal bone growth is the result of endochondral bone formation which takes place in the growth plate. The rate of chondrocyte proliferation and hypertrophy, vascular invasion with the formation of primary ossification centers and cartilage replacement by bone tissue are all important processes required for normal growth. We have shown a role for the PI3K signaling pathway in chondrocyte hypertrophy and bone growth in tibia explant cultures. In this current study, we aimed to investigate the role of Akt1, an important target of PI3K, in endochondral ossification. Akt1 KO mice showed reduced size compared to their littermates throughout life, but the largest difference in body size was observed around 1 week of age. Focusing on this specific developmental stage, we discovered delayed secondary ossification in the long bones of Akt1 KO mice. A delay in formation of a structure resembling a secondary ossification center was also seen in tibia organ cultures treated with the PI3K inhibitor LY294002. The expression of matrix metalloproteinase-14 (MMP-14), the main protease responsible for development of secondary ossification centers, was decreased in the epiphysis of Akt1 KO mice, possibly explaining the delay in secondary ossification centers seen in the Akt1 KO mice. Bone mineral density (BMD) and bone mineral content (BMC) measured in the proximal tibia of 1-year-old mice were decreased in Akt1 KO mice, suggesting that the original delay in ossification might affect bone quality in older animals.

Sonic hedgehog alleviates the inhibitory effects of high glucose on the osteoblastic differentiation of bone marrow stromal cells

2009-09-14

Abstract: To assess the influence of high extracellular glucose levels on the osteogenic differentiation of bone marrow stromal cells (BMSCs) and to determine if Sonic hedgehog (Shh) protein can alleviate those effects. BMSCs were incubated with NG (normal glucose), NG+Shh (200 ng/ml Shh in normal glucose), NG+Shh+Gan (200 ng/ml Shh and 5 μmol/L GANT61 in normal glucose), HG (high glucose), HG+Shh (200 ng/ml Shh in high glucose), and HG+Shh+Gan (200 ng/ml Shh and 5 μmol/L GANT61 in high glucose). The expression levels of Shh signaling pathway genes Patched 1 (PTCH1) and osteogenesis-related genes were tested, which included bone morphogenetic protein 4 (BMP4), runt-related transcription factor 2 (Runx2), and osteopontin (OPN). Alkaline phosphatase (ALPase) activity and mineralized matrix formation were also investigated. Immunofluorescent staining of Gli1 was tested for Shh signaling activation. We found that recombinant Shh in normal-glucose medium could promote osteogenic differentiation of BMSCs, while inhibiting Shh signaling by GANT61 could antagonize this differentiation. Besides that high glucose impaired the Shh signaling as well as osteogenic differentiation of BMSCs, reactivation of Shh signal pathway by addition of Shh protein could mitigate the inhibition while further deactivation by Shh inhibitor GANT61 could retain their osteogenic inhibitions. The above data suggest that Shh pathway activity is involved in the HG condition mediated osteoblastic differentiation deficiency for BMSCs and that recombinant Shh could alleviate this inhibitory effect.

The level of ATP analog and isopentenyl pyrophosphate correlates with zoledronic acid-induced apoptosis in cancer cells in vitro

Laura M. Mitrofan, Jukka Pelkonen, Jukka Mönkkönen — 2009-09-07

Abstract: Bisphosphonates are potent inhibitors of osteoclast function widely used to treat excessive bone resorption associated, e.g., with bone metastases. They have also antitumor activity. However, it is unclear whether this reflects an indirect effect via inhibition of bone resorption or a direct antitumor effect.Nitrogen-containing bisphosphonates (N-BPs), including zoledronic acid (ZOL), act by inhibiting farnesyl pyrophosphate synthase (FPPS). The mevalonate pathway is blocked and the accumulation of isopentenyl pyrophosphate (IPP) consequently occurs. IPP is conjugated to AMP to form a novel ATP analog (ApppI). The present study was undertaken to clarify whether IPP and/or ApppI has a direct involvement in apoptosis caused by ZOL in different cancer cell lines.There are marked differences in ZOL-induced ApppI formation between different cancer cell lines. On this basis, we selected three cancer cell lines that differ significantly from each other in their ZOL-induced IPP and ApppI accumulation: human estrogen-dependent (MCF7) and estrogen-independent (MDA-MB 436) breast cancer cell lines and a human myeloma cell line (RPMI 8226).The amount of IPP/ApppI correlated with the capacity of cells to undergo apoptosis. Geranylgeraniol (GGOH), an intermediate of mevalonate metabolism, blocks both IPP and ApppI formation and to some degree ZOL-induced apoptosis in a cell line-dependent manner. In addition, lovastatin (LOV), an inhibitor of the enzyme HMGCoA reductase, completely blocks IPP/ApppI formation as determined by mass spectrometry analysis, but enhances apoptosis.In conclusion, the current data suggest that ZOL-induced IPP/ApppI formation can contribute to ZOL-induced apoptosis. This mechanism and the inhibition of protein prenylation, both outcomes of FPPS inhibition in mevalonate pathway, seem to act in concert in ZOL-induced apoptosis in cancer cells.

Patterns of FGF-23, DMP1, and MEPE expression in patients with chronic kidney disease

2009-09-07

Abstract: Fibroblast growth factor 23 (FGF-23), dentin matrix protein 1 (DMP1), and matrix extracellular phosphoglycoprotein (MEPE) are skeletal proteins involved in the regulation of phosphate homeostasis and bone metabolism. Circulating FGF-23 levels are increased in patients with chronic kidney disease (CKD); however, FGF-23 skeletal expression and its regulation by DMP1 and MEPE have yet to be evaluated. Thus, expression of these three proteins was characterized by immunohistochemistry in 32 pediatric and young adult patients with CKD stages 2–5. When compared to normal controls, bone FGF-23 and DMP1 expression were increased in all stages of CKD; significant differences in bone FGF-23 and DMP1 expression were not detected between pre-dialysis CKD and dialysis patients. Bone MEPE expression in CKD did not differ from controls. FGF-23 was expressed in osteocyte cell bodies located at the trabecular periphery. DMP1 was widely expressed in osteocyte cell bodies and dendrites throughout bone. MEPE was also expressed throughout bone, but only in osteocyte cell bodies. Bone FGF-23 expression correlated directly with plasma levels of the protein (r=0.43, p<0.01) and with bone DMP1 expression (r=0.54, p<0.01) and expression of both proteins were inversely related to osteoid accumulation. Bone MEPE expression was inversely related to bone volume. In conclusion, skeletal FGF-23 and DMP1 expression are increased in CKD and are related to skeletal mineralization. The patterns of expression of FGF-23, MEPE, and DMP1 differ markedly in trabecular bone, suggesting that individual osteocytes may have specialized functions. Increases in bone FGF-23 and DMP1 expression suggest that osteocyte function is altered early in the course of CKD.

Effects of polymorphisms of the sex hormone-binding globulin (SHBG) gene on free estradiol and bone mineral density

2009-09-28

Abstract: Background: Polymorphisms of the sex hormone-binding globulin (SHBG) gene are associated with differences in SHBG levels, influencing the risk for breast cancer and polycystic ovarian syndrome, but no association has been reported for osteoporosis in postmenopausal women.Objective: To determine the effect of G to A substitution in the 5′UTR (rs1799941) and the Asp356Asn (rs6259) polymorphisms of the SHBG gene on bone mineral density (BMD).Methods: This is a cross-sectional study in a university-based research center from May, 2002 to December, 2007. A total of two hundred and thirteen healthy postmenopausal Caucasian women ≥ 1 year from last menstrual period participated to this study. Serum estradiol by ultrasensitive radioimmnunoassay, serum sex hormone-binding globulin by immunoradiometric assay, and urinary NTx by enzyme-linked immunoassay were measured. BMD measurements were performed by dual energy X-ray absorptiometry and genotyping by Pyrosequencing.Results: There were no significant differences in SHBG levels associated with either rs1799941 or rs6259. Using a p value of <0.00625 for significance, we found that subjects with the A allele (GA+AA) for the rs1799941, had a trend for lower free estradiol index (FEI) compared to the GG genotype (p=0.04). They also had significantly lower BMD at the intertrochanter (p=0.003) and trend for lower BMD at the total hip (p=0.02). There was no significant difference in FEI levels between the genotypes for the rs6259 polymorphism, but women with the Asn allele (Asp/Asn+Asn/Asn), had significantly lower BMD in the total femur (p=0.004) and intertrochanter (0.002) compared to those with the Asp/Asp genotype.Conclusions: Our data suggest that polymorphisms of the SHBG gene are associated with significant differences in BMD at the proximal femur sites. Thus, genetic variations in the SHBG gene may influence BMD at the hip in postmenopausal women.

Chloride intracellular channel 1 regulates osteoblast differentiation

2009-09-07

Abstract: We have identified chloride intracellular channel 1 (CLIC1) through proteomic approach, which was increased in response to canonical wnt signaling while being almost shut-off by adipogenic treatment in mouse mesenchymal C3H10T1/2 cells. We found that CLIC1 was expressed in mouse (MC3T3-E1), rat (ROS 17/2.8 and UMR-106) or human (MG63 and SaOS2) osteoblastic cell lines as well as primary culture of mouse calvarial cells by RT-PCR or Western blot analysis. The expression level of CLIC1 is increased upon treatment of osteogenic medium, whereas it almost disappeared in adipogenic condition, confirming the proteomic data. The expression of CLIC1 was localized mainly in nuclear membrane and vesiculo-cytoplasmic, the latter of which was colocalized with mitochondria. Retroviral overexpression of CLIC1 did not increase whole-cell current but induces hyperpolarization of mitochondrial membrane potential estimated using the fluorescent dye TMRE. Moreover, overexpression of CLIC1 resulted in increase in osteoblastic differentiation of C3H10T1/2 cells as measured by ALP activities or osteoblastic gene expression (osterix, ALP and osteocalcin), although it did not result in induction of Runx2 transcription activities at mouse osteocalcin (OG2) promoter. Finally, in vitro knock-down of CLIC1 using stable siRNA CLIC1 significantly suppressed osteoblastic differentiation. Taken together, these results suggest that CLIC1 may play a role in the regulation of osteoblastic differentiation from mesenchymal progenitors, although its physiologic role in osteoblasts remains to be determined.

Grizzly bears (Ursus arctos horribilis) and black bears (Ursus americanus) prevent trabecular bone loss during disuse (hibernation)

2009-09-07

Abstract: Disuse typically causes an imbalance in bone formation and bone resorption, leading to losses of cortical and trabecular bone. In contrast, bears maintain balanced intracortical remodeling and prevent cortical bone loss during disuse (hibernation). Trabecular bone, however, is more detrimentally affected than cortical bone in other animal models of disuse. Here we investigated the effects of hibernation on bone remodeling, architectural properties, and mineral density of grizzly bear (Ursus arctos horribilis) and black bear (Ursus americanus) trabecular bone in several skeletal locations. There were no differences in bone volume fraction or tissue mineral density between hibernating and active bears or between pre- and post-hibernation bears in the ilium, distal femur, or calcaneus. Though indices of cellular activity level (mineral apposition rate, osteoid thickness) decreased, trabecular bone resorption and formation indices remained balanced in hibernating grizzly bears. These data suggest that bears prevent bone loss during disuse by maintaining a balance between bone formation and bone resorption, which consequently preserves bone structure and strength. Further investigation of bone metabolism in hibernating bears may lead to the translation of mechanisms preventing disuse-induced bone loss in bears into novel treatments for osteoporosis.

Erratum to “Mechanical loading enhances the anabolic effects of intermittent parathyroid hormone (1–34) on trabecular and cortical bone in mice” [Bone 43 (2008) 238–248]

2009-09-03

In this article, we assessed the separate and combined effects of intermittent parathyroid hormone (iPTH) (1–34) and mechanical loading at trabecular and cortical sites of mouse long bones. The data suggest that, at some but not all bone sites, there is a synergistic relationship between the osteogenic effects of iPTH (1–34) and mechanical loading.

Corrigendum to “Effects of silencing connective tissue growth factor (CTGF) by RNA interference on expression of matrix metalloproteinase-1 and -2 in human osteoblasts” [Bone 43 (2008) S41–S41]

Yiqun Peng, Guoliang Sui, Eryuan Liao — 2009-10-05

The author regrets that the institution was inadvertently omitted from the affiliation line. The corrected affiliation appears above.

Announcement

2009-12-01

Relationship in the musculo-skeletal syndrome Prader–Willi

V. Montangero, R. Capiglioni, E. Roldan — 2009-12-01

It is well known and little studied osteopenia and subsequent osteoporosis sufferers carry Prader–Willi syndrome. This syndrome is characterized by muscular atony, insatiable appetite for obesity, hypogonadism, incomplete sexual development, and mental retardation. This syndrome is due to the denunciation of interstitial chromosome 15q. The aim of this study was to observe and quantify the musculo-skeletal patients with this syndrome. Materials and methods: 25 patients of both sexes, whose ages were between 7 and 25 years, were studied by radiological methods, vertebral morphometry, index radiogrammetry, and determination of bone age (Rx palmar). The muscle–bone was examined by pQCT tibia tomography cut 50% of the length. Results: The vertebral morphometry showed a trend towards greater wedging to the normal reference values, the index indicates radiogrammetic osteopenia, and bone age differs from the chronological variables. The rates of pQCT studies indicate decreased muscular mass, increased subcutaneous fat, and muscle infiltration fat, and osteopenia with decreased bone strength index in some cases could be seen rising in the internal remodeling. Conclusion: It can be concluded that a possible cause of osteopenia is due to the lack of adequate stimulation of bone mechanostat. The lack in muscular mass due to sedentary lifestyle and overweight are important factors to consider in this syndrome.

Extracellular ATP activates MAP kinase cascades and increases intestinal Caco-2 cell proliferation

N. Buzzi, P. Scodelaro Bilbao, R. Boland, A. Russo de Boland — 2009-12-01

We examined the role of ATP in the activation of the MAP kinases (MAPKs) ERK1/2, JNK1/2, and p38 and their involvement in the modulation of transcription factors and proliferation of human colon cancer Caco-2 cells. ATP induces the phosphorylation of MAPKs in a time- and dose-dependent manner. Moreover, UTP and ATPgS but not ADP or ADPβS increased phosphorylation of MAPKs, indicating the involvement of, at least, P2Y2/P2Y4 purinergic receptor subtypes. RT–PCR studies and PCR product sequencing supported the expression of P2Y2 and P2Y4 receptors in this cell line. Spectrofluorimetric measurements showed that cell stimulation with ATP induced transient elevations in intracellular calcium concentration. In addition, Western blot analysis revealed that ATP-induced phosphorylation of MAPKs in Caco-2 cells was dependent on Ca2+ influx and intracellular Ca2+ release, Src-family tyrosine kinases, and partially dependent on the cAMP/PKA and PKC pathways. The epidermal growth factor receptor (EGFR) specific inhibitor AG1478 decreased ERK1/2, JNK1/2, and p38 MAPK phosphorylation by ATP, suggesting that EGFR transactivation is important for ATP-mediated stimulation of MAPKs in this intestinal cell line. We demonstrate, by confocal microscopy, that stimulation of Caco-2 cells with ATP results in the translocation of active MAPKs to the nucleus where they induce the expression of c-Fos and Jun family proteins, the phosphorylation of ATF-1, ATF-2, and JunD transcription factors and stimulate Caco-2 cell proliferation. Moreover, MAPKs participate in the induction and phosphorylation of the dual phosphatase MKP-1, which is known to inactivate ERK, JNK, and p38 MAPK by dephosphorylating two critical sites in the activation loop. These findings provide new molecular basis for further understanding the mechanisms involved in ATP functions, as a signal transducer and activator of MAP kinase cascades, in Caco-2 cells derived from human colon adenocarcinoma.

25-OH-Vitamin D serum levels in patients with osteogenesis imperfecta from Venezuela

E.Y. Herrera González, E. Fradinger, A.L. Negri — 2009-12-01

Osteogenesis imperfecta (OI) is an autosomic dominant disease caused by a defect in the gene that codify collagen type I that produces an increase in skeletal fragility. Bisphosphonates has been frequently used in this disease to reduce bone pain and fracture rate, but these drugs are capable of producing hypocalcemia in the presence of vitamin D deficiency. The objective of the present study was to evaluate 25-OH-vitamin D levels in 41 patients from Venezuela with OI randomly selected from the total population of affected patients in Venezuela; 31 of them had received treatment with pamidronate before, but none had received oral vitamin D supplements. 25-OH-Vitamin D levels were measured in Buenos Aires by a commercial radioimmune assay (Diasorin, Stillwater, MN, USA). Patients were classified according to Holick in deficient (< 20 ng/ml), insufficient (20–32 ng/ml), sufficient (32–100 ng/ml), or with normal levels (54–90 ng/ml). Of the 41 patients, 26 (63%) were women and 15 were men (37%). The mean age of the patients was 15.7 ± 13.2 years (with a range of 2 to 45 years). Of the patients, 39% presented OI type Ia (mild), 32% presented OI type III (moderate), and 22% presented OI type IV (severe). The mean serum values of 25-OH-vitamin D of the studied simple was 29.83 ± 8.85 ng/ml (range = 13.2 to 59.4 ng/ml). Of the patients, 51% were insufficient, while 5% were deficient. Of the patients, 35% had values of sufficiency and only 2% had normal values. We did not find a significant correlation between 25-OH-vitamin D levels and age of the patients (r=0.18) or height from the sea level at which the patients lived (r=0.22; NS). We conclude that 25-OH-vitamin D deficiency (<20 ng/ml) is infrequent in the sample of patients studied with osteogenesis imperfecta probably because of the geographical latitude from where the population studied came.

Prevalence of deficit of vitamin D in patients with overweight and obesity

M. Larrroude, M. Moggia, R. Díaz, M. Pérez Sainz, G. Macías, Z. Man — 2009-12-01

Vitamin D (VD) is essential for bone and muscle health. Obesity is a risk factor for hypovitaminosis D because obese patients often have less exposure to UV radiation either by making physical activity less or the possible kidnapping of VD by the adipocytes. This determined lower synthesis of 25(OH) VD due to a negative feedback with 1.25(OH)2D and PTH. A recent survey shows that 10.6% of people between 20 and 49 years were overweight and obese and have VD values ≥36 ng/ml. Objective: To establish the prevalence of VD deficiency in postmenopausal (PM), osteoporotic, overweight, and obese women. Materials and methods: 397 PM women (mean age=65years, range=53–98 years) with osteoporosis by DXA in Buenos Aires (latitude 35°S) in Centro TIEMPO between OCT–DEC were recruited. Excluded: thyroid disease, liver and kidney failure, and corticosteroids, anticonvulsants, and anticoagulants treatments. BMI <19 was considered as low weight (LW), 19–25 was considered as normal weight (NW), 25–30 was considered as overweight (OW), and >30 was considered as obese (O). VD measured by RIA (kit DiaSorin) divided in <20, 20–30, 30–40, and >40 ng/ml. Results: LW was recorded in 2% (8) of the patients (mean age=66 years, range=54–80 years), NW in 37.8% (150) of the patients (mean age=64 years, range=53–79 years), OW in 45.3% (180) of the patients (mean age=66 years, range=55–98 years), and O in 14.9% (59) of the patients (mean age=67 years, range=54–80 years). LW: 12.5% (1) had values <20 ng/ml, 75% (6) had values between 20 and 30 ng/ml, and 12.5% (1) had values between 20 and 30 ng/ml. NW: 22.7% (33) had values <20 ng/ml, 47.3% (71) had values between 20 and 30 ng/ml, 24.7% (37) had values between 30 and 40 ng/ml, and 5.5% (8) had values >40 ng/ml. OW: 23.9% (43) had values <20 ng/ml, 53.3% (96) had values between 20 and 30 ng/ml, 22.2% (40) had values between 30 and 40 ng/ml, and 0.6% (1) had values >40 ng/ml. O: 44.1% (26) had values <20 ng/ml, 42.4% (25) had values between 20 and 30 ng/ml, and 13.5% (8) had values between 30 and 40 ng/ml. Conclusion: Although obesity has been regarded as a protective factor for osteoporosis, we detected 60.2% of osteoporotic PM, overweight and/or obese in which 79.5% had values under 30 ng/ml 25(OH)D.

Experimental models of nutritional deficiencies and hormonal status that influence bone mass

2009-12-01

Elderly people often had a low food intake and sun exposure which could contribute to the high prevalence of osteopenia and fracture risk observed in this population. To evaluate the influence of the low protein intake and/or vitamin D (vit. D) status in the pathogenesis of osteoporosis, we developed the present experimental models. Wistar rats (300±50 g) were OVX (n=20) or SHAM operated (n=20). During 15 days after surgery, all rats were fed a diet containing 15% protein and 200 IU% of vit. D. Then, rats were divided into 5 groups, fed a specified diet during an additional period of 45 days: G1: SHAM+15% casein and 200 IU% vit. D (control); G2: SHAM+2.5% casein and 200 IU% vit D; G3: SHAM+2.5% casein and 0 IU% vit. D; G4: OVX+2.5% casein and 200 IU% vit. D; G5: OVX+2.5% casein and 0 IU% vit. D. Total skeleton BMD and BMC (BMDet and BMCet, respectively) were measured by DXA (Lunar); BMD of proximal tibia (BMDtp) was evaluated; and serum Ca, 25OHD, bALP, and BGP were determined at baseline and at the end of the study (Tf). At Tf tibias were removed to measure total bone volume (BV/TV %) histologically. Results (mean±SE), G1 to G5, respectively: 25OHD (ng/dl): 29.4±6.1a, 50.0±0.2b, 29.4±1.5a, 8.6±2.5c, 10.3±4.5c. Ca (mg/dl): 10.00±0.2a, 10.0±0.1a, 9.9±0.6a, 9.3±0.1b, 9.8±0.1a. bALP (IU/l): 39.7±5.4a, 39.7±4.6a, 58.0±4.9b, 57.0±4.0b, 66.4±6.5b. BMDet (mg): 2.3±0.3a, 2.0±0.3a, 0.4±0.4b, 0.5±0.1b, 0.2±0.2c. CMOtp (mg/cm2): 55.0±16.2a, 23.0±10.1b, −25.8±11.7c, −3.0±0.4d, −40.7±13.7e. BV/TV (%): 25.0±4.0a, 9.4±1.4b, 3.1±1.5c, 5.3±2.6c, 1.0±0.4d. Different letters correspond to p<0.05. The negative changes observed in bone mass and bone volume paralleled the increment of deficiencies.

Protective role of sex hormones in apoptosis of skeletal muscle

L. Pronsato, A. Ronda, L. Milanesi, A. Vasconsuelo, R. Boland — 2009-12-01

The loss of muscle mass and strength with aging, also referred to as sarcopenia, is a prevalent condition among elderly and predicts adverse outcomes, including disability, institutionalization, and mortality. Sarcopenia has been associated to a deficit of sex hormones since the levels of estrogens and/or testosterone decline upon ageing. Although the mechanisms underlying sarcopenia are far for being clarified, evidence suggests that an age-related acceleration of myocyte loss via apoptosis might represent a mechanism responsible for muscle loss performance. Furthermore, increased levels of apoptosis have also been reported in old rats undergoing muscle atrophy. We previously demonstrated that 17β-estradiol (E2) inhibits apoptosis in C2C12 murine skeletal muscle cells through estrogen receptors (ERs) with non classical localization involving PI3K/Akt, MAPKs, and HSP27. Here, using siRNAs to silence ER isoforms, we show that E2 activates ERK2 through ERa and p38 MAPK stimulation is independent of ERs. We confirmed that E2 is able to abrogate apoptosis through MAPKs in primary cultures of neonate mouse skeletal muscle. Also, we prove that testosterone blocks apoptosis as E2. Typical changes of apoptosis such as nuclear fragmentation, cytoskeleton disorganization, mitochondrial reorganization/dysfunction, and cytochrome c release induced by H2O2 were abolished when C2C12 cells were preincubated with testosterone. Further studies are required to establish whether there is a parallelism between the mechanisms triggered by both hormones which might be involved in muscle pathologies associated to apoptosis. The data presented deepen the knowledge on the molecular basis of sex hormone-dependent sarcopenia.

Involvement of caveolin-1 in 1α,25(OH)2-vitamin D3 regulation of c-Src, MAPK cascades, and VDR localization in skeletal muscle cells

C. Buitrago, R. Boland — 2009-12-01

Previously, we demonstrated that 1,25-dihydroxi-vitamin D3 [1,25(OH)2D3] induces non-transcriptional rapid responses through activation of MAPKs in the skeletal muscle cell line C2C12. However, there is no information on the molecular mechanism underlying the initiation of 1,25(OH)2D3 signaling through these pathways. Lipid raft components have been involved in steroid non-genomic effects. In this work we investigated the role of caveolae and caveolin-1 (cav-1) in phosphorylation of MAPKs and c-Src activation by 1,25(OH)2D3. When proliferating C2C12 cells were pre-treated with methyl-beta-cyclodextrin (MbCD), a caveolae disrupting agent, under conditions in which cell morphology is not affected and no signs of apoptosis are observed, 1,25(OH)2D3-dependent activation of ERK 1/2, p38 MAPK, and c-Src was suppressed. Similar results were obtained by siRNA technology where silencing of cav-1 expression abolished phosphorylation of MAPKs and c-Src induced by 1,25(OH)2D3. Confocal immunocytochemistry and co-immunoprecipitation assays showed that cav-1 colocalizes with c-Src in the periplasma membrane zone at basal conditions. Hormone treatment redistributed these proteins into cytoplasm and nucleus and disrupted their colocalization. Confocal microscopy also revealed that 1,25(OH)2D3 induces translocation of the VDR to the plasma membrane in C2C12 cells, and this effect is abolished by MbCD. Preliminary studies suggested a 1,25(OH)2D3-dependent VDR–c-Src association. Altogether, these data indicate that intact caveolae participate in an early upstream step in 1,25(OH)2D3 signal transduction via c-Src–MAPKs and that the VDR and cav-1 are involved in the rapid events triggered by the hormone in skeletal muscle cells.

Osteonecrosis of the jaw (ONJ) in chronically treated patients with bisphosphonates (BPS)

S. Picardo, G.G. Pellegrini, E. Rey, S.N. Zeni — 2009-12-01

BPs inhibit osteoclastic activity and are the election drug for osteoporosis treatment. BPs may play a role in ONJ development. We report our experience in patients under BPs chronic treatment who were referred to us since 2007 by Buenos Aires suburban hospitals to the Dentomaxillofacial Department, School of Dentistry, Buenos Aires University. One man and fifteen women (62.75±4 years old) with ONJ diagnosis under BPs treatment administered during at least 6 months were evaluated. A total of 12 patients were treated with one BPs (alendronate: 56.25%; pamidronate: 12.50%; zolendronate: 25%; risendronate: 6.25%); the remaining were treated with two BPs: pamidronate/zolendronate, 1; alendronate/zolendronate, 2; and alendronate/pamidronate, 1. Two patients were discarded for lacking inflammatory signs after invasive dental procedures. Fourteen patients had ONJ signs (bone exposed: 62.50%; inflammation: 100%; osteomyelitis: 43.75%; delayed healing: 81.25%; oral mucosal changes: 56.25%; sequester: 50%). Reported cases were manifested in maxilla (37.50%) and in jaw (50%). While 87.50% of cases occurred after an invasive dental procedure (extractions: 87.50%; implants: 12.50%; endodontic treatments: 50%), the remaining 12.50% occurred spontaneously.

Calcium intake and its relationship to overweight and obesity. Data obtained from the National Survey of Nutrition and Health (ENNyS)

L. Kogan, E. Abeya-Gilardon, G. Mangiolavori, A. Biblieri, S.N. Zeni — 2009-12-01

Several studies performed in Argentina showed high prevalence of deficient calcium (Ca) intake independently of age and socioeconomic conditions. Overweight (OW) and obesity (OB) are increasing worldwide; Argentina is not an exception. In this regard, several epidemiological studies showed an inverse relationship between adiposity and Ca intake expressed as a quotient of caloric intake. The ENNyS during 2004–2005 detected high prevalence of inadequate Ca intake in women between 18 and 49 years old. However, the interrelationship between Ca intake and OW or OB has not been evaluated yet. The present study determined the Ca intake in 4819 women (18 to 49 years) using data of ENNyS on body mass index (BMI; low W <18.5, normal W 18.5–24.9, OW 25–29.9, and OB ≥30). In all groups, mean Ca intake was 426.5 mg [95% CI=408.1–445.5 mg]. Data showed a low Ca intake in OB (n=929: 318.6 [287.8–349.5]) and OW women (n=1164: 385.5 [386.8–414.3]) compared to those with low (n=196: 514.7 [422.6–606.8]) or normal W (n=2530: 476.7 [451.8–501.5]) (p<0.001). These results showed an inverse relationship between OW and/or OB prevalence and Ca intake, reaching statistically differences between women who presented a Ca intake included into the 1st quartile (≤166.6 mg Ca) and those included into 3rd and 4th quartiles (≥ 347.5 mg Ca). Conclusions: These results obtained in a large number of women confirmed the deficient Ca intake of Argentine women in the fertile period and suggest a possible interrelationship between OB and/or OW and a low Ca intake.

Doses-dependent effect of propranolol on bone biomechanical performance in an animal model of growth retardation

C.h.E. Lezón, M.I. Olivera, C. Bozzini, G. Champín, R.M. Alippi, P.M. Boyer — 2009-12-01

Previous studies performed in our laboratory showed that propranolol (P) attenuates the impairment of bone biomechanical performance in an animal model of growth retardation (NGR). The aim of the present work was to study the effect of different doses of P on morphometric parameters and bone structural properties in NGR rats. Weanling male Wistar rats were randomly assigned to eight groups: Control (C), C+P3.5, C+P7, C+P10.5, NGR, NGR+P3.5, NGRP+P7, and NGRP+P10.5. C and CP rats were fed freely with the standard diet. NGR and NGRP rats received, for 4 weeks (W4), 80% of the amount of food consumed by C and CP, respectively. P 3.5, 7, and 10.5 mg/kg/d was injected IP 5 d/wk, for 4 weeks in CP3.5 and NGRP3.5, CP7 and NGRP7, CP10.5, and NGRP10.5, respectively. C and NGR received saline injections at an identical dosage regimen. At W4 morphometric and mechanical studies were performed. Food restriction induced detrimental effects on body and femur growth and bone structural properties confirming previous studies. However, only P 7 and 10.5 mg/kg/d could overcome the deleterious effect of nutritional stress on biomechanical competence with a maximal response at 7 mg/kg/d. At any dosage, anthropometric and bone morphometric parameters were not affected by P. Results suggest that propranolol, depending on the dosage, markedly attenuated the impaired bone status in NGR rats, exerting its effects on spatial distribution of bone material. The different response of mechanical competence in NRG rats at different doses could be the result, at least in part, of changes in the OPG/RANKL ratio related to intrinsic activity of the drug and/or up-down regulation receptor process. UBACyT O004.

Vitamin D insufficiency in two groups of women from Southern Argentina: Seasonal effect

2009-12-01

Although it is frequent in elderly people, young adults may also be at risk of having inadequate vitamin D status. On this basis, 25(OH)D was evaluated at the end of winter and summer in the same group of women to observe seasonal changes in vitamin D status and its effect on PTH and bone markers levels. Healthy women from two Southern cities of Argentina: Cro. Rivadavia (45-S) and Ushuaia (54-S) (20 pre- and 20 postmenopausal) were studied in March and August. In fasting blood 25(OH)D (ng/mL) (Diasorin), PTHmm (pg/mL), b-ALP (IU/L), and CTX (ng/L) (Crosslaps, Osteometer Bio Tech) were evaluated. Results: Mean±SD levels at the end of winter and summer were as follows: Premenopausal: 25(OH)D: 13.8±5.3, 23.9±10.4⁎; PTH: 51.6±17.8, 52.6±37.3; b-ALP: 56±10, 61±11; CTX: 223.0±69.2, 217.8±77.1. Postmenopausal: 25(OH)D: 14.2±7.2, 23.1±10.6⁎; PTH: 59.3±25.2, 54.8±36.1; b-ALP: 62±15, 73±20; CTX: 313.1±119.4⁎⁎, 335.8±137.9⁎⁎. (⁎) P<0.05: summer vs. winter within the same group, (⁎⁎) P<0.05: pre- vs. postmenopausal in the same season. In summary, 100% of the pre- and 95% of the postmenopausal women had inadequate levels of 25(OH)D at the end of winter, while at the end of summer, reached 65% in pre- and 78% in postmenopausal women. For all women, PTH levels presented a negative correlation with 25(OH)D levels (r=−0.313, P>0.024). Bone markers increase in pre- vs. postmenopausal in both seasons without differences between winter and summer for the same group. Conclusion: Even at the end of the summer, premenopausal women had a high 25(OH)D insufficiency percentage. Grant from Buenos Aires University M099.

Strontium ranelate for the treatment of osteoporosis: Starting at which age? Two clinical cases

J. Chiarpenello, A. Sánchez — 2009-12-01

Strontium ranelate has emerged in the last few years as a first-line treatment for postmenopausal and senile osteoporosis. Its dual mechanism of action, which combines osteoblastic stimulation and osteoclastic inhibition (anabolic and antirresorptive at the same time), makes it an important element in the therapeutic armamentarium for osteoporosis. There is no published information about its use in young patients with osteopenia. We herein show the experience in two patients with secondary osteoporosis: a young male (aged 27 years) with hypopituitarism who persisted osteoporotic after replacement with testosterone and a premenopausal woman (aged 32 years) with idiopathic hypercalciuria. Both showed good biochemical and densitometric response to strontium ranelate after 1 year of treatment.

Recovery in serum 25OHD by dietary vitamin D2 or D3 in an experimental model of vitamin D. Insufficiency and established osteopenia

M. Gonzales Chaves, C. Marotte, G. Pellegrini, S. Friedman, S. Zeni — 2009-12-01

Previous studies suggested that vitamin (vit) D3 is almost twice more potent than vitD2 in raising 25OHD levels (J Clin Met Endoc 93:3015, 2008). To confirm experimentally this suggestion we performed a study using our model of vitD insufficiency and established osteopenia (Bone 39: 837–844, 2006). Female Wistar rats (200±50 g) were ovariectomized (OVX) (n=32) or sham-operated (SHAM) (n=32). During 15 days after surgery all rats fed a diet containing 15% protein, 200 IU% vitD, and 0.6% calcium. For an additional period of 45 days a diet without vitD (0 IU%) were supplied to obtain vitD insufficiency. After 105 days rats were divided into 4 groups that fed a diet containing 200 UI% of vitD2 (OVX+vitD2 and SHAM+vitD2) or vitD3: (OVX+vitD3 and SHAM+vitD3). Basal 25OHD levels were randomly obtained in 9 rats. At 60, 85, and 105 days of experience serum 25(OH)D (ng/ml) was determined in each group by employing the methodology considered to assess both 25OHD3 and 25OHD2 (125I RIA Kit, DiaSorin, Stillwater, MN, USA). The results of 25OHD were (x±ES): basal 19.2 ± 0.3: OVX+D2 T:60=5.0±0.9a, T:85=16.6±1.5b, T:105=15.5±0.3b; OVX+D3 T:60=5.0±0.6a, T:85=15.1±0.6b, T:105=15.9±1.0b; SHAM+D2 T:60=4.8±0.5a, T:85=14.7±1.4b, T:105=15.5±0.9b; SHAM+D3 T:60=6.0±1.2a, T:85=16.6±2.1b, T:105=18.0±1.3b. Different letters indicate p< 0.05. Results indicate no differences in 25(OH)D levels because of feeding vitD2 or vitD3. Conclusions: Under our experimental conditions, vitD2 and vitD3 supplied daily had the same power to maintain and to increase 25(OH)D levels, in both estrogen conditions. PIP 6483.

Decrease in femoral neck (FN) bone mineral density (BMD) after menopause: Relative importance of changes in bone mineral content (BMC) and area (A)

H. Claus Hermberg, M. Lozano, M. Rey, M. Pozzo — 2009-12-01

Bone mineral density (BMD) of the femoral neck (FN) suffers a rapid loss during the first 5–7 years after menopause. We investigated the changes of BMC and A (both determine BMD) along life in women after menopause. Materials and methods: In a cross-sectional study, BMD, BMC, and A of left FN were measured with DXA (Lunar Prodigy) in 191 women which required bone routine evaluation. Pearson regression test was used to evaluate the influence of age on the three variables on the whole population (WP) and group (G) 1<60 and G2≥60 years old, assuming that at 60 years old most women are 5–7 years after menopause. Mean differences between groups were evaluated by Student's t test. Results: Age (X±1 SD) was 63±11, 51±6, and 70±7 years old for the WP, G1, and G2, respectively. Correlation coefficients (r) of BMD, BMC, and A vs. age were −0.49, −0.32, and 0.26 for the whole population (p<0.001). In G1 the correlation was significant (negative) for BMD and BMC but not for A, while in G2 the correlation was significant for BMD (negative) and A (positive) but not for BMC. The regression coefficient (RC) of BMD was explained in 92% by decrease in BMC in G1 and in 75% by increase in A in G2. RC of BMD in G1 (−0.0076) is greater than in G2 (−0.004) in absolute values. In a multivariate analysis including height and weight, age was maintained significant in WP and G2. Mean differences between G1 and G2 for DMO (0.897±0.11 vs. 0.80±0.10), CMO (4.2±0.7 vs. 3.8±0.5), and A (4.69±0.3 vs. 4.81±0.3) were significant (p<0.01). Conclusions: BMD of FN decreases with age after menopause. Loss of bone mass could determine this reduction during the first years, while increase in area could be responsible later.

Effect of aluminum toxicity on biomechanical bone quality in post-hypoxic immature rats

G. Dmytrenko, M.I. Conti, M.I. Olivera, C. Bozzini, G.M. Champin, M.P. Martínez — 2009-12-01

Aluminum (Al) is a nonessential element to which humans are exposed. Chronic administration induces a negative effect on bone tissue, affecting collagen synthesis and matrix mineralization. Its toxic effects are cumulative. Hypobaric hypoxia (HX) induces stress erythropoiesis leading to hypertrophy of the erythropoietic marrow and may affect bone. The aim of this investigation is to evaluate the chronic effect of aluminum on the architectural properties and the biomechanical quality of femoral diaphysis under hypoxic conditions. Female Sprague-Dawley rats, aged 21 days, received IP doses of 27 mg/kg of elemental Al, as Al(OH)3, 3 times a week, during 3 months. Control rats were injected with vehicle (20% glycerol). Half of the animals of each group (n=9) were exposed to simulated high altitude (SHA, 506.5 mbar). Morphometric femur studies were performed measuring the distance between stable anatomical points. Biomechanical performance was estimated by the three-point yielding method. Al administration did not affect general or femoral anthropometries although it significantly depressed structural strength (load capacity parameters). The indicators of bone material intrinsic properties, elastic modulus and stress at the yielding point, were significantly reduced either by Al or by HX with no additive effect in the AlHX group (p<0.01). Results suggest that chronic intoxication with Al or exposure to SHA induced negative effects on bone material quality but significantly enhanced the moment of inertia. This is presumably an adaptive response to compensate the impaired diaphyseal strength. However, no additive effects were observed when both treatments were assayed together. UBACyT O407.

Regulation of endothelial cell function by phytoestrogens

M. Sandoval, P. Cutini, M.B. Rauschemberger, V. Massheimer — 2009-12-01

Epidemiological studies propose that phytoestrogens rich diet may attenuate bone loss in post-menopausal women. In this study, we evaluated whether the isoflavone genistein (Gen) would modulate the cellular and molecular events associated with vascular disease. We employed rat endothelial cell (EC) cultures. After 24 hours of treatment, Gen increased DNA synthesis in synchronized EC (70.52±6.77 vs 41.24±4.49 cpm103/mg prot, Gen vs Control). This action was prevented by the presence of estrogen receptor antagonist ICI 182780 or l-NAME (nitric oxide synthase inhibitor), suggesting that the stimulation of EC growth involves ER and nitric oxide production. To study Gen's effect on leukocyte adhesion to EC, isolated monocytes were added to EC previously exposed to Gen or to bacterial lipopolysaccharide (LPS). Maximal adhesion was detected in LPS group, meanwhile Gen prevented monocyte adhesion (267±36, 417±21.5, 160±9.8 cells/μl for control, LPS, and Gen, p<0.01). Since leukocyte adhesion depends on cellular adhesion molecules (CAMs) expression, we evaluated Gen regulation of CAM expression using RT-PCR. VCAM-1, P-selectine, and E-selectine mRNA levels were diminished in cells exposed to Gen, compared to control or LPS group, suggesting that the inhibition of monocyte adhesion would be due to Gen's effect on CAM–RNA expression. To investigate the action of the Gen on EC apoptosis (DNA laddering) we used H2O2 as an apoptosis inducer. In the presence of H2O2, EC DNA was completely fragmented, but if EC were incubated with Gen 24 hours prior to H2O2 addition, DNA laddering was partially inhibited. In summary, the results obtained shows that Gen stimulates EC proliferation and reduced monocyte adhesion and EC apoptosis induced by cytotoxic agents, suggesting a potential benefit action of the phytoestrogen at the vascular level.

Melatonin reverses apoptosis through which menadione inhibits intestinal calcium absorption

2009-12-01

We have previously demonstrated that menadione (MEN) inhibits intestinal calcium (Ca2+) absorption through the mitochondria apoptotic pathway (BBA, 2008). Afterwards, we have observed that this inhibition could be reversed by melatonin (MEL) administration. The aim of this study was to elucidate the molecular mechanisms of this protection caused by MEL. Four-week-old chicks were divided in four groups: 1) controls, 2) treated i.p. with 2.5 μmol MEN/kg of b.w., 3) treated i.p. with 10 mg MEL/kg of b.w., and 4) treated i.p. with MEL after i.p. MEN administration. Glutathione (GSH) content and the activities of superoxide dismutase (SOD), catalase (CAT), and caspase 3 were measured by spectrophotometric methods. Apoptosis was evaluated by the TUNEL technique and cytochrome c localization. Data indicate that GSH depletion produced by MEN was completely reversed with MEL treatment. The increment in the SOD and CAT activities provoked by MEN was abolished. The release of cytochrome c from mitochondria and the increase in caspase 3 activity, both effects triggered by MEN, returned to the control values after MEL treatment. To conclude, MEL reverses the alteration in the intestinal Ca2+ absorption produced by MEN, counteracting the oxidative stress and the activation of the mitochondrial apoptotic pathway. Therefore, MEL has anti-apoptotic properties that protect the intestine against apoptosis produced by MEN and maybe by other oxidants, which suggests that MEL is a potential drug for reversing the inhibition of the intestinal Ca2+ absorption produced by oxidative stress. Dr. Tolosa de Talamoni and Dr. Carpentieri are Members of the Investigator Career from CONICET.

Dual hip densitometry: Analysis of densitometric and geometrical differences

H. Claus Hermberg, M. Lozano, M. Rey, M. Pozzo — 2009-12-01

Bone mineral density (BMD) of both femurs is often requested in spite of that World Health Organization does not recommend it for assessment of densitometric categories. We used data of patients who realized dual femur densitometry to analyze the variations in densitometric and geometrical properties between both hips. Materials and methods: BMD was measured with DXA (Lunar Prodigy). BMD (mg/cm2) disparities of femoral neck and total hip were analyzed by Z score. Differences in BMD, bone mineral content (BMC, g), area (A, cm2), cross-sectional moment of inertia (CSMI, mm4), hip axis length (HAL, mm), hip axis/diaphysis axis angle (angle, degrees), and calculated volumetric BMD (vBMD, g/cm3) were computed by subtracting the left hip values to those of the right. Results: Median (upper quartile) BMD disparities in Z score were femoral neck 0.28 (0.4) and total hip 0.2 (0.3). No woman had a difference higher than 0.8. The following significant differences from null (reference value) were observed in the neck (mean ±SD): A=0.074±0.16, Vol=0.264±0.69 (both p<0.001), CSMI=313±1343 (p=0.036), and marginal significant vBMD=−0.007±0.03 (p=0.051), CMO=0.051±0.25 (p=0.06). Bigger A in the right femoral neck was observed in 58 women (68%), significantly higher than expected if the distribution were random (p<0.001). Conclusions: Little BMD variations between both hips were observed. Right femoral neck was modestly bigger (A) and showed a trend to higher BMC. Since both have opposite effects on the determination of BMD, BMD did not show significant difference, whereas CSMI, which is positively influenced by them, did.

Calcemia and tetany in hypocalcemic models in the rat

M. Lupo, A. Rigalli — 2009-12-01

Low PTH levels decrease bone remodeling and calcemia. This situation can be obtained by ablation of either thyroid and parathyroid glands (TPTX) or parathyroid glands (PX). Both surgeries produce calcemia lower than 7.5 mg/dl. After the mentioned surgeries, hypocalcemia and tetany make difficult the follow-up of rats under treatments and surgeries. The objective of this work was the development of hypocalcemic models that avoid tetany. After PX, TPTX and ablation of one parathyroid gland (1/2PX), calcemia (before and 5 and 20 days after surgery), tetany, and survival were assessed. Results were expressed as mean±SEM and differences were considered significant when p<0.05. Experimental groups (n=8 each): G1: PX, G2: PX+calcium gluconate (CaG) in the drinking water. After 5 days rats of G2 were subjected to general anesthesia and divided into as follows: G2a, without treatment, and G2b, with intramuscular CaG administration. G3, PX+CaG in the drinking water+water without CaG; G4, 1/2PX+CaG in the drinking water; G5, TPTX. Calcemia (mg/dl) before surgery was not different among groups. Calcemia after 5 days was lower in all groups when compared to the day of surgery (G1: 4.8±0.7, G2: 7.6±0.8, G2a: 7.2±0.5, G2b: 7.7±1.3, G3: 5.9±0.8, G4: 3.5±4.9, G5: 7.6±0.3). Rats of G1 had tetany, which was not present in the other groups. G2a and G2b had tetany after anesthesia, which produced 100% of death in rats of G2a. The intramuscular injection of CaG in G2b produced 100% of survival. Calcemia at day 5 were lower in G1 and G4 compared to the other groups. Conclusions: 1. PX and 1/2PX produced a more severe hypocalcemia than TPTX. 2. The administration of CaG in the drinking water abolished tetany, but not under anesthesia. 3. ½ PX is a model of transitory severe hypocalcemia. 4. The intramuscular administration of CaG increased survival during general anesthesia.

Histological differences in bones of rats treated with sodium fluoride (NaF) and sodium monofluorophosphate (MFP)

L.R. Brun, S.M. Roma, F. Pérez, A. Rigalli — 2009-12-01

NaF and MFP increase bone mass but the quality of bones under both treatment have not been compared. The objective of this work was to evaluate the quality of bone of rats under treatment for 30 days with 80 μmol of NaF (n=8) or MFP (n=8) in 21-day-old female rats. The right tibia was subjected to histomorphometric and histopathologic analysis of trabecular bone. Bone volume (BV%), trabecular number (Tn), and trabecular thickness (Tt) were measured. Cellular proliferation, organization, and inflammation were evaluated simultaneously. Endosteal and periosteal perimeters were measured on the diaphysis of femur. BV% (mean±SEM) was higher in NaF and MFP when compared with controls (C: 22.1±2.7, NaF: 35.2±8.2, MFP: 33.3±7.1). The same result was observed in Tt and Tn. Periosteal and endosteal perimeters were higher in MFP compared with C and NaF. The bone histological appearance NaF-treated rats, showed increased cellularity in the bone marrow and in bone, irregular trabeculae with unevenly distributed osteocytes and reactive osteoblasts, arrayed in two or more layers, with conspicuous osteoclasts, indicating a disorganized and immature cancellous bone structure. Multiple foci of acute inflammation and peritrabecular bands of fibrosis was also noticeable. Rats treated with MFP showed prominent bone formation but in a more organized fashion, with osteoblasts disposed in a continuous layer, one cell thick, lacking inflammatory changes and fibrosis. Conclusions: NaF and MFP increased BV% as a consequence of the increase in Tn and Tt. In the case of NaF inflammation, fibrosis, disorganized, and immature bone were observed. This pattern was not present in MFP treated rats. At the cortical level, MFP increased the sectional area of femoral diaphysis.

Osteoclastic recruitment in bisphosphonates-treated animals and the relation with splenic and medullary macrophages

N.D. Escudero, N.F. Mina, P.M. Mandalunis — 2009-12-01

Antiresorptive effects of bisphosphonates (BPs) have been extensively studied. However, in vivo data regarding the osteoclast (Oc) and other cells of the mononuclear phagocytic system (MPS) remains controversial. The aim of this work was to study Oc recruitment, number, morphology, and its association with the macrophage population in animals treated with two BPs: alendronate and olpadronate (Gador SA). Wistar rats were divided into three groups: ALN: received alendronate; OPD: received olpadronate; both in an i.p. dose of 0.3 mg/kg/wk during 5 weeks. The sham group received vehicle. All animals received 5-bromo-2-deoxyuridine i.p. (BrdU; 0.1 mg/g bw). Within each experimental group, animals were divided into sub-groups according the day of BrdU administration: day 28 or 34. After sacrifice, spleen and femur were processed for ED1 and BrdU IHCh detection. Data were analyzed statistically. Femur:Oc number increased in BPs treated animals. The number of nuclei per cell was also higher (sham 4.5±1.7, OPD 8.1±5.1, ALN 8.9±5.5; p<0.01), showing giant Ocs and loss of cell polarity. The number of positive nuclei per Oc was evaluated by BrdU detection, showing a rise in the number of BrdU+ nuclei in BPs treated animals in both groups (days 28 and 34). ED1 detection showed macrophage depletion (No. macrophages/mm2: sham 1.5±1.2, OPD 0.6±0.8, ALN 0.3±0.6; p<0.05) in bone marrow and spleen (sham 25.7±10.3, OPD 15.4±6.4, ALN 15±5.2; p<0.01). Conclusion: the increase in Oc number and the presence of giant Ocs during BPs treatment are due to an increase in the fusion of mononuclear preosteoclasts. Macrophage depletion suggests that BPs detour the monocyte/macrophage pathway towards the monocyte/preosteoclastic lineage.

Pseudohypoparathyroidism in children

C. Tau, G. Viterbo, N. Geniuk — 2009-12-01

Pseudohypoparathyroidism (PHP) is an hereditary disease characterized by hypocalcemia and hyperphosphatemia due to resistance to parathyroid hormone. Patients with PHP-Ia often present other hormone resistances and show a physical phenotype called Albright's hereditary osteodistrophy (AHO), brachydactyly, short stature, obesity, round face, mental retardation, and subcutaneous calcifications. We analyzed 13 patients (8 girls) with PHP. The age of beginning of the disease was variable: (X±SD) 4.7±4.4 years old (0.1–12.4). The age of consultation was 7.9.±3.9 years old (2.2–14). The mean Z-score of height was −0.11±1.5 (−2.66 to 2.30) and weight was 0.93± 1.93 (−1.51 to 4.7). Two patients had short stature. The body mass index Z-score was 2.2±2.0 (−0.60 to 5.59). Six patients had obesity, 1 had overweight. All patients had at least 2 stigma of AHO. Every patient had hypocalcemia (7.0±1.5 mg/dl, 4.2–8.7 mg/dl), hyperphosphatemia (8.2 ±1.8 mg/dl, 6.2–11 mg/dl), and elevated PTH (483±543 pg/ml, 88–2100). Patients were treated with calcitriol 0.25 to 1 mg/d (26 ±12 ng/kg/d) and 1 to 3 g of calcium/d. Calcemia and phosphatemia normalized in 1 month. Calcitriol dose was monitored by calciuria. Twelve patients had hypothyroidism due to TSH resistance. They were treated with levothyroxine. Growth hormone (GH) deficiency was identified in 4 of 7 patients explored, due to resistance to GHRH, but only 2 patients (10.3 and 11.8 years old) received GH therapy. Two patients had evidence of luteinizing hormone/follicle–stimulating hormone (LH/FSH) resistance. Early diagnosis, the study of other hormone resistances, and periodic control of therapy are mandatory to promote a correct growth and to decrease the consequences of hypocalcemia and hyperparathyroidism.

Sporadic hypocalciuric hypercalcemia

M. Pozzo, M. Lozano, M. Rey, H. Claus Hermberg — 2009-12-01

The presence of hypercalcemia, relative hypocalciuria, and a normal or modestly elevated parathyroid hormone (PTH) should raise a suspicion of familial hypocalciuric hypercalcemia (FHH). Without a positive family history, it is very difficult to separate FHH from mild primary hyperparathyroidism (PHPT). The ratio clearance calcium/clearance creatinine (Clca/Clcr) has been proposed as a useful tool for separating both entities. About 80% of patients with FHH have a clearance ratio of less than 0.01, whereas almost all patients with typical PHPT have a value above 0.02. Hypocalciuric hypercalcemia has rarely been caused by autoantibodies against the calcium sensing receptor (CASR) associated to other autoimmune features (thyroiditis or sprue). We report four female patients who presented hypercalcemia and hypocalciuria. Conditions which usually contribute to low rates of calcium excretion were excluded (lithium or thiazides administration, vitamin D deficiency). Serum calcium (RV: 8.8–10.2 mg/dl), PTH (RV: 10–65 pg/ml), and Clca/Clcr for each patient were as follows: P1: 11.2, 100, 0.006; P2: 10.4, 107, 0.009; P3: 11.1, 77, 0.005; P4: 11.1, 84.7, 0.003, respectively. Family screening was negative in all patients. In P1 a sequencing test of the CASR was realized and no mutation was found. P2 and P3 underwent neck exploration which confirmed a single parathyroid adenoma (1.5 g and 0.24 g, respectively). Serum calcium level normalized after surgery in both cases. P4 had autoimmune thyroiditis and rheumatoid arthritis with positive antibodies. Conclusion: relative hypocalciuria is probably not so an unusual finding in PHPT. PTH levels in our patients were higher than those reported in FHH, which suggests PHPT. This diagnosis was confirmed in two patients. The relationship between hypocalciuria and autoimmunity in P4 remains speculative.

Body composition and bone markers in obese (β) growing rats: Influence of the dietary calcium content

2009-12-01

Osteoporosis and obesity are interrelated. We compare the effect of feeding different dietary Ca levels on body weight (BW) and composition and its relation to bone markers levels, in genetically modified obese (βO) rats during growth. Rats were mated and fed diets varying Ca content (g/100 g): high, 0.9 (GβH); normal, 0.5 (GβN); low, 0.2 (GβL). A Wistar group was run simultaneously (GCW) (fed according to AIN93). At weaning, male pups continued feeding the same diet till 50 days of age. Food consumption and BW were recorded. At the end of the experience, body composition, BAP, BGP, and CTX were determined. Results (mean±SD): Food intake was similar in all groups. BW was lower in GβH vs. N and H groups (162±18 vs. 217±31 and 221±37; p<0.01). Ash content (g/100 g) increased in Gβ as dietary Ca content increases (1.26±0.42, 2.04±0.25, 2.51±0.17; p<0.01), without reaching GCW values (2.90±0.40). Lipid percentage was higher in GβL and GβN (14.7±1.3 and 13.6±2.7) vs. GCW (10.9±2.6) and in GβH (12.6±2.2; p<0.05). BAP (IU/L) did not show differences among Gβ groups (186±27, 193±39 and 184±30), being higher vs. GCW (67.8±10.0). BGP showed an inverse correlation with dietary Ca level in Gβ (447±45, 375±46, and 279±73), being lower than GCW (825±106; p<0.01). CTX (mg/ml) did not show differences between GCW (88±16) and GβH (87±4) but they were higher than GβN and GβL (69±12 and 70±3, respectively; p<0.01). Conclusions: In βO rats, the increase in dietary Ca content decreased BW changes and body lipids content and increased total BMC. Bone markers suggest an imbalance in bone metabolism related to the adipose tissue. UBACYT B 091.

Does a new source of monounsaturated fatty acids improve lipid profile and body mineral content in nutritional hypercholesterolemia?

2009-12-01

In recent years, food industries has demonstrated increased interest in food rich in high-oleic-acid sunflower oil (HOSO, ω9) made from sunflower oil (SO, ω6) to substitute trans-fatty acids. Diets containing HOSO are often promoted to treat nutritional hypercholesterolemia (NHC); however, there are limited data about the effect of this new source of monounsaturated fatty acids (MUFA; HOSO, ω9) on lipid profile and body mineral content (BMC). We performed the study in an experimental model of NH. Weaning male Wistar rats (n=34) were randomly assigned to one of 5 groups (C, Control; E, Experimental). All groups underwent an atherogenic diet (AD, pellets+saturated fat+cholesterin) for 3 weeks (T3), followed by 3 weeks (T6) of the following: C remained on AD; E1 and E2 replaced AD by HOSO and SO, respectively; E3 and E4 replaced saturated fat by HOSO and SO, respectively. At T3 serum total CHO (TCHO) and at T6, TCHO, triglycerides, TBARS, HDL-CHO, n-HDL-CHO, and BMC were evaluated by DEXA (Lunar DPX-L). Results (median±SD, Pearson's correlation coefficients were calculated; p=0.05). At T3, all groups showed HC (p<0.001). At T6, E3 increased in TCHO and TBARS vs. E2 and C (p<0.001). n-HDL-CHO decreased in rats fed UFA (p<0.001). The correlation coefficients between lipid profile and BMC for TCHO, n-HDL-CHO, and HDL-CHO were as follows: HC rats (n=20), −0.78 (0.0001), −0.78 (0.0001), +0.44 (0.06) vs. normo rats (n=14), 0.03 (ns), −0.60 (0.02), +0.48 (0.08). No correlation with triglycerides was found. The findings suggest that NHC individuals may not benefit from eating diets rich in HOSO. Efforts are needed to improve diets. We suggest to follow-up BMC. UBACyT O008 and O015.

Late onset of X-linked hypophosphatemic osteomalacia

L. Plantalech, M. Buttazzoni, S. González, M.A. Redal — 2009-12-01

X-linked Hipophosphatemic rickets (Rk) and osteomalacia (Om) is a familiar dominant inheritance disease (PHEX gene mutation) that begin in childhood. Recent studies show sporadic forms and late manifestations. A clinical sporadic case of late onset will be described: A 35 year old Paraguayan woman had bone pains in both hips since her first childbirth (22 years old) and progress after the 2nd delivery. She has no family history of Rk or Om. Hips screw was fixed in both femurs. At physical examination: weight, 70 kg; height, 1.60 m; BMI, 27.3. Inferior limbs are shortened (L), in abduction (R). She had “duck-like” gait and pain at superior limbs. The complementary studies shown: (1) Rx: Looser's zone in right fibula and humerus, Fx ileopectineal branches. (2) Bone scintigram with hypercaptation in hip, ribs, pelvis. (3) BMD: spine: z −2.5, femoral neck: z −2.8. (4) Biochemical: Cap 8.4 mg%, Pp 2.0 mg% (2.7–4.5), Ca/cru 0.03, Cl P 24 ml/min, RTP 64%, PTHmm 298 pg/ml (100), 25OHD 26 ng/ml, 1.25(OH)2D3 <4 pg/ml (25–45). (5) NMR reject rumor induced Om (TIO). Late onset of pseudo vitamin D Om was considered and calcium 1.5 g/d and calcitriol 0.75 μg/d was started. Two years later, clinical, radiological, and bone densitometric parameters were improved, but persistence of hyperphosphaturia was seen and genetic studies were started. CYP27B1 (1α hydroxylase), no mutations in exons 1-9 (GeneDx): FGF23: no mutations in exons 1–3. PHEX: A reported mutation (A>G) in 41 position of intron 8 was shown (abnormal “splicing”). The high requirements of calcium and phosphorus during the pregnancies exposed Om. The genetic research is imposed in patients with hypophosphatemic Om of late and sporadic appearance. Mutations in genes alleviate the long-term search for TIO.

Genotypes of the vitamin D and the estrogen receptors in three osteoporotic members of a family

M. Echecury, E. Nebel, A. Pérez, G. Picotto, N. Tolosa de Talamoni, A. Sánchez — 2009-12-01

There is a relationship between some polymorphic sites of the vitamin D receptor and the estrogen receptor genes, and bone mineral density (BMD). The goal of this report is to show this relationship in three members of one family. Patient PC is a 28-year-old male, BMI 19.2 kg/m2. He smokes 10 cigarettes/d. He sustained five peripheral fractures between the ages of 8 and 20 years. When he was 23, radiographs taken for a pre-occupational examination showed osteopenia. Bone densitometry (DXA) showed T-scores of −3.3 (spine) and −3.4 (femur). Causes of secondary osteoporosis were ruled out. Treatment with oral strontium ranelate has been started. Patient LB is a 53-year-old housewife, 3 years postmenopausal, without past history of fractures. Osteoporosis was diagnosed at the age of 50 after the finding of osteoporosis in her son. Her mother (78) is alive and well; her father died with dementia at age 80. Vertebral BMD (QCT) is low: T-score was −2.73. Patient NB is a 31-year-old female clerk, without past history of fractures, BMI 23.2 kg/m2. Osteoporosis was diagnosed at age 27, after the diagnosis of the disease in her half brother. BMD (DXA): T-scores were −2.4 (spine) and −1.6 (femur). She presents no risk factors for osteopenia. Treatment with strontium ranelate and calcium supplements has been started. The three patients are related: PC and NB are LB's children, but were fathered by different men. Polymorphisms were assessed by RFLP–PCR technique using BsmI and FokI for vitamin D receptor gene (VDR) and PvuII for estrogen receptor-alpha gene (ERalpha) as restrictases. Results were as follows: Patient PC: Pp, bb, Ff. Patient LB: Pp, bb, Ff. Patient NB: pp, bb, FF. Of interest is the finding that the three patients are homozygous for the bb allele, which we found to be most frequently associated with low femoral neck BMD in postmenopausal osteoporotic women from the city of Cordoba (Argentina) and surroundings (cf. Pérez A, et al. JBMM 26:358, 2008).

Low fat-free soft mass in legs of Duchenne's disease carriers

F.D. Saraví, A. Mampel, M.I. Echeverría, A.L. Vargas — 2009-12-01

Duchenne/Becker muscular dystrophy (DMD/BMD) is an X-linked disease caused by mutations in the dystrophin gene at Xp21 and therefore affects males. In these males, lower bone mineral content and lean mass, and higher fat mass, have been found by several methods, including dual x-ray absorptiometry (DXA). While women are carriers, subtle biochemical and structural alterations have been reported in them (e.g., elevation of serum creatine phosphokinase and muscle weakness). Aiming to assess whether DXA might be used for screening women with suspected DMD/BMD carrier status, whole body scans (GE Lunar Prodigy) were performed in eight known carriers and eight healthy women matched by age. Three carriers had 2 affected sons each. The other five women had one affected son each, and their carrier status was confirmed by a molecular method (Multiplex Ligation Probe Amplification). In this sample, no significant difference was found in height, body mass, total bone mineral density, total bone mineral content, total fat mass, or total body fat-free soft tissue mass (FFSTM). An analysis focused on the lower legs (region of interest set between knees and ankles) showed no significant difference for bone mineral content, bone mineral density, or fat mass. However, linear regression showed for this region a high correlation between leg FFSTM and bone mineral content in carriers (r=0.89; p=0.003) which was absent in controls. Importantly, leg FFSTM, known to correlate with muscle mass, was 3005 (SD 424) g in carriers but 3484 (SD 300) g in controls; p=0.021. These findings (not previously reported) suggest that leg DXA analysis merits further study as an additional screening tool for DMD/BMD carrier status.

Calcium nutritional status and its relation to oral health, exercise, and lifestyle in a group of students ranging 18 to 34 years old

O. Antonenko, G.G. Pellegrini, G. Brito, J. Somoza, S.N. Zeni — 2009-12-01

Calcium (Ca) nutritional status, oral health, exercise and lifestyle was evaluated in 25 college students (ISALUD University): 28% of males and 72% women (18 to 34 years old). Weekly consumption, frequency, dietary practices, exercise, and lifestyle (consumption of cigarettes, soft drinks, contraceptives, etc.) were recorded. Blood and 2-h urine sample was obtained in a fasting state. Dental–oral status and total skeletal BMD by DXA was assessed. Body weight was 59.8±12.6 kg. Dairy products consumption was quite low, so 96% had Ca intake below the adequate intake and 48% <500 mg/d; 16% did not consume fruits and 4% vegetables. Good quality protein intake (<0.66 g/kg) was deficient in 36% of the students. The 100% of the students had chronic gingivitis, 68% oral soft tissue injuries, 72% bruxism, and 84% tightening. Of the students, 68% had high consumption of sugar (MA)>4. Also, 48% showed partial tooth loss (83% had MA >4 and 92% consumed soft drink daily). Only 20% exercised every day, 24% exercised 2–3 times per week, and 20% did not exercise at all. The remaining hiked regularly. The 39% of women took contraceptives and 39% women and 14% men were smokers. Conclusion: Based on the results obtained, there is a high Ca deficiency in the studied group with dietary and lifestyle habits that would affect oral health and, in the future, skeleton status. A nutrition education would benefit the nutritional status and would improve life quality of subjects.

Connexin43 modulates PTH-induced survival signaling in osteoblasts through its interaction with β-arrestin

V. Lezcano, N. Bivi, T. Bellido, L.I. Plotkin — 2009-12-01

Connexin (Cx) 43 expression in osteoblasts is necessary for the cAMP-mediated anti-apoptotic effect of parathyroid hormone (PTH) in vitro and for bone anabolism induced by the hormone in vivo. We found that PTH response in osteoblastic cells silenced for Cx43 using shRNA was restored by expressing wild type Cx43 or a Cx43 mutant unable to form gap junctions, but not by Cx43 mutants lacking the C-terminus tail (Cx43D245) or unable to be phosphorylated at Ser368 (Cx43S368A). These results indicate that channel activity is not required whereas phosphorylation of Ser368 is indispensable for survival by PTH. We have previously shown that β-arrestin, a protein that interacts with PTH receptor (PTHR) reducing intracellular signaling downstream of cAMP, also associates with Cx43 in osteoblastic cells. Thus, we hypothesized that Cx43 sequesters β-arrestin, thereby allowing PTH-induced survival signaling. To test this hypothesis, we investigated whether phosphorylated Cx43 C-terminus was also required to sequester β-arrestin and, consequently decrease β-arrestin/PTHR association, using an enzyme fragment complementation assay in CHO cells (DiscoveRx). As expected, PTH increased β-arrestin/PTHR interaction, which was reduced by transfecting Cx43, whereas transfection of Cx43D245 or Cx43S368A had no effect. Moreover, α-glycyrrhetinic acid increased Cx43 phosphorylation in Ser368 and also reduced β-arrestin/PTHR interaction. Our studies suggest that the role of Cx43 on osteoblast survival induced by PTH is due to sequestration of β-arrestin by pSer368Cx43, showing a new scaffolding function of Cx43.

Rat mandible bone response to atherogenic diet

2009-12-01

We observed that an atherogenic diet in active growth phase did not induce macroscopic alterations in mandible. However, diminution in bone accretion and alterations in mandible skeletal units could lead to structure alterations. In this study we hypothesized that atherogenic diets could alter bone remodeling in mandible. Male weanling Wistar rats (n=11), randomly divided into control (C) and experimental (E). C received rodent stock diet (pellets) and E, atherogenic diet (pellets+saturated fatty acids+cholesterol) for 7 weeks (Tf), monitoring anthropometry and diet intake. At Tf, blood (mg/dL) lipid–lipoprotein atherogenic profile: total cholesterol (T-cho), triglycerides (TG), high density lipoprotein-cholesterol (HDL-cho), HDL-non-cholesterol (n-HDL-cho). Dissected hemimandibles, fixed in buffered formalin–PBS 10%, decalcified in EDTA (pH 7.2), embedded in paraffin for mesio-distal oriented sections of first mandibular molar, stained with hematoxylin–eosin, where histomorphometric bone volume (BV) was measured. Statistics: Student's t test and Pearson. Results (mean±SD) at Tf: no anthropometric or intake differences (p>0.05); In E, lipid profile showed significant increases in T-cho (96.2±8.9 vs 56.4±2.5: p<0.0001), n-HDL-cho (57.8±6.9 vs 21.0±3.5: p<0.0001), but the opposite in TG (76.5±16.4 vs 163.2±47.4: p=0.002). BV in E was significantly reduced (35.9±6.8 vs 47.7±7.4: p=0.022). T-cho vs BV correlation was found highly significant (p=0.0095). These results suggest that an atherogenic diet alters bone remodeling of interradicular bone in mandible, accounted by diminished BV. UBACyT O008.

Sacral fractures: Report of two cases

C. Gómez Acotto, E. Santini Araujo, C. Galloso, E. Roldán — 2009-12-01

Sacral fractures are relatively rare and can constitute a debilitating condition causing low back pain. The etiology is diverse: sacral insufficiency type fractures in elder osteoporotic people, fatigue fractures in young active individuals, metastasis and consequent irradiation therapy in gynaecological malignancies. We hereby describe 2 cases: (a) a 64-year-old woman suffering osteopenia who is under treatment with calcium tablets. She reported lower back pain and difficulty walking in the absence of trauma. Plain radiographs showed a linear fracture in the alae sacrum, while bone scinthigraphic 99mTc methylene disposphonate showed linear pattern uptake, suggesting osteolitic lesion. She had suffered breast adenocarcinoma 14 years earlier. We decided to perform bone biopsy and the pathology examination diagnosed metastasis of adenocarcinoma. (b) An 82-year old female patient with history of visual disease (macular degeneration). She had been diagnosed with osteopenia 3 years ago and was being treated with alendronate. At presentation, she reported moderate back pain. A sacral fracture was detected by computerized tomography at the emergency room. Bone biopsy was carried out trying to detect possible metastasis or primary tumor. Anatomopathology examination reported no cellular malignancy, hence the injury is interpreted as a fracture by insufficiency. Afterwards, we realized that she had previously had high serum alkaline phosphatase. The biopsy was reviewed, and it revealed Paget's disease of bone. The first patient improved after receiving radiotherapy in the fracture site and pamidronate at 90 g i.v. doses. In the second case we advised resting and pamidronate intake; the patient also improved.

Antioxidant and antiapoptotic properties of quercetin prevent oxidative stress caused by menadione in chick intestine

A.M. Marchionatti, N. Tolosa de Talamoni — 2009-12-01

In a previous work we have demonstrated that menadione (MEN) inhibits intestinal Ca2+ absorption by mitochondrial dysfunction, as a consequence of oxidative stress generated via glutathione depletion (GSH) and alteration of inner mitochondrial membrane, which leads to cytochrome c release and DNA fragmentation (Marchionatti et al., Biochim Biophys Acta 2008). Afterwards, we have observed that quercetin (QT) protects the enterocyte against oxidative stress caused by MEN, maintaining the intactness of the glutathione (GSH) content and the enzyme activity of GSH peroxidase (GPX) (Marchionatti et al., Bone 2008). The aim of this study was to elucidate the apoptotic mechanisms through which QT restores the intestinal function altered by MEN. Four week old chicks were employed; their intestinal mature enterocytes were isolated and exposed to either 500 μM MEN or 50 μM QT or both drugs for 30 min. Caspase-3 activity was measured by spectrophotometric procedure and FAS expression was evaluated by Western blot analysis. Data indicate that both caspase-3 activity and FAS expression were increased by MEN treatment, but not with QT alone or the combined treatment. These findings, together to the previous data, suggest that QT exerts antioxidant and antiapoptotic roles through which prevents the induction of proteins involved in the extrinsic and intrinsic pathways caused by MEN. This protective effect could be due to the maintenance of the GSH content and the activity of antioxidant enzymes, avoiding the oxidative stress caused by MEN and restoring the thiol redox status from the enterocytes, and therefore, the intestinal function.

Long term effects of normal calcium–high fat diets on body composition and bone mass, in rats

2009-12-01

Obesity and osteoporosis share a common cell progenitor. High adipocyte count in bone marrow is directly related to bone loss, as fat cells replace osteoblasts. We investigated long-term intake effect of normal calcium (Ca) diets—with different fat content—on body composition and bone mineral content (BMC), in healthy rats. At weaning (21 days), female Wistar rats (n=30) were randomly assigned one of the following 3 groups (n=10 each). G1 was sacrificed in order to get basal values (P0); G2 and G3 were fed ad libitum one of the two isocaloric diets (A; fat=7% wt./wt. or B; fat=15% wt./wt.). Mothers (M1A and M1B) were mated and mothers (M1A and M1B) and pups (P1A and P1B) were evaluated at weaning. At day 70, 20 rats from P1A and P1B (M2A and M2B, respectively) were mated and mothers (M2A and M2B) and pups (P2A and P2B) were studied at weaning. Body weights (BW), body fat (% BF) by chemical method (AOAC), total skeleton BMD, and BMC by DXA (Lunar DPX-L) in mothers and pups were evaluated. Results (mean±SE): BW increased across reproductive cycles being significant in group B pups (P2B: 53.6±1.2>P1B: 40.7±0.6>P0: 37.4±0.5; p<0.01). BW increase because of the increment in %BF (P2B: 14.9±0.5>P1B: 11.6±0.7>P0: 7.7±0.3; p<0.01); BMC decrease significantly in group B pups (C2B: 2.27±0.08>C0: 3.97±0.24; p<0.01). BMC and BMD in mothers did not reach significance. Conclusions: A long-term normal Ca–high fat diet leads to a progressive change in body composition with an increase in BF content and a decrease in BMC in pups. Awarded by UBACyT O 008.