bluuebunny/crossref_metadata_2025 huggingface dataset:
Serum insulin-like growth factor binding protein-3 in the hypophosphatemic mouse: Decreased activity and abnormal modulation by dietary phosphate
Authors: Moriwake T. , Abribat T. , Brazeau P. , Ecarot B.
Published: February 2010

The hypophosphatemic mouse, the murine homologue of X-linked hypophosphatemia, is characterized by renal defects in phosphate reabsorption and 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) production and by an osteoblast dysfunction. In view of the potential importance of insulin-like growth factors (IGFs) in the regulation of these processes and the role of IGF-binding proteins (IGFBPs) as modulators of IGF action, we asked whether Hyp mice have alterations in IGFs or IGFBPs. Using specific radioimmunoassays and Western ligand blot analysis, we evaluated serum levels of IGFs (IGF-I and IGF-II) and IGFBPs, respectively, in normal and Hyp mice. We also examined the effect of dietary phosphate on these parameters. Serum levels of IGF-I and IGF-II in Hyp mice were not significantly different from those in normal mice, but IGFBP-3 levels were significantly lower (70% of normal, p < 0.05) in the mutant strain. The other IGFBP species appear unchanged. Phosphate supplementation normalized serum phosphate levels in Hyp mice and elicited a significant decrease in serum IGF-I levels (23%, p < 0.05) and a further reduction in IGFBP-3 (22%, p < 0.02). Phosphate deprivation induced hypophosphatemia in normal mice to a degree similar to that in Hyp mice and did not alter serum levels of IGFBP-3 and IGF-I and IGF-II. The present results indicate that the low serum IGFBP-3 activity in Hyp mice is not related to hypophosphatemia per se. Based on the documented effects of parathyroid hormone (PTH) on IGF-I and IGFBP-3, we propose that the secondary hyperparathyroidism displayed by Hyp mice and its exacerbation by phosphate supplementation may contribute to low IGFBP-3 levels in control Hyp mice and to the decreases in serum IGF-I and IGFBP-3 in phosphate-supplemented Hyp mice.
Ultrastructural immunodetection of osteopontin and osteocalcin as major matrix components of renal calculi
Authors: McKee M. D. , Nancl A. , Khan S. R.
Published: February 2010

The organic matrix of renal calculi has long been considered to influence the crystal growth that occurs in these pathological mineral deposits. Recent advances in characterizing individual organic moieties from mineralized tissues in general and the combined use of antibodies raised against these molecules with different immunocytochemical approaches have allowed their precise distribution to be visualized in a variety of normal and pathological mineralized tissues. The present ultrastructural study reports on the epithelial expression and extracellular localization of several noncollagenous proteins in rat and human kidney stones using high-resolution colloidalgold immunocytochemistry. To this end, we have examined in an ethylene glycol-induced calcium oxalate model of urolithiasis in the rat, and in human kidney stones, the distribution of certain noncollagenous and plasma proteins known to accumulate in bone and other mineralized tissues that include osteopontin, osteocalcin, bone sialoprotein, albumin, and α2HS-glycoprotein. Of these proteins, osteopontin (uropontin) and osteocalcin (or osteocalcin-related gene/protein) were prominent constituents of the calcium oxalate-associated crystal “ghosts” found in the nuclei, lamellae, and striations of the organic matrix of lumenal renal calculi in the rat and of small crystal ghosts found within epithelial cells. Immunocytochemical labeling for both proteins of the content of secretory granules in tubular epithelial cells from treated rats, together with labeling of a similarly textured organic material in the tubular lumen, provides evidence for cosecretion of osteopontin and osteocalcin by epithelial cells, their transit through the urinary filtrate, and ultimately their incorporation into growing renal calculi. In normal rat kidney, osteopontin was localized to the Golgi apparatus of thin loop of Henle cells. In human calcium oxalate monohydrate stones, osteopontin was similarly detected in the lamellae and striations of the organic matrix. Based on these data, it is proposed that during urolithiasis, secretion of osteopontin (uropontin) and osteocalcin (or osteocalcin-related gene/protein), and the subsequent incorporation of these proteins into kidney stone matrix, may influence the nucleation, growth processes, aggregation, and/or tubular adhesion of renal calculi in mammalian kidneys.
End labeling studies of fragmented DNA in the Avian growth plate: Evidence of apoptosis in terminally differentiated chondrocytes
Authors: Hatori Masashi , Klatte Kevin J. , Teixeira Cristina C. , Dr. Shapiro Irving M.
Published: February 2010

The chondro-osseous junction has been the subject of considerable scrutiny, especially in terms of the fate and role of the terminally differentiated chondrocyte. Although it has been proposed that these cells change their phenotype and survive in the epiphysis, possibly as osteoblasts, evidence from a number of other studies suggests that chondrocytes may undergo apoptosis or programmed cell death. A useful test for programmed cell death is to end label DNA in cryosections using the commercial reagent ApopTagTM and detect antibody binding to fragmented DNA by epifluorescence; more direct assessments include examination of the nucleus for condensation of chromatin, evaluating fragmentation through alkaline and pulsed field agarose gel electrophoresis of DNA, and measuring apoptosis by flow cytometry. We found that we could label cells in the proliferative and the hypertrophic region of the proximal tibial growth plate of the chick with ApopTag. Most of the chondrocytes in the hypertrophic region were labeled by the reagent; in contrast, few proliferative chondrocytes were stained by the end-labeling procedure. Both agarose and pulsed field electrophoresis were used to confirm that there was fragmentation of chondrocyte DNA. Alkaline gel electrophoresis indicated that there was more fragmentation of DNA from hypertrophic cells than from proliferative chondrocytes. Further evidence in support of apoptosis was provided by electron microscopic observation of cells in the hypertrophic region of the growth plate. We noted that many of the cells in this region of the growth plate appeared to be undergoing programmed cell death since their nuclei contained condensed chromatin. Finally, we used flow cytometry to analyze chondrocytes isolated from the proliferating and hypertrophic regions of the growth plate for apoptosis. Dual parameteric flow cytometric contour plots of Hoechst and 7-amino-actinomycin D fluorescence showed that about 8% of cells in the plate were apoptotic. Most of these cells were in hypertrophic cartilage. In summary, the results of this investigation indicate that chondrocytes terminate their life history by apoptosis. While it is possible that the terminal labeling studies may overestimate the number of cells undergoing this event, the data lend credence to the view that cells are removed from the epiphysis through apoptosis. If this is the case, then chondrocytes probably enter the terminal phase of their life as fully functioning cells and genomic, and/or local environmental conditions provide termination signals that initiate events that lead to programmed cell death.
Aluminum inhibits both initiation and progression of mineralization of osteoid nodules formed in differentiating rat calvaria cell cultures
Authors: Dr. Bellows C. G. , Aubin J. E. , Heersche J. N. M.
Published: February 2010

Osteoid nodules form in cultures of fetal rat calvaria (RC) cells grown in medium containing 10% fetal bovine serum (FBS) and 50 μg/ml of ascorbic acid. When 10 mM β-glycerophosphate (β-GP) is added, the nodules mineralize in two phases: an initiation phase that is dependent upon alkaline phosphatase activity for cleavage of β-GP to inorganic phosphate (P1) and a progression phase that proceeds independently of the activity of alkaline phosphatase and does not require exogenous phosphate. We have used this system to investigate the effects of aluminum (Al3+) on mineralization. When AlCl3 was added to culture medium at concentrations of 0, 3, 10, 30, 100, and 300 μM, the total concentrations of aluminum were 0.98, 6.07, 16.82, 40.19, 88.45, and 284.52 μM, respectively. The corresponding free Al3+ concentrations, assessed after ultrafiltration, were found to be 1.11, 1.75, 3.40, 6.22, 5.38, and 12.11 μM. In cultures in which osteoid was formed and mineralization initiated in the presence of added Al3+ (3–300 μM), a dose-dependent inhibition of mineralization occurred. Osteoid formed in the presence of added Al3+ mineralized normally when Al3+ was removed from cultures at the time of initiation of mineralization with β-GP. In osteoid nodules grown in the absence of Al3+, addition of Al3+ (3–300 μM) at the start of the initiation phase of mineralization resulted in a dose-dependent inhibition of mineralization. Addition of Al3+ to cultures after mineralization had been initiated in the absence of Al3+ inhibited progression of mineralization at added Al3+ concentrations of 10 μM and above. Al3+ did not decrease the conversion of β-GP to Pi and caused a small but significant increase in alkaline phosphatase activity at added concentrations of 100 μM or greater. The data show that Al3+ inhibits both the initiation and progression phases of mineralization starting at added concentrations of 3–10 μM (∼1.7–3.4 μM free Al3+) and that mineralization of osteoid formed in the presence of Al3+ is unaffected if Al3+ is removed prior to the initiation of mineralization.
Heterogeneity of the skeleton: Comparison of the trabecular microarchitecture of the spine, the iliac crest, the femur, and the calcaneus
Authors: Amling Michael , Herden Stephan , Pösl Martin , Hahn Michael , Ritzel Holger , Delling Günter
Published: February 2010

The objective of this study was to elucidate the structure of cancellous bone and its age-related changes at different skeletal sites. Therefore, the lumbar spine, iliac crest, femur, and calcaneus of 12 age- and sex-matched skeletal healthy autopsy cases (6 females, 6 males, aged 28–84 years, mean 54 years) were removed. The following analysis includes an evaluation of the trabecular bone volume (BV/TV, %) and the trabecular interconnection (TBPf, mm−1) as well as a qualitative investigation of the structure of trabecular bone. BV/TV shows the following mean values: lumbar spine, 8.3% (±0.8%); iliac crest, 11.5% (±1.6%); intertrochanteric, 10.2% (±1.2%); femoral neck, 15.8% (±1.6%); and calcaneus, 15.4% (±2.0%). There are significant differences between the BV/TV of the femoral neck and that of the lumbar spine as well as between that of the calcaneus and the lumbar spine (p < 0.01). However, a positive correlation can be seen between the bone mass of the spine and that of all other investigated sites (r = 0.67 tor = 0.80; Pr < 0.1). The trabecular interconnection of the lumbar spine (2.7 mm−1, SEM ± 0.2 mm−1) and the femoral neck (0.3 mm−1, SEM ± 0.3 mm−1) differs significantly. Only these two sites show a significant positive correlation of TBPf (r = 0.60; pr < 0.1). Age-dependent alteration of the spine and the femoral neck in bone mass and bone structure is nearly the same. The trabecular microarchitecture of the iliac crest varies systematically. A region 4–10 cm behind and 1–3 cm below the anterior superior iliac spine turns out to be the most suitable biopsy site because of its closest relation to the lumbar bone mass. However, drawing information about the trabecular interconnection within the lumbar spine by measurement of the iliac crest at any site seems to be impossible. The horizontal specimens reveal a vertical running tubular spongiosa pattern that is arranged in concentric rings starting from the dorsal shell like a honeycomb. The comparison of TBPf in horizontal and vertical planes and its age-related changes indicates the age-related bone loss to be predominantly a loss of horizontal trabeculae. Thus, the presented data provide further information about the skeletal distribution and heterogeneity of the trabecular microarchitecture.
Prostaglandin E2 stimulates osteoclast-like cell formation and bone-resorbing activity via osteoblasts: Role of cAMP-dependent protein kinase
Authors: Kaji Hiroshi , Sugimoto Toshitsugu , Kanatani Masanori , Fukase Masaaki , Kumegawa Masayoshi , Chihara Kazuo
Published: February 2010

Prostaglandin E2 (PGE2) is an important local regulator in bone. The present study was performed to investigate the effect of PGE2 on osteoclast-like cell formation and bone-resorbing activity of mature osteoclasts in the presence or absence of osteoblasts. PGE2 (10−8 to 10−6 M) significantly stimulated osteoclast-like cell formation in osteoblast-containing mouse bone cell cultures, although it did not affect osteoclast-like cell formation from hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor in osteoblast-free mouse spleen cell cultures. The conditioned medium from osteoblastic UMR-106 cells pretreated with PGE2 (10−8 to 10−6 M) significantly stimulated osteoclast-like cell formation from hemopoietic blast cells. PGE2 also significantly stimulated the bone-resorbing activity of mature osteoclasts in osteoblast-containing mouse bone cell cultures. In contrast, PGE2 significantly inhibited the bone-resorbing activity and osteopontin mRNA expression in isolated rabbit osteoclasts. Rp-cAMPS, a direct protein kinase (PKA) antagonist, significantly inhibited PGE2-stimulated osteoclast-like cell formation and the bone-resorbing activity of mature osteoclasts, although protein kinase C inhibitors, dantrolene (an inhibitor of calcium release from the intracellular calcium pool) and voltage-dependent calcium channel blockers did not affect PGE2-stimulated osteoclast-like cell formation. In conclusion, PGE2 stimulated osteoclast-like cell formation and bone-resorbing activity in mouse bone cell cultures presumably through osteoblasts. The activation of PKA is linked to PGE2-stimulated osteoclast-like cell formation and bone-resorbing activity.
Estrogen inhibits interleukin-6 production and gene expression in a human osteoblastic cell line with high levels of estrogen receptors
Authors: Kassem Moustapha , Harris Steven A. , Spelsberg Thomas C. , Riggs B. Lawrence
Published: February 2010

Some studies suggest that estrogen acts on bone by decreasing the production of interleukin-6 (IL-6), a cytokine that increases bone resorption, by osteoblasts or bone marrow cells. However, other studies have not confirmed this, possibly because of a low and variable number of estrogen receptors (ER) in the model systems used. Thus, we employed a recently developed human fetal osteoblast cell line with high levels of ER. Treatment (n = 4 experiments) with 0.01 to 10 nM of 17β-estradiol had no effect on the constitutive production of IL-6. However, stimulated production, induced by treatment with IL-1β plus tumor necrosis factor-α (TNF-α), was reduced in a dose-dependent manner to 74 + 3% (mean + SEM) of control (p < 0.01). This response was blocked by cotreatment with the type II antiestrogen ICI 182,780. Treatment with hydrocortisone (1 μM), a known inhibitor of IL-6 production in many cell types, reduced IL-6 production to 17 + 1% of control (p < 0.001). As assessed by Northern analysis, treatment (n = 3 experiments) with 0.01-10 nM of 17β-estradiol decreased steady-state levels of IL-6 mRNA in a dose-dependent manner. These data support the hypothesis that at least part of the antiresorptive action of estrogen in humans is mediated by decreased production of IL-6 by osteoblastic cells.
Post-transcriptional stimulation of transforming growth factor β1 mRNA by TGF-β1 treatment of transformed human osteoblasts
Authors: Liu Changbao , Wallace Kathleen , Shi Congzhu , Heyner Susan , Komm Barry , Dr. Haddad John G.
Published: February 2010

Following exogenous administration of transforming growth factor-β1 (TGF-β1) polypeptide to the human osteosarcoma cell line TE-85, we observed a 2- to 6-fold stimulation of steady-state TGF-β1 mRNA. The stimulation was dose- and time-dependent, as judged from Northern blot hybridization analyses. A 2- to 6-fold increase of the TGF-β1 polypeptide was also found in the media of these cells after TGF-β1 treatments. The autostimulation of TGF-β1 mRNA was nullified by cycloheximide treatment of the cells. The in vitro transcription rates of the TGF-β1 gene by isolated nuclei were not altered by TGF-β1 treatment. Under conditions of transcriptional inhibition, the stability of TGF-β1 mRNA was enhanced nearly two-fold by TGF-β1 treatment. Our findings indicate that TGF-β1 can stimulate autologous gene expression and subsequent polypeptide translation by a post-transcriptional mechanism requiring protein synthesis in human osteoblast-like cells. The recognized versatility of TGF-β1 autostimulation mechanisms (transcriptional and post-transcriptional) in other mesenchymal cells may apply also to skeletal cells, further underscoring the broad and potent activities of this cytokine.
Effects of sodium fluoride and alendronate on the bone mineral in minipigs: A small-angle X-ray scattering and backscattered electron imaging study
Authors: Fratzl Peter , Schreiber Sabine , Roschger Paul , Lafage Marie-Helene , Rodan Gideon , Klaushofer Klaus
Published: February 2010

Sodium fluoride (NaF), which stimulates bone formation, and bisphosphonates, which reduce bone resorption, are both used in the treatment of osteoporosis, and are binding to bone mineral. In this study, using small-angle X-ray scattering and backscattered electron imaging, we analyzed the bone mineral in the vertebrae of minipigs treated with fluoride, with the bisphosphonate alendronate (ALN), or with vehicle. All specimens were investigated blindly. A slight increase in the average thickness of the mineral crystals as well as changes in the structure of the mineral/collagen composite were found in the case of fluoride-treated animals. No differences were found between ALN-treated animals and controls. The changes produced by fluoride are in the same direction as seen in bones from patients treated with NaF, albeit much smaller. They also correlate quantitatively with the reduction in biomechanical properties of bone in fluoride-treated minipigs found in an earlier study with the same animals. These findings suggest that small changes in the structure of the mineral/collagen composite in bone may considerably affect its biomechanical properties. It also emphasizes the delicate balance between the increase of bone mass and deterioration of bone material properties for the effect of fluoride on the biomechanical properties of bone.
Lack of an effect of sodium zeolite A on rat tibia histomorphometry
Authors: Firling Conrad E. , Evans Glenda L. , Wakley Glenn K. , Sibonga Jean , Turner Russell T.
Published: February 2010

Cell culture studies suggest that the aluminum silicate polymer sodium Zeolite A (SZA) increases bone cell proliferation and extracellular matrix production. This study in rats investigated the short-term (2 weeks) and long-term (18 weeks) in vivo effects of SZA on growth rate (weight gain) and tibia histomorphometry. In separate short-term experiments, female (experiment 1) or male (experiment 2) Sprague-Dawley rats grown and maintained on normal calcium diets were gavaged daily during a 2 week treatment period with 30 mg/kg, 100 mg/kg, or 500 mg of SZA/kg of rat body weight. In the long-term study (experiment 3) ovariectomized (OVX) rats were fed a low calcium diet containing 0, 1.80, and 9.00 g of SZA/kg of diet (0, 125, and 617 mg/kg of body weight, respectively). Short- and long-term treatment of growing rats with SZA resulted in a dose-dependent increase in bone aluminum. In the first experiment, growing intact female rats showed no significant SZA dose-dependent response in growth rate (weight gain) or histomorphometry of cortical bone in the tibial diaphysis or cancellous bone in the secondary spongiosa of the tibial metaphysis. In the second experiment, growing male rats, with right hind limbs immobilized by unilateral sciatic neurotomy, showed no SZA dose-dependent response in growth rate. The longitudinal growth of cancellous bone in the tibia of the denervated limb and the intact contralateral limb were not influenced by sciatic neurectomy and/or by treatment with SZA. Histomorphometry demonstrated that cortical bone mass and formation was reduced in the sciatic-sectioned limb when compared with the contralateral intact limb of vehicle-treated rats, as evidenced by significant reductions in static measurements of cortical bone area (-8.5%) and cross-sectional area (-4.8%) and in calculations of the periosteal formation rate (-33.8%) and mineral apposition rate (-31.6%), and the endocortical formation rate (-35.5%) and mineral apposition rate (-37.9%). The cancellous bone mass of denervated limbs of vehicle-treated rats was also deficient, as evidenced by decreased cancellous bone area (-39.1%) and perimeter (-31.9%). The bone mineral apposition rate was decreased (-26.7%) indicating reduced osteoblast activity. Treatment with SZA did not influence these indices in the tibiae of either sciatic-sectioned limbs or contralateral intact limbs. In the long-term experiment, OVX resulted in a dramatic 88% decrease in cancellous bone volume which was prevented by treatment with 17/β-estradiol and not influenced by treatment with Zeolite A. The increases in osteoblast and osteoclast number following OVX were not influenced by SZA. The results indicate that SZA treatment has no anabolic effect on cortical and cancellous bone formation and mass in normal growing female rats and that this compound does not protect against osteopenia due to reduced load bearing in the growing male rat or gonadal hormone deficiency in adult female rats.
Association of bone mineral density with polymorphism of the estrogen receptor gene
Authors: Kobayashi Shinji , Inoue Satoshi , Dr. Hosoi Takayuki , Ouchi Yasuyoshi , Shiraki Masataka , Orimo Hajime
Published: February 2010

PvuII and XbaI restriction fragment length polymorphisms (RFLPs) of the estrogen receptor (ER) gene and its relation to bone mineral density (BMD) were examined in 238 postmenopausal healthy women aged 45–91 years (66.3 ± 0.6 years, mean ± standard error of the mean [SEM]) in Japan. The RFLPs were represented as Pp (PvuII) and Xx (XbaI), with capital letters signifying the absence of and small letters the presence of restriction sites. In the PPxx genotype (n = 18), Z score values of BMD were significantly lower than those for other genotypes (n = 220) (lumbar spine, −0.746 vs. −0.065 [p = 0.022]; total body, −0.482 vs. 0.308 [p = 0.002]). We classified the subjects into three genotypes with allelic haplotype: homozygote of the Px haplotype was expressed as the 11 genotype, heterozygote of the Px haplotype as the 10 genotype, and the one lacking the Px haplotype as the 00 genotype. The PpXx genotype was not included in this analysis because the allelic haplotypes are uncertain. The Px haplotype was associated with a low BMD in postmenopausal women (Z score for the lumbar spine, −0.746 vs. −0.279 vs. 0.083, for the 11, 10, 00 genotypes, respectively [p = 0.029]; Z score for the total body, −0.482 vs. 0.164 vs. 0.427, respectively [p = 0.003]). We suggest that some variation of the ER gene linked to these RFLPs is associated with low BMD and that this at least partly explains the cause of postmenopausal osteoporosis in Japanese women.
Increased bone turnover in late postmenopausal women is a major determinant of osteoporosis
Authors: Dr. Garnero Patrick , Sornay-Rendu Elizabeth , Chapuy Marie-Claire , Delmas Pierre D.
Published: February 2010

Changes of bone turnover with aging are responsible for bone loss and play a major role in osteoporosis. Although an increase of bone turnover has been documented at the time of menopause, the subsequent abnormalities of bone resorption and formation and their potential role in determining bone mass in the elderly have not been investigated. To address this issue, we have measured a battery of new sensitive and specific markers of bone turnover in a population-based study of 653 healthy women analyzed cross-sectionally, including 432 women postmenopausal from 1 to 40 years, and the data were correlated with bone mineral density (BMD) measured by dual-energy X-ray absorptiometry (DXA) at different skeletal sites. Bone formation was assessed by serum osteocalcin (OC), serum bone-specific alkaline phosphatase (B-ALP), serum C-propeptide of type I collagen (PICP), and bone resorption by the urinary excretion of two pyridinoline cross-linked peptides (NTX and CTX). Bone turnover increased in perimenopausal women with both irregular menses and elevated serum follicle stimulating hormone (FSH). Menopause induced a 37–52% and 79–97% increase in the bone formation and bone resorption marker levels, respectively (p < 0.0001 except for PICP). In postmenopausal women, bone formation markers did not decrease with age. When resorption markers were corrected by whole body bone mineral content (BMC), the fraction of bone resorbed per day was not correlated with age in postmenopausal women and remained elevated for up to 40 years after menopause. In premenopausal women, the bone turnover rate accounted for only 0–10% of the variation in whole body BMC, total hip, distal radius, and lumbar spine BMD. With increasing time after menopause, the importance of the bone turnover rate as a determinant of bone mass increased at all sites and accounted for up to 52% of the BMD variance in elderly women. Thus, in women 20 years or more postmenopause, bone turnover was higher in those in the lowest quartile than in those in the highest quartile of BMD. In elderly women, 20 years since menopause and over, but not in younger ones, serum PTH was negatively correlated with serum 25-hydroxyvitamin D (r = −0.22,p < 0.05) and explained only 5–8% of the bone turnover variance (p < 0.01-0.001). These data indicate that the overall rates of both bone formation and bone resorption remain high in elderly women. The rate of bone turnover appears to play an increasing role as a determinant of bone mass with increasing time since menopause with a high bone turnover rate being associated with a low bone mass. Thus assessing bone marker levels may be useful in the evaluation of osteoporosis risk. In elderly women, secondary hyperparathyroidism caused in part by reduced serum 25-hydroxyvitamin D appears to be a marginal determinant of an increased bone turnover rate.
Calcitonin prevents bone loss but decreases osteoblastic activity in ovariohysterectomized beagle dogs
Authors: Monier-Faugere Marie-Claude , Geng Zhaopo , Qi Quanle , Arnala Illka , Malluche Hartmut H.
Published: February 2010

The antiresorptive effects of calcitonin are well documented. Recent in vitro and in vivo evidence points to an anabolic effect of calcitonin on osteoblasts. To assess the value of calcitonin in preventing the rapid and early bone loss after cessation of ovarian function and to investigate its effects on osteoblasts in vivo, 32 dogs were ovariohysterectomized (OHX) and 32 dogs were sham-operated (Sham). After the surgeries, half of the OHX and Sham dogs received every-other-day subcutaneous injections of human calcitonin (0.25 mg/dog/d), and the remaining dogs were given vehicle. Half of the animals had a bone biopsy at week 2 and were euthanized thereafter; the other half of the animals underwent a bone biopsy at month 1 and were euthanized at month 4. Blood drawings were done at baseline and at the time of each bone biopsy. Calcitonin prevented the increase in erosion depth seen in OHX animals and prevented the cancellous bone loss observed at 2 weeks and at 1 and 4 months. Calcitonin did not affect bone volume in Sham dogs. However, treatment with calcitonin induced a decrease in mineralizing surfaces and bone formation rates at the bone surface and cell level and an increase in mineralization lag time in both Sham and OHX animals without significantly affecting osteoblast number. This finding indicates that the negative effect of calcitonin on bone mineralization is not solely the result of a decrease in bone turnover. The data show that calcitonin, because of its antiresorptive effects, can prevent bone loss after cessation of ovarian function. However, short-term treatment with calcitonin does not stimulate osteoblast activity; on the contrary, it exerts a negative effect on osteoblastic bone formation and mineralization. Long-term studies are needed to investigate whether this unwanted effect of calcitonin on osteoblasts in vivo represents a transitory or persistent phenomenon.
The heritability of bone mineral density, ultrasound of the calcaneus and hip axis length: A study of postmenopausal twins
Authors: Arden N.K. , Baker J. , Hogg C. , Baan K. , Spector T.D.
Published: February 2010

Population based studies have demonstrated that having a first degree relative with a hip fracture is predictive of future hip fractures. Postmenopausal bone mineral density (BMD), ultrasound of calcaneus and hip axis length are associated with hip fracture, with the association for ultrasound and hip axis length being independent of BMD. The aim of this study was to determine the genetic component of these three important risk factors. We performed a classical twin study using 500 normal female twins, 128 identical and 122 non-identical pairs, aged 50 to 70 years. We measured bone mineral density at multiple sites, hip axis length (distance from the inner rim of the acetabulum to the greater trochanter), broadband ultrasound attenuation and velocity of sound of the calcaneus. Bone density had a strong genetic component at all sites with estimates of heritability ranging from 0.46 to 0.84. Hip axis length and velocity of sound had major genetic components with estimates of 0.62 and 0.61 respectively, which remained virtually unchanged after adjustment for bone mineral density. Broadband ultrasound attenuation had a moderate genetic component with an estimate of 0.53, which was reduced further to 0.45 after adjustment for BMD. In summary, all three bone measurements, which are independently associated with hip fracture, are independently heritable. This study suggests that a combination of different genetic factors acting on the structure, dimensions and density of bone may explain the importance of family history as a risk factor for hip fracture.
Age-related changes in osteogenic stem cells in mice
Authors: Bergman R.J. , Gazit D. , Kahn A.J. , Gruber H. , Mcdougall S. , Hahn T.J.
Published: February 2010

Osteoblasts arise from partially differentiated osteogenic progenitor cells (OPCs) which in turn arise from undifferentiated marrow stromal mesenchymal stem cells (MSCs). It has been postulated that age-related defects in osteoblast number and function may be due to quantitative and qualitative stem cell defects. To examine this possibility, we compared osteogenic stem cell number and in vitro function in marrow cells from 4-month-old and 24-month-old male BALB/c mice. Histologic studies demonstrated that these mice undergo age-related bone loss resembling that seen in humans. In primary MSC cultures grown in media supplemented with 10 nM dexamethasone, cultures from older animals yielded an average of 41% fewer OPC colonies per given number of marrow cells plated (p < 0.001). This implies that for a given number of marrow cells there are fewer stem cells with osteogenic potential in older animals than there are in younger animals. The basal proliferative rate in cultures from older animals, as measured by 3H-thymidine uptake, was more than three times that observed in cultures from young animals (p < 0.005). However, the increase in proliferative response to serum stimulation was 10-fold in the younger cultures (p < 0.001) and insignificant (p < 0.4) in the older cultures. Colonies in both age groups became alkaline phosphatase positive at the same rate, and virtually all colonies were positive after 12 days of culture. Cultures from both age groups produced abundant type I collagen. These studies suggest that defects in the number and proliferative potential of MSCs may underlie age-related defects in osteoblast number and function.
Bone quality factor analysis: A new noninvasive technique for the measurement of bone density and bone strength
Authors: Abbasi-Jahromi S. Hasan , Matayoshi Alicia , Kimble Robert , Dimarogonas Andrew , Pacifici Roberto
Published: February 2010

The sensitivity of bone mineral density (BMD) as a predictor of fracture risk is limited by the fact that this index does not take into account the geometrical and material characteristics of bone. In contrast, both BMD and bone architecture influence the quality factor (QF), the fraction of the inverse of the energy lost in one cycle of deformation. In this study we have compared the sensitivity of a QF analyzer and dual-energy X-ray absorptiometry (DXA) in detecting the changes induced by ovariectomy (OVX) on the QF, impact strength, and BMD of the femur of mature rats. QF and BMD were measured noninvasively before and 4 weeks after OVX or sham operation using a QF analyzer developed in our laboratory and a Hologic QDR 2000 bone densitometry, respectively. Impact strength was measured in excised femurs at the end of the study. The in vivo short-term precision (coefficient of variation) of the QF analyzer was 1.9%. BMD and QF measurements were highly correlated (r = 0.80, p < 0.0001). At baseline, QF and BMD were similar in OVX and sham-operated rats. At 4 weeks, BMD was 14.7 + 0.9% lower than at baseline (p < 0.001) in OVX rats and 5.3 + 1.3% lower in sham-operated rats (p < 0.05). QF decreased 36.0 + 2.8% (p < 0.0001) in OVX and 10.6 + 3.6% in sham rats (p < 0.01). As a result, at 4 weeks the difference between sham-operated and OVX rats was larger (p < 0.05) by QF than by BMD. Moreover, QF correlated better than BMD with impact strength and the difference in impact strength between sham and OVX mice was closer to that in QF than that in BMD. These data demonstrate that QF analysis is a precise technique that is more sensitive than DXA in detecting the changes in bone density and strength induced by OVX. QF analysis may represent a new, simple, and economic technique for predicting fracture risk.
Dual-energy X-ray absorptiometry derived structural geometry for stress fracture prediction in male U.S. marine corps recruits
Authors: Beck Thomas J. , Ruff Christopher B. , Mourtada Firas A. , Shaffer Richard A. , Maxwell-Williams Karen , Kao George L. , Sartoris David J. , Brodine Stephanie
Published: February 2010

A total of 626 U.S. male Marine Corps recruits underwent anthropometric measurements and dual-energy X-ray absorptiometry (DXA) scans of the femoral midshaft and the distal third of the tibia prior to a 12 week physical training program. Conventionally obtained frontal plane DXA scan data were used to measure the bone mineral density (BMD) as well as to derive the cross-sectional area, moment of inertia, section modulus, and bone width in the femur, tibia, and fibula. During training, 23 recruits (3.7%) presented with a total of 27 radiologically confirmed stress fractures in various locations in the lower extremity. After excluding 16 cases of shin splints, periostitis, and other stress reactions that did not meet fracture definition criteria, we compared anthropometric and bone structural geometry measurements between fracture cases and the remaining 587 normals. There was no significant difference in age (p = 0.8), femur length (p = 0.2), pelvic width (p = 0.08), and knee width at the femoral condyles (p = 0.06), but fracture cases were shorter (p = 0.01), lighter (p = 0.0006), and smaller in most anthropometric girth dimensions (p < 0.04). Fracture case bone cross-sectional areas (p < 0.001), moments of inertia (p < 0.001), section moduli (p < 0.001), and widths (p < 0.001) as well as BMD (p < 0.03) were significantly smaller in the tibia and femur. After correcting for body weight differences, the tibia cross-sectional area (p = 0.03), section modulus (p = 0.05), and width (p = 0.03) remained significantly smaller in fracture subjects. We conclude that both small body weight and small diaphyseal dimensions relative to body weight are factors predisposing to the development of stress fractures in this population. These results suggest that bone structural geometry measurements derived from DXA data may provide a simple noninvasive methodology for assessing the risk of stress fracture.
Calbindin-D9k and calbindin-D28k expression in rat mineralized tissues in vivo
Authors: Berdal A. , Hotton D. , Saffar J.L. , Thomasset M. , Nanci A.
Published: February 2010

Following their terminal differentiation, highly specialized cells, ameloblasts, odontoblasts, and osteoblasts sequentially elaborate mineralized tissues. While the developmental expression pattern of matrix proteins has been studied extensively, less attention has been paid to the molecules involved in calcium handling, such as calcium-binding proteins. This shortcoming, as well as previous conflicting data, led us to conduct studies on calbindin-D9k and calbindin-D28k in rat mandibular bone and incisor based on several methods established on rat ameloblasts in vivo. Radioimmunoassays showed that calbindin-D28k accounts for approximately 0.1% of cytosolic proteins in the ectomesenchymal fraction and 1% in the epithelial fraction of the rat incisor and is 100-fold more concentrated than calbindin-D9k in both tissue types. Western blot analysis confirmed that the anticalbindin-D28k reactive species corresponded to the well characterized renal calbindin-D28k in the ectomesenchyme. In this tissue, calbindin-D28k was ultrastructurally immunolocalized in the odontoblasts. Quantitative immunocytochemistry showed that labeling was distributed throughout their nucleus and cytoplasm. The similar cytoplasmic distribution of both calbindin-D proteins and mRNAs suggests that their expression is regulated at the subcellular level. In particular, immunoreactive calbindin-D28k appeared to be associated with rough endoplasmic reticulum. Calbindin-D9k antisense probe showed negligible labeling in odontoblasts, in parallel with the protein quantities measured (∼10 ng/mg of total protein). Finally, in situ hybridization showed transcripts for both calbindins-D in ameloblasts and also in osteoblasts. In summary, the present results support the concept that an elevated expression of these vitamin D–dependent calcium-binding proteins may characterize the phenotype of cells directly involved in the elaboration of mineralized tissues, enamel, dentine, and bone.
Changes in biological activity of bone cells in ovariectomized rats revealed by in situ hybridization
Authors: Ikeda Tohru , Yamaguchi Akira , Yokose Satoshi , Nagai Yumiko , Yamato Hideyuki , Nakamura Toshitaka , Tsurukami Hiroshi , Tanizawa Tatsuhiko , Yoshiki Shusaku
Published: February 2010

Twelve-week-old female rats were ovariectomized (OVX) and compared with sham-operated control rats at 3, 5, 7, 10, 14, 30, and 60 days postoperation with respect to the expression of type I collagen and osteopontin mRNAs, as well as bone structure and the number of osteoclasts. The trabecular number and separation were significantly decreased and increased, respectively, in the metaphyseal trabecular bones of OVX rat femurs. The number of osteoclasts was significantly increased in the same region of OVX rats at 3 and 5 days postoperation. Type I collagen mRNA was expressed in osteoblasts, and osteopontin mRNA was expressed in some osteoclasts, in mononuclear cells on the bone resorption surface, and in osteocytes near the resorption surface. In the metaphyseal trabecular bone, type I collagen and osteopontin mRNA expression levels in individual cells was initially increased in OVX rats from 7 to 10 days postoperation, and this was sustained for 60 days. The number of osteopontin mRNA-expressing osteocytes was also significantly increased at 10 days postoperation, which lasted until 60 days. In the epiphysis, an increase in type I collagen mRNA expression was initially observed in OVX rats at 14 days postoperation, which lasted until 60 days. The number of osteopontin mRNA-expressing osteocytes was virtually identical until 30 days postoperation in the epiphysis. These findings indicated that the biological activities of osteoblasts and osteocytes are stimulated in bones of the OVX rat and that the response for OVX differs between the metaphysis and epiphysis. Furthermore, the number of osteopontin mRNA-expressing osteocytes was increased only in bones that tended to be resorbed after OVX. This indicates that some osteocytes were stimulated to express osteopontin mRNA by estrogen deficiency and suggests that these osteopontin mRNA-expressing osteocytes may be involved in regulation of bone metabolism.
Differential regulation of receptor-stimulated cyclic adenosine monophosphate production by polyvalent cations in MC3T3-E1 osteoblasts
Authors: Hartle James E. , Prpic Veronica , Siddhanti Suresh R. , Spurney Robert F. , Quarles L. Darryl
Published: February 2010

Extracellular cations have paradoxical trophic and toxic effects on osteoblast function. In an effort to explain these divergent actions, we investigated in MC3T3-E1 osteoblasts if polyvalent cations differentially modulate the agonist-stimulated cyclic adenosine monophosphate (cAMP) pathway, an important regulator of osteoblastic function. We found that a panel of cations, including gadolinium, aluminum, calcium, and neomycin, inhibited prostaglandin E1 (PGE)-stimulated cAMP accumulation but paradoxically potentiated parathyroid hormone (PTH)-stimulated cAMP production. In contrast, these cations had no effect on forskolin- or cholera toxin–induced increases in cAMP, suggesting actions proximal to adenylate cyclase and possible modulation of receptor interactions with G proteins. Phorbol 12-myristate 13-acetated (PMA) mimicked the effects of cations on PGE1-and PTH-stimulated cAMP accumulation in MC3T3-E1 cells, respectively, diminishing and augmenting the responses. Moreover, down-regulation of protein kinase C (PKC) by overnight treatment with PMA prevented gadolinium (Gd3+) from attenuating PGE1- and enhancing PTH-stimulated cAMP production, indicating involvement of PKC-dependent pathways. Cations, however, activated signal transduction pathways not coupled to phosphatidylinositol-specific phospholipase C (PI-PLC), since there was no corresponding increase in inositol phosphate formation or intracellular calcium concentrations. In addition, pertussis toxin treatment failed to prevent Gd3+-mediated suppression of PGE1-Stimulated cAMP, suggesting actions independent of Gαi. Thus, polyvalent cations may either stimulate or inhibit hormone-mediated cAMP accumulation in osteoblasts. These differential actions provide a potential explanation for the paradoxical trophic and toxic effects of cations on osteoblast function that occur in vivo under different hormonal conditions.
β-Adrenergic receptor kinase-like activity and β-arrestin are expressed in osteoblastic cells
Authors: Bliziotes Michael , Murtagh James , Wiren Kristine
Published: February 2010

Biologic responses to peptide calciotropic hormones, such as parathyroid hormone (PTH) and calcitonin, exhibit desensitization. As with most hormones, however, the mechanisms of desensitization are not completely understood. For the β2-adrenergic receptor (β2AR) system, which is coupled to adenylyl cyclase via the stimulatory guanine nucleotide-binding regulatory (Gs) protein, homologous desensitization is mediated in part by a receptor-specific kinase (βARK) and a soluble cofactor (β-arrestin). Recently, this system has been reported to be involved in rapid homologous desensitization of the PTH/parathyroid hormone receptor protein (PTHrP) receptor. We have identified the presence of this system in bone using reverse-transcriptase PCR. Nucleotide sequence of PCR fragments from ROS 17/2.8 cells revealed 100% identity with rat brain βARK1 and β-arrestin 1 sequences. Northern analyses with RNA from ROS 17/2.8, UMR 106-H5 cells, and primary cultures of nontransformed neonatal rat calvariae demonstrated two mRNA species of 4 and 2.6 kilobases (kb) for βARK and 7.5 kb for β-arrestin, comparable to those found in bovine brain. βARK-like activity was demonstrated in cytosolic extracts of the UMR 106-H5 cells by assessing phosphorylation of the retinal photoreceptor, rhodopsin, by the extracts. Phosphorylation was enhanced with light-activated rhodopsin and by bovine brain Gβγ subunits; heparin inhibited phosphorylation. These findings are characteristic of βARK. Expression of β-arrestin in the UMR 106-H5 cells was confirmed by immunoblot. Thus, osteoblastic cells express proteins, βARK, and β-arrestin, which may regulate desensitization of calciotropic hormone receptors.
A controlled trial of raloxifene (LY139481) HCl: Impact on bone turnover and serum lipid profile in healthy postmenopausal women
Authors: Draper Michael W. , Flowers David E. , Huster William J. , Neild Julie A. , Harper Kristine D. , Arnaud Claude
Published: February 2010

This randomized, double-blind, placebo-controlled, multicenter, 8-week study evaluated short-term effects of raloxifene on bone turnover, serum lipids, and endometrium in healthy, postmenopausal women. A total of 251 women received either placebo, raloxifene HCl 200 or 600 mg/day, or conjugated estrogens (Premarin, 0.625 mg/day). Bone turnover (serum alkaline phosphatase, serum osteocalcin, urinary pyridinoline cross-links, urinary calcium excretion, urinary hydroxyproline) and serum lipids (total serum cholesterol, high- and low-density lipoprotein cholesterol [HDL-C and LDL-C]) were evaluated at weeks 0, 2, 4, and 8. Endometrial biopsies were performed at weeks 0 and 8. Treatment groups were compared for each parameter for baseline-to-endpoint changes. The estrogen and raloxifene groups experienced similar decreases in serum alkaline phosphatase (range 10–11%), serum osteocalcin (range 21–26%), urinary pyridinoline cross-links (range 20–26%), and urinary calcium excretion (range 45–72%). These decreases differed significantly compared with placebo-treated subjects for all markers except serum osteocalcin, the raloxifene HCl 200 mg group. LDL-C decreased significantly in the estrogen and both raloxifene groups (range 5–9%) compared with placebo-treated subjects. HDL-C increased significantly in the estrogen group (16%) but was unchanged in the raloxifene groups. HDL-C:LDL-C ratios increased significantly in the estrogen and raloxifene groups (range 9–29%). Serum cholesterol decreased significantly in both raloxifene groups (range 4–8%) but was unchanged in the estrogen group. Uterine biopsies of raloxifene-treated subjects showed no change in the endometrium during this short-term treatment. Biopsies of the estrogen group showed significant endometrial stimulation. The only adverse event possibly related to raloxifene was vasodilatation (hot flashes) which was most common in the raloxifene HCl 600 mg group. Study results indicate that raloxifene may provide beneficial effects to bone and serum lipids in humans without uterine stimulatory effects.