bluuebunny/crossref_metadata_2025 huggingface dataset:
PCR detection of cytokines in normal human and pagetic osteoblast-like cells
Authors: Birch M.A. , Ginty A.F. , Walsh C.A. , Fraser W.D. , Gallagher J.A. , Bilbe G.
Published: February 2010

We investigated the expression of cytokine transcripts in osteoblast-like cells derived from explants of pagetic and normal bone. A reverse transcription-linked PCR was used that allowed the simultaneous analysis of a range of cytokines. Normal osteoblast-like cells were found to contain the transcripts for IL-1β, IL-6, and TGF-β1. For the first time we detected in bone cells the two other mammalian isoforms of TGF-β, β2, and β3. Furthermore, we have also identified mRNA for IL-3 and the novel chemotactic factor, IL-8. Using this sensitive technique it was not possible to detect mRNA for IL-1α, IL-2, IL-4, IL-5, IL-7, TNF-α, or interferon-γ. The human osteosarcoma cell line Saos-2 also showed a similar pattern of expression of these cytokines to primary osteoblast-like cells, with the exception that TNF-α was also identified. Cells isolated from pagetic bone showed essentially the same profile of cytokine expression as normal bone except that TNF-α was also detected in two of four samples. The cytokine profile of successive populations of cells harvested from one explant culture at 9, 22, and 57 days showed a consistent pattern of cytokine expression, demonstrating the phenotypic stability of the osteoblast-like cells in long-term cultures.
Preliminary characterization of porcine bone marrow stromal cells: Skeletogenic potential, colony-forming activity, and response to dexamethasone, transforming growth factor β, and basic fibroblast growth factor
Authors: Dr. Thomson B.M. , Bennett J. , Dean V. , Triffitt J. , Meikle M.C. , Loveridge N.
Published: February 2010

Neonatal pig bone marrow stromal cells (PBMSC) were tested in vivo and in vitro to establish their use as a large-animal model for the study of skeletogenesis. When implanted in diffusion chambers in athymic mice for 6–8 weeks, both freshly isolated pig bone marrow and passage 2 PBMSC formed partially mineralized cartilage, bone-like material, and fibrous tissue. The cartilage showed metachromatic, perilacunar staining with toluidine blue and safronin O, alcian blue staining for chondroitin and keratan sulfate, and intense immunostaining for type II collagen. Osteocalcin was immunolocalized to the mineralized regions, consistent with the formation of bone. Alkaline phosphatase was primarily observed in cell layers at boundaries between tissue types. Unstimulated monolayer cultures of PBMSC produced type I but not type II collagen, responded to dexamethasone (10−8 M) with a 1.7-fold increase in alkaline phosphatase activity, and were stimulated to divide by basic fibroblast growth factor (1.5-fold; EC50 1 ng/ml). Transforming growth factor β (TGF-β) blocked both dexamethasone-induced alkaline phosphatase expression (EC50, 1 ng/ml of TGF-β) and the mitogenic effects of bFGF (EC50 0.06 ng/ml of TGF-β). When incubated for 10–14 days in medium containing dexamethasone, β-glycerophosphate and ascorbate PBMSC formed mineralized nodules. Calcification occurred in the middle of the aggregates and was associated with intensely alkaline phosphatase positive cells and a dense type I collagen-rich matrix. PBMSC also displayed colony-forming unit-fibroblastic activity, with approximately 1 in 80 of the plated cells formed colonies > 128 cells over 14–21 days. PBMSC therefore mimic the known activities of stromal cells from other species, including the human, suggesting that they are a valid model for skeletal research.
Tumor necrosis factor α modulates parathyroid hormone action in UMR-106–01 osteoblastic cells
Authors: Hanevold C.D. , Yamaguchi D.T. , Jordan S.C.
Published: February 2010

Tumor necrosis factor (TNF-α) has been shown to play an important role in local control of bone remodeling. The interaction of TNF-α and PTH was evaluated in UMR-106–01 cells, a phenotypic osteoblastic osteosarcoma cell line. We examined the influence of TNF-α on the two signal transduction systems triggered by PTH in UMR-106–01 cells, adenylate cyclase and free cytosolic calcium ([Ca2+]i). cAMP generation was inhibited in TNF-α-pretreated cells by 69, 61, 34, and 21% at PTH concentrations of 0.1, 1, 10, and 100 nM, respectively. Inhibition was seen at TNF-α doses of 100–1500 units/ml after a minimum incubation time of 12 h. TNF-α inhibition of the PTH-stimulated increase in [Ca2+], was even more pronounced: treated cells showed no change in baseline [Ca2+]i, after stimulation with 40 nM PTH. Treatment with TNF-α was also found to inhibit both arms of the PTH response in the nontransformed osteoblastic cell line, MC3T3-El. TNF-α treatment did not alter cAMP generation in response to PGE2. TNF-α inhibition of the PTH-stimulated cAMP response was reversed completely by addition of cholera toxin (5 μg/ml) and partially by forskolin (10 μM) but not pertussis toxin (100 and 500 ng/ml). Scatchard analysis using PTHrP revealed that TNF-α treatment reduced the number of receptors but had no effect on KD. These findings suggest that TNF-α inhibits the osteoblastic response to PTH at least in part because of a reduction in receptor number. Further investigation is indicated to provide insight into the interaction of calciotropic hormones and cytokines in vivo.
Long-term fracture prediction by bone mineral assessed at different skeletal sites
Authors: Dr. Melton L. Joseph , Atkinson Elizabeth J. , O'Fallon W. Michael , Wahner Heinz W. , Riggs B. Lawrence
Published: February 2010

Bone mineral density (BMD) was measured at the lumbar spine and cervical and intertrochanteric regions of the proximal femur by dual-photon absorptiometry and bone mineral content was assessed at the distal and midradius by single-photon absorptiometry in an age-stratified random sample of 304 Rochester, Minnesota women aged 30–94 years. Over follow-up extending to 10 years (median 8.3 years), 93 women experienced 163 new fractures. After adjusting for age, these bone mineral measurements predicted the likelihood of any incident fracture due to moderate trauma, with relative hazards varying from 1.4 to 1.6 per SD decrease in baseline bone mineral. A 1 SD decrease in lumbar spine BMD increased the risk of a new vertebral fracture comparably to a 17 year increase in age; a 1 SD decrease in femoral BMD was comparable to a 13–14 year increase in age on the risk of a hip fracture. We conclude that bone mineral measurements made at a variety of skeletal sites can predict the occurrence for at least 8–10 years of moderate trauma fractures of the sort that might be related to osteoporosis.
Stimulatory effect of TGF-β on anionic glycoconjugate synthesis by rat calvarial cells: Specificity, uncoupling of cell density dependence, and modulation by chondroitin sulfate
Authors: Professor Anastassiades Tassos P. , Chan Catherine
Published: February 2010

Anchorage-dependent cultures of a population of cells derived from the outer part of the rat calvaria demonstrated decreased net accumulation of radiolabeled chondroitin sulfate (CS) and hyaluronic acid (HA) per cell as the cell density of the cultures increased. The addition of TGF-β1 resulted in large stimulations of the net CS, but not of the net HA, accumulating in the medium at all cell densities and an abolition of the density-dependent effect. These effects were largely due to increases in newly synthesized CS appearing in the medium. Supplementation of the culture media with CS had complex but relatively small effects on the stimulation of the net accumulation of radiolabeled medium CS by TGF-β1. The addition of TGF-β1 also resulted in a biphasic effect on cell growth that depended on the plating density, but cell growth differences could not account for the marked stimulation of CS synthesis by TGF-β1. Experiments with cycloheximide and β-xyloside and isolation of the intact anionic glycoconjugates (AG) indicated that although synthesis of core protein was the limiting factor in CS synthesis, TGF-β1 stimulated the synthesis of CS chain when sufficient β-xyloside acceptor was available. The overall results suggest that, in this cell system, the action of TGF-β1 on the synthesis of the major extracellular AGs is characterized by a relatively specific upregulation of CS proteoglycan (PG) synthesis and an uncoupling of the inhibitory effect of high cell density on CS PG synthesis.
Effects of interleukin-4 on the formation of macrophages and osteoclast-like cells
Authors: Riancho José A. , Zarrabeitia María T. , Mundy Gregory R. , Yoneda Toshiyuki , González-Macías Jesus
Published: February 2010

The lymphokine interleukin-4 (IL-4) is an important lymphocyte growth factor, and it also has a modulatory role on hematopoiesis. It was recently reported that IL-4 has an inhibitory effect on bone resorption in vitro, but the underlying mechanisms are not well known. We studied its effects on the formation of osteoclast-like cells in mouse bone marrow cultures and in cocultures of spleen cells and stromal cells. The addition of recombinant mouse IL-4 (0.01–10 ng/ml) induced a marked dose-dependent inhibition on the formation of TRAP-positive multinucleated cells (MNC) in bone marrow cultures. The effect was blocked by anti-IL-4 antibodies and was not related to a decreased production of IL-6. The inhibitory effect required the presence of IL-4 during the second half of the culture period. Time course experiments showed that IL-4 impaired the formation of osteoclast-like cells rather than inducing the disappearance of previously formed cells. This inhibitory effect was associated with increased numbers of esterase-positive cells. Moderately high doses of IL-4 (1–10 ng/ml) also induced the formation of abundant macrophage polykaryons that did not form resorption pits. IL-4 had a similar inhibitory effect on the formation of osteoclast-like cells in cocultures of mouse spleen cells and stromal cells. Our results suggest that IL-4 acts on uncommitted macrophage-osteoclast precursors, inducing a preferential differentiation toward the macrophage lineage and thus decreasing the formation of osteoclast-like cells.
Characterization of radioiodinated recombinant human TGF-β1 binding to bone matrix within rabbit skull defects
Authors: Richardson Louise , Zioncheck Thomas F. , Amentoè Edward P. , Deguzmanè Leo , Leeè Wyne P. , Xuè Yvette , Beckè L. Steven
Published: February 2010

Bone healing is regulated in part by the local production of TGF-β1 and other growth factors produced by cells at the site of injury. The single application of recombinant human TGF-β1 (rhTGF-β1) to calvarial defects in rabbits induces an accelerated recruitment and proliferation of osteoblasts within 3 days. This ultimately results in the formation of new bone and the complete closure of the defect within 28 days. The persistence and localization of [125I]rhTGF-β1 within an osseous defect was investigated after applying a single dose of [125I]rhTGF-β1 formulated in a 3% methylcellulose vehicle. Normal bone encompassing the defect site, the periosteum, and the gel film covering the dura were harvested at 0, 4, 8, and 24 h and 3, 7, and 16 days after [125I]rhTGF-β1 application. The defect site-associated radioactivity was quantitated, visualized by autoradiography, and characterized by TCA precipitation and SDS-PAGE. Radioactivity was observed in autoradiographs of gross specimens, histologic sections of the bone matrix, and periosteal tissue surrounding the defect. There was a time-dependent decrease in TCA-precipitable radioactivity; however, radioactivity was still associated with the bone matrix 16 days after application of [125I]rhTGF-β1. SDS-PAGE and autoradiography of the radioactivity in homogenized bone and periosteal samples revealed a 25 kD band, suggesting that the radioactivity remaining at the defect site represented intact [125I]rhTGF-β1. Results of this study indicate that rhTGF-β1 may bind tightly to bone matrix in its active form, and this binding may be associated with the observed increase in osteoblast number and bone matrix within the calvarial defects.
Thyroid hormones increase insulin-like growth factor I content in the medium of rat bone tissue
Authors: Lakatos Peter , Caplice Matthew D. , Khanna Vikram , Stern Paula H.
Published: February 2010

The mechanism of action of thyroid hormones on bone is still not clear. At low concentrations, they stimulate bone formation; at high concentrations, they elicit bone resorption in vitro and in vivo. In the present study we investigated the effect of T3 and T4 as well as their active and inactive analogs (TRIAC, SKF L-94901, rT3, and DIT) on the IGF-I and TNF-α content in the medium of UMR-106 rat osteoblastic cells and fetal rat limb bones. In the dose-response studies, a biphasic increase in medium IGF-I was observed in both cells and limb bones, with peak stimulatory concentrations of 10−8 M for T3 and 10−7 M for T4 in both systems. At higher concentrations, at which thyroid hormones elicit bone resorption, the stimulatory effect diminished and finally was no longer detectable. The active analogs TRIAC and SKF L-94901 also enhanced IGF-I release in UMR-106 cells. The inactive compounds rT3 and DIT failed to increase IGF-I in these cultures. The protein content of the cell culture wells exposed to high concentrations of thyroid hormones was similar to those containing low concentrations, indicating that the decrease in IGF-I content at high doses was not due to toxic effects. This was also confirmed by trypan blue exclusion. Time course studies with UMR-106 cells revealed a significant increase in medium IGF-I after 2 days of incubation. No significant further increase was observed after this up to 5 days of culture. In contrast, the medium of limb bone cultures showed a linear increase in IGF-I content up to 7 days of culture. No TNF-α production was observed in either UMR-106 cells or fetal limb bones. Also, no increase in medium TNF-α levels was seen in response to thyroid hormones. Based on our results, we conclude that IGF-I may be responsible for some of the anabolic effects of thyroid hormones in bone tissue, but TNF-α, at least in the models we used, does not play a role in the mediation of thyroid hormone action.
Effects of the bisphosphonate tiludronate on bone resorption, calcium balance, and bone mineral density
Authors: Dr. Ammann Patrick , Rizzoli René , Caverzasio Joseph , Shigematsu Takashi , Slosman Daniel , Bonjour Jean-Philippe
Published: February 2010

Bone resorption inhibitors, such as bisphosphonates, are potentially useful in treatments aimed at increasing bone mass. Among bisphosphonates, tiludronate has proven efficacious in preventing bone loss in postmenopausal women. However, it is not clearly established whether bisphosphonates are more potent when given intermittently or continuously. We investigated the effects of tiludronate on (1) retinoid-stimulated bone resorption in thyroparathyroidectomized rats, (2) calcium balance in intact rats, and (3) bone mineral density (BMD) as measured by dual-energy x-ray absorptiometry at the levels of the lumbar spine, tail, and tibia in 6-month-old rats made osteoporotic by ovariectomy (OVX), in which an intermittent cyclic schedule of treatment was compared to continuous administration. Tiludronate induced a dose-dependent decrease in retinoid-stimulated bone resorption. It increased the intestinal absorption and body retention of calcium. In OVX rats it caused a time- and dose-dependent increase in BMD at the level of the three investigated sites, the effects being maintained for at least 8 weeks after the end of therapy. Continuous and intermittent cyclic regimens appeared to induce similar increases in BMD. These results indicate that tiludronate is efficacious in decreasing bone resorption and increasing calcium balance and bone mineral density in rats.
Evidence that a non-RGD domain in rat osteopontin is involved in cell attachment
Authors: van Dijk Simon , D'Errico John A. , Somerman Martha J. , Farach-Carson Mary C. , Butler William T.
Published: February 2010

The bone sialoprotein osteopontin (OPN) promotes cell attachment and spreading through its RGD (Arg-Gly-Asp) sequence. To study additional regions of OPN involved in cell attachment, peptides of rat OPN were evaluated for their capacity to mediate cell binding to wells in vitro. Human gingival fibroblasts were incubated on microtiter plates coated with either OPN or OPN peptides. A peptide of Mr 28 kD, obtained after digestion with endoproteinase Arg-C and isolated by reversed-phase HPLC, enhanced cell attachment to a similar degree as OPN. Sequence analysis showed that the amino terminus of the 28 kD peptide starts at Ser142 and therefore does not contain the RGD cell attachment sequence (residues 128–130). Cell attachment mediated through both OPN and the 28 kD peptide was blocked by the addition of GRGDSPA peptides or LM-609, a monoclonal antibody to the integrin αVβ3, a receptor for vitronectin. A variant peptide, GRG-ESPA, did not alter cell attachment. Based on these observations, we conclude that (1) binding of OPN and the 28 kD peptide to fibroblasts involves binding to αVβ3, (2) a site other than the RGD sequence on OPN is also involved in binding to integrins, and (3) the binding of this second site to αVβ3 is inhibited by RGD-containing peptides.
Journal of Bone and Mineral Research
Authors: Robey Pamela Gehron , Fedarko Neal S. , Hefferan Theresa E. , Bianco Paolo , Vetter Ulrich K. , Grzesik Wojciech , Friedenstein Alexander , van der Pluijm Gabri , Mintz Keith P. , Young Marian F. , Kerr Janet M. , Ibaraki Kyomi , Heegaard Anne-Marie
Published: February 2010

The organic matrix of bone contains several protein families, including collagens, proteoglycans, and glycoproteins, all of which may be extensively modified by posttranslational events, such as phosphorylation and sulfation. Many of the glycoproteins contain Arg-Gly-Asp (RGD), the integrin-binding sequence, within their structure, whereas other constituent proteins contain gamma-carboxyglutamic acid. The deposition of bone matrix by cells in the osteoblastic lineage is regulated by extrinsic factors, such as systemic and local growth factors and physical forces, and factors that are intrinsic to the cell, such as position in the cell cycle, maturational stage, and developmental age of the donor. Recent studies of several bone matrix gene promoters have identified cis- and trans-acting elements that are responsible for gene activity, although the precise sequence of regulatory events is not known. Development of in vitro assays, coupled with studies of the appearance of these proteins during development in vivo, provides insight into the functions of these proteins during the various stages of bone metabolism. Potential roles for these proteins include proliferation and maturation of stem cells, formation of matrix scaffolding elaborated by bone-forming cells, modeling, and remodeling. Changes in the functional properties of the extracellular matrix may be involved in a variety of disease processes, including osteoporosis and oral bone loss.
Journal of Bone and Mineral Research
Authors: Mundy Gregory R.
Published: February 2010

Osteoporosis and periodontal disease both represent examples of abnormal bone remodeling. As knowledge of the cellular and molecular events in the normal bone remodeling process has accumulated in the last decade, better understanding of the pathophysiology of bone loss associated with periodontal disease and with aging has occurred. This short review does not attempt to include all aspects of this topic but covers specific areas in which there have been recent advances. (1) Observations made in the last few years have indicated that a hierarchy of both receptor and nonreceptor tyrosine kinases may be involved in normal osteoclastic bone resorption and that certain members of these tyrosine kinase families may mediate cytokine effects. Studies in the op/op variant of murine osteopetrosis have shown that normal production of monocyte-macrophage colony-stimulating factor 1 (M-CSF, also called CSF-1) and activation of its receptor (the receptor tyrosine kinase c-fms) are required for normal osteoclast formation. (2) Studies in mice made deficient in nonreceptor tyrosine kinase by gene knockout have shown that expression of this nonreceptor tyrosine kinase is required for normal osteoclast action and ruffled border formation, although not for osteoclast formation. (3) Recent studies have shown that in addition to protaglandins of the E series, other arachidonic acid metabolites may be involved in normal and abnormal osteoclastic bone resorption. 5-Lipoxygenase metabolites, the leukotrienes, stimulate isolated osteoclasts to form resorption pits as well as cause osteoclastic bone resorption in organ cultures of neonatal mouse calvariae. These compounds, which are unstable in tissue culture media, are readily inhibitable by agents that inhibit 5-lipoxygenase enzymes. Since they accumulate in chronic inflammatory cells, they may be important in bone remodeling associated with periodontal disease. (4) Recently, a new growth regulatory factor for osteoclasts has been described. This factor, called the osteoclastpoietic factor (OPF), is likely akin to the colony-stimulating factors for the formed elements of the blood, since the osteoclast shares a common precursor with these cells. OPF enhances the differentiation of osteoclasts from committed precursors. Its role in normal and abnormal bone remodeling is still to be determined. (5) Recent studies using prolonged cultures of fetal rat calvarial cells have shown that members of the TGF-β superfamily (TGF-β and the BMPs) may work in concert to stimulate normal bone formation. In this in vitro model culture system, TGF-β enhances cell proliferation but inhibits differentiated function. In contrast, BMP-2, a prototype for the BMP family, enhances differentiated function in these cells, including expression of osteocalcin, alkaline phosphatase, and the formation of bone nodules. For more comprehensive reviews of the role of cytokines in bone remodeling, the reader is referred to Mundy (1993), Manolagas and Jilka (1992), and Canalis et al. (1989).
Journal of Bone and Mineral Research
Authors: Kimmel Donald B.
Published: February 2010

This article integrates engineering principles with skeletal biology to describe skeletal strength homeostasis. Skeletal strength revolves around its perceived mechanical usage. Mass, geometric properties, and fatigue damage burden are the principal determinants of structural strength. Bone cells form sensor and effector systems that monitor usage and adjust strength and stiffness by changing mass, geometric properties, and fatigue damage burden. The bone lining cell–osteocyte complex is the sensor; the bone modeling and remodeling systems are the effectors. Deformation and fatigue damage in bone are the signals received by the sensor. Accumulated energy in the sensor's cytoskeleton determines the rate at which the sensor sends messages to the effectors. The activity of both effector systems is proportional to the rate of incoming messages. Modeling raises bone strength and stiffness by improving geometric properties as it adds bone where customary deformation is greatest. Remodeling improves bone strength by replacing fatigue-damaged areas without mass changes. Bone removed during modeling and remodeling comes from sites where the impact on bone strength and stiffness is least. Hormones and agents alter the rigidity of the cytoskeleton and, thus, its capacity to deform and store energy. Osteopenic agents make it more rigid, causing detection of fewer deformations and transmission of fewer loading signals to the effector. Osteotropic agents decrease the rigidity of the cytoskeleton, causing detection of more strain events and transmission of more loading signals to the effector. Agent treatment thus establishes false conditions of disuse or hyperuse.
Journal of Bone and Mineral Research
Authors: Aufdemorte Thomas B. , Boyan Barbara D. , Fox W. Casey , Miller Dennis
Published: February 2010

Recent data have suggested that there may be a relationship between osteoporosis and oral bone loss. Both diseases are major public health problems of enormous magnitude, but their relationship is poorly understood. This is due to their complex pathogenesis and the fact that coexisting periodontal disease, loss of the dentition, site-specific variability in anatomy and bone density, as well as aging and many other factors complicate the development and progression of bone loss relative to each. The focus of this article is to review the characteristics of animals that are suited for studies of the relationship of osteoporosis and oral bone loss to better understand their relationship.
Journal of Bone and Mineral Research
Authors: Webber Richard L.
Published: February 2010

Practical use of images for diagnosis of osseous, abnormalities presumes cost-effective technology that targets known physiologic processes. The most obvious modalities for such applications are radiographic, and the newest are characterized by quantitative methods expressed both tomographically and by means of digital subtraction registered in two and three dimensions. Quantitative nuclear medicine and possibly electron spin resonance spectroscopy also appear promising. New analytic techniques for interpreting osseous images include characterization of trabecular patterns by fractal descriptors and micromorphometry.
Journal of Bone and Mineral Research
Authors: Baylink David J. , Finkelman Richard D. , Mohan Subburaman
Published: February 2010

During the past decade we and others have shown that bone is a storehouse for growth factors. Accordingly, bone contains a number of growth factors including insulin-like growth factors I and II (IGF-I, IGF-II) transforming growth factor (TGF-β1, TGF-β2), platelet-derived growth factor, basic and acidic fibroblast growth factor, and bone morphogenetic proteins (BMPs). Osteoblasts have been shown to produce many of these growth factors, which then act in an autocrine and paracrine fashion. The production of these growth factors is regulated by both systemic hormones and local mechanical stress. Recent studies on the relative distribution of bone growth factors during different physiologic and pathologic situations indicate that the concentration of bone growth factors is not invariant and provide indirect evidence that growth factors deposited in bone have physiologic significance. In addition, many of these bone growth factors have been shown to increase bone formation either systemically or locally in vivo. Based on the past findings, we propose that different growth factors may have a specific role in regulating proliferation and differentiation of different stages of osteoblast lineage cells and play important roles in the local regulation of bone formation.
Journal of Bone and Mineral Research
Authors: Ott Susan M.
Published: February 2010

The bisphosphonates are analogs of naturally occurring pyrophosphate. They appear to block bone resorption much more than mineralization. A series of compounds has been investigated. These have antiresorptive potencies ranging from 1 to 10,000. So far, structure-function relationships have eluded definition. These drugs increase the bone density in women with postmenopausal osteoporosis by about 5%-10% over 1 year. The bone density then appears to plateau, but most studies are of short duration. Improved bone density does not necessarily result in stronger bone, but no studies have had adequate power to assess fracture incidence. Bone formation rates decrease as measured histomorphometrically. This occurs while the bone density is improving, which implies an inhibition of bone resorption that is probably due to direct toxicity to the osteoclasts. Etidronate blocks mineralization as well as resorption and can cause osteomalacia, which is not seen with low-dose cyclical etidronate or with the second-generation compounds. The long-term effects of interfering with the remodeling cycle are still not known. Subtle side effects may be those on the bone itself. These might go undetected, since bone pain or fractures usually are atributable to the underlying disease.
Changes in bone mass early after kidney transplantation
Authors: Horber F.F. , Casez J.P. , Steiger U. , Czerniak A. , Montandon A. , Jaeger PH
Published: February 2010

Renal transplant patients exhibit increased rates of trabecular bone fractures, probably due to glucocorticoid-induced osteopenia, which is known to occur within 6 months after kidney grafting. This mineral loss at a mostly trabecular site (lumbar spine) contrasts with a gain at the radius, which consists mainly of cortical bone. However, the early effects of kidney transplantation on the other parts of the human skeleton and the time course of these changes during the first 5 months after transplantation remain unknown. Therefore, 34 kidney transplant recipients were prospectively followed immediately after kidney grafting (12 ± 1 days, mean ± SEM, and then on a monthly basis up to 152 ± 3 days) and compared with 34 normal healthy volunteers matched for age, sex, and body mass index. Bone mineral measurements of whole body (n = 34), lumbar spine (n = 32), and upper femur (n = 23) were performed using dual-energy x-ray absorptiometry (Hologic QDR 1000W). At time of transplantation, lumbar bone mineral density (BMD) and BMD of the upper femur were lower (p < 0.01) in female but not male patients compared with controls. Lumbar BMD decreased by 1.6 ± 0.2% per month in both sexes (p < 0.01), whereas BMD of upper femur further decreased in males (p < 0.01) but only tended to decrease in females. At time of transplantation, whole-body bone area (BA), bone mineral content (BMC), and BMD were decreased by about 8, 15, and 9%, respectively, in patients compared with controls (p < 0.01). Compartmental analysis revealed similar reductions in BMD and BMC at trunk, head, and limbs, whereas BA was reduced only at limbs (p < 0.01). During the 152 ± 3 days of observation, BMC and BMD of limbs remained essentially unchanged, whereas BA of limbs increased (p < 0.01) and BA, BMC, and BMD of trunk and head decreased (p < 0.01). In conclusion, within 5 months after successful kidney transplantation resulting in good renal function, about 41 g bone mineral is lost, mostly due to losses in the trabecular bone compartment (i.e., about 10%). Longitudinal trials to prevent trabecular bone loss during the phase of terminal renal failure and during the first half-year following transplantation are urgently needed to develop rational and effective therapeutic strategies in patients on dialysis and kidney transplant patients.
Grip strength and bone mineral density in older women
Authors: Kritz-Silverstein Donna , Dr. Barrett-Connor Elizabeth
Published: February 2010

Most previous literature on muscle strength and bone density examined the association between specific muscle groups and adjacent bones. This study examined the association of grip strength with bone density at distant sites, such as the spine and hip, as well as at the wrist and radius. Subjects were 649 postmenopausal women aged 65 years and older. Bone mineral density was measured at the ultradistal wrist and midshaft radius in the nondominant arm using single-photon absorptiometry and at the lumbar spine and hip using dual-energy x-ray absorptiometry. Grip strength was measured in both the dominant and nondominant hands using a dynamometer. Overweight women had significantly stronger grip strength in both hands (p < 0.01 for dominant hand, p < 0.05 for nondominant hand grip strengths). Exercisers had significantly stronger grip strength in the nondominant hand than nonexercisers (p < 0.05). Women reporting arthritis had significantly weaker grip strength in both hands than women not reporting arthritis (p < 0.001). There were no significant differences in grip strength by estrogen use, cigarette smoking, or thiazide use. Regression analyses adjusted for age, obesity, exercise, cigarette smoking, thiazide use, arthritis, years postmenopausal, and estrogen use indicated significant positive associations between grip strength and bone density at all sites. Regression analyses stratified by exercise indicated that only women who exercised had significant associations of grip strength with bone mineral density (all p < 0.05). In this cohort of older women, grip strength was an independent indicator of general bone density. These results are compatible with the thesis that grip strength is a marker for overall fragility and general health and for the adequacy of skeletal loading exercise.
Long-term (3 years) prevention of trabecular postmenopausal bone loss with low-dose intermittent nasal salmon calcitonin
Authors: Reginster J.Y. , Denis D. , Deroisy R. , Lecart M.P. , de Longueville M. , Zegels B. , Sarlet N. , Noirfalisse P. , Franchimont P.
Published: February 2010

The long-term effect of intermittent low-dose nasal salmon calcitonin on trabecular early postmenopausal bone loss was assessed as follow-up to a previously published study. Randomized controlled group comparison was made of 287 healthy women with 6–36 months of natural menopause and no treatment interfering with calcium metabolism at an outpatient clinic for research in bone and cartilage metabolism. The 287 women were randomly allocated to 3 years of treatment with either 500 mg/day, 5 days/week of calcium or the same amount of calcium plus 50 IU/day, 5 days per week of nasal salmon calcitonin. A total of 186 women complied with the study protocol throughout. The main outcome measures were bone mineral density of the lumbar spine (DPA) and biochemical parameters reflecting bone turnover (serum alkaline phosphatases, urinary calcium/creatinine, and hydroxyproline/creatinine ratio). The average changes in bone mineral density after 36 months showed a positive (p < 0.05) outcome (1.8 ± 5.7%; mean ± SD) in the group treated with salmon calcitonin and calcium and a significant (p < 0.01) loss (-5.8 ± 4.8%) in patients receiving calcium alone. The difference between the evolution of the two groups was significantly (p < 0.01) different after 6 months of treatment and remained so until the end of the study. No significant changes were recorded in biochemical parameters reflecting bone turnover. As previously shown during a 1 year follow-up, nasal salmon calcitonin given at low dose and intermittently, in association with calcium, can counteract trabecular postmenopausal bone loss.
Mechanical loading thresholds for lamellar and woven bone formation
Authors: Dr. Turner Charles H. , Forwood M.R. , Rho J.-Y. , Yoshikawa T.
Published: February 2010

Bone formation was measured in rat tibiae after 12 days of applied loading. Bending forces were applied using a four-point loading apparatus. Sham loads were applied at the same magnitudes as bending forces but the loading pads were arranged so that bending was minimized. Bending and sham loading were applied to the right tibiae of rats and the left tibiae served as contralateral controls. Loading was applied as a sine wave with a frequency of 2 Hz for 18 s (36 cycles) per day. The peak magnitude of applied load was 27, 33, 40, 52, and 64 N. Woven bone was observed on the periosteal surface in all animals subjected to loads of 40 N or greater. Periosteal woven bone formation occurred in both bending and sham loading groups. Woven bone formation on the periosteal surface was either absent or responded at a maximal rate if the stimulus threshold was surpassed. The amount of new woven bone and the woven bone-forming surface were independent of the magnitude of applied strain. Bone formation on the endocortical surface was exclusively lamellar. Lamellar bone formation was stimulated by applied bending of the tibia but not by sham loading. Bending strains above a loading threshold of 40 N or about 1050 μstrain increased both bone-forming surface and the mineral apposition rate and subsequently increased the bone formation rate as much as sixfold. No evidence of increased bone formation was seen for applied strains below 1050 μstrain. Examination of bulk stained sections from animals exposed to the highest applied loads showed no evidence of microcracks. Thus, microcracks have not been implicated as a causal factor for the observed increases in bone formation rate.
Pp60 c-src expression and activity in MG-63 osteoblastic cells modulated by PTH but Not required for PTH-mediated adenylate cyclase response
Authors: Izbicka E. , Niewolna M. , Yoneda T. , Lowe C. , Boyce B. , Mundy G.
Published: February 2010

A role for pp60c-src (c-src) tyrosine kinase in bone resorption was recently demonstrated in vivo. However, it is not known whether expression of this ubiquitous tyrosine kinase is essential in osteoblasts or osteoclasts. In this work, we have examined the expression of c-src in osteoblast-like cells. We found that c-src was expressed in the MG-63 osteoblastic osteosarcoma cell line as assessed by immunocytochemistry. When MG-63 cells were treated for 30 minutes with parathyroid hormone (PTH), the levels of tyrosine phosphorylation of c-src were increased in comparison with the untreated control. On the other hand, no changes in total c-src protein were observed after PTH treatment. The increase in c-src tyrosine phosphorylation due to PTH treatment was paralleled by an increase in c-src tyrosine kinase activity. Calcitonin had no effect on c-src phosphorylation, c-src protein level, or tyrosine kinase activity. To determine if c-src tyrosine kinase was required for normal bone cell function and PTH responsiveness, osteoblastic cells were isolated from the calvaria of a src-deficient mouse. The cyclic AMP response to PTH in this cell was identical to that seen in freshly isolated calvarial cells from normal mice. These results suggest that the activity of c-src in MG-63 cells is regulated by PTH as a result of tyrosine phosphorylation. Expression of src is not required for PTH responsiveness in osteoblastic cells as assessed by adenylate cyclase activation. The mode of signal transduction from the PTH receptor to nonreceptor c-src tyrosine kinase is not known at present.
Synthetic peptide containing Arg-Gly-Asp inhibits bone formation and resorption in a mineralizing organ culture system of fetal rat parietal bones
Authors: Gronowicz Gloria A. , Derome Mary E.
Published: February 2010

The role of integrins, cell surface receptors involved in cell adhesion to the matrix, was studied in a mineralizing organ culture system. Integrin-mediated cell attachment to matrix proteins has been shown to depend partially on the amino acid sequence Arg-Gly-Asp (RGD), present in the extracellular matrix proteins. Therefore, the effect of RGD peptides on bone formation and resorption was studied in the mineralizing organ culture system derived from 18 day fetal rat parietal bones. Addition of 0.1–50 μM GRGDSPK to bones cultured for 4 days inhibited mineralization in a dose-dependent manner as determined by measuring calcium content and % bone/unit area of tissue. A maximal decrease in calcium content and % bone/unit area of 32.5 and 42.9%, respectively, was found with 50 μM GRGDSPK. With 10 and 50 μM GRGDSPK, bone morphology was dramatically altered, with a disruption of osteoblast and mineralized matrix organization. To assess the effect of the peptides on bone resorption, fetal bones were prelabeled in vivo with 45Ca and resorption was stimulated in vitro with parathyroid hormone in the presence or absence of the peptide. A significant decrease in 45Ca release was found with 10 and 50 μM GRGDSPK. Osteoclast number was also significantly decreased on the bone surface. The peptide was not cytotoxic, since no effect on DNA content, dry weight, or collagen synthesis was found. GRADSP, a control peptide, had no significant effect on mineralization, resorption, or other parameters of bone growth. Visualization of β1 and α2 integrin in GRGDSPK-treated bones by indirect immunofluorescence demonstrated a decreased in integrin staining, particularly in the osteoblast layer, compared to control bones and bones treated with GRADSP. The inhibition of bone formation and resorption by an RGD-containing peptide in a mineralizing organ culture system suggests that integrins have an important role in osteoblast and osteoclast-mediated bone remodeling.
Proton-induced physicochemical calcium release from ceramic apatite disks
Authors: Bushinsky David A. , Sessler Nelson E. , Glena Richard E. , Featherstone John D.B.
Published: February 2010

When bone is cultured in acid medium there is net calcium efflux (JCa) and proton influx (JH) relative to the mineral. The acid medium appears to induce physicochemical mineral dissolution as well as cell-mediated bone resorption. To determine the independent effect of acid medium on physicochemical dissolution, we utilized cell-free synthetic ceramic apatite (CAP) disks, which contain carbonate (5.5%) in an apatite structure chemically similar to mammalian bone. CAP disks were cultured in control (Ctl, pH ≅ 7.44) or acid (Met, pH ≅ 7.11) medium for 48 h and compared to similarly treated neonatal (4–6 days old) mouse calvariae. Medium was changed and analyzed at 3, 24, and 48 h. At 3, 24, and 48 h there was significantly greater JCa from the CAP disks and calvariae incubated in Met compared to Ctl; over the entire 48 h time period there was a greater progressive increase in JCa from the CAP disks than the calvariae incubated in Met. There was no significant JCa at 3, 24, or 48 h from CAP disks or calvariae incubated in Ctl. At 3 h there was significantly greater JH into the CAP disks and calvariae incubated in Met compared to Ctl; JH was greater into the CAP disks than the calvariae. Utilizing a synthetic model of bone mineral we demonstrated that acid medium induces physicochemical calcium efflux and proton influx relative to the mineral.
Use of a rat model for the simultaneous assessment of pharmacokinetic and pharmacodynamic aspects of bisphosphonate treatment: Application to the study of intravenous 14C-labeled 1-hydroxy-3-(1-pyrrolidinyl)-propylidene-1,1-bisphosphonate
Authors: Koopmans Sietse J. , Van Der Wee-Pals Lianne , Löwik Clemens W.G.M. , Papapoulos Socrates E.
Published: February 2010

Bisphosphonates are drugs that suppress osteoclast-mediated bone resorption and are used with increasing frequency in the treatment of skeletal disorders. Therapeutic regimens are largely based on pharmacodynamic information because of difficulties in obtaining and interpreting pharmacokinetic data. We describe here the application of a permanently cannulated rat model, previously used in other areas of endocrine research, to the simultaneous study of pharmacokinetic and pharmacodynamic properties of the newly developed bisphosphonate EB-1053 [1-hydroxy-3-(1-pyrrolidinyl)propylidene-1,1-bisphosphonate]. Two groups of five rats each received daily intravenous injections of [14C]EB-1053 (0.025 and 0.1 mg/day, respectively); a third group (n = 7) received only normal saline injections and served as control. Treatment was given for at least 20 days. A fourth group (n = 3) received IV injections of the bisphosphonate on three separate occasions. Following IV administration, EB-1053 was rapidly cleared from the circulation. Urinary excretion of radioactivity reached about 55% of the daily administered dose within 48 h and remained at this level during the whole treatment period, indicating continuing retention of the bisphosphonate. Bone resorption, assessed biochemically as the hydroxyproline to creatinine ratio in urine, was suppressed effectively with both doses used. Suppression reached a maximum around day 4 and remained at the same level until the end of treatment. Accumulation of the bisphosphonate in the skeleton was therefore not associated with a cumulative effect on bone resorption. This strongly suggests that in treatment planning a distinction should be made between surface-bound and hence biologically active bisphosphonate from the drug which is incorporated in bone during bone turnover. The rat model used in these studies may also allow the examination of other issues of bisphosphonate pharmacology, such as sequential changes in biochemical parameters of bone metabolism in plasma and urine of the same animal, which are necessary for the planning of long-term therapy with uninterrupted administration of these compounds.
Characterization of immunoreactive forms of human osteocalcin generated in vivo and in vitro
Authors: Garnero Patrick , Grimaux Marc , Seguin Patrick , Dr. Delmas Pierre D.
Published: February 2010

Three monoclonal antibodies recognizing the 5–13, 25–37, and 43–49 sequence of the human osteocalcin were used in competitive and two-site radioimmunoassays (RIA) to characterize specifically various immunoreactive forms of circulating human osteocalcin. The intact molecule accounts for 36% of total in normals (2.6 nM), 46% in patients with osteoporosis (3.1 nM), and 26% in chronic renal failure (6.9 nM). Four fragment were detected in addition to the intact molecule in the serum of healthy adults and patients with metabolic bone disease. N-terminal, mid, and mid C-terminal fragments were present in minute amounts (each accounting for 5–14% of the total circulating osteocalcin immnoreactivity). In contrast, the N-terminal mid-fragment, probably resulting from the cleavage around amino acids 43–44, represents about 30% (2 nM) of the total osteocalcin immunoreactive level in normals and patients with osteoporosis and up to 50% (13 nM) in patients with chronic renal failure. This large N-terminal midfragment, representing 75–80% of the intact osteocalcin level, is not lower when the plasma assay is performed immediately after sampling (within 20 minutes at 4°C with proteinase inhibitors), indicating that it circulates in vivo. In addition, this fragment was detected in the supernatant of osteoblastic cells, representing about 28% of the intact peptide. Levels of N-terminal midfragment were not changed after treatment of patients with metabolic bone disease (Paget's disease, reflex sympathetic dystrophy, fibrous dysplasia, and osteoporosis) by bisphosphonate, suggesting that it is not released during bone resorption. The osteocalcin level measured with the two-site immunoradiometric assay specific for the intact molecule or with a conventional bovine RIA was rapidly decreased after incubation of serum at room temperature (-20 and −15%, respectively, after 3 h), whereas the total level of intact osteocalcin plus N-terminal midfragment was not changed. Intact osteocalcin loss can be partially avoided by proteinase inhibitors and by incubating serum at 4°C. In conclusion, we characterized multiple immunoreactive forms of osteocalcin that circulate in addition to the intact molecule, none of them being specifically altered in osteoporosis. The N-terminal midfragment circulates in a large amount, probably resulting from cleavage of the intact molecule in the circulation and/or at peripheral sites. These fragments can also be generated in vitro by proteolytic degradation of the intact molecule. To obtain reliable intact osteocalcin values but also reliable levels measured with conventional competitive RIA, careful control of the sampling conditions is warranted.