bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Mac-2 antigen in the preosteoclast and osteoclast identified in the op/op mouse injected with macrophage colony-stimulating factor
Authors: Niida Shumpei , Amizuka Norio , Hara Fumiko , Ozawa Hidehiro , Kodama Hiroaki
Published: February 2010

Osteoclast deficiency in op/op mice is cured by a single injection of 5 μg recombinant human macrophage colony-stimulating factor (rhM-CSF). In this study, we found that mouse osteoclasts are positive for Mac-2 antigen, but not for F4/80, MOMA-2, Mac-1, or BM8 antigen. By using F4/80 and MOMA-2 monoclonal antibodies, we confirmed the absence of mature macrophages in the femora of op/op mice and found that multiple injections of rhM-CSF are required for the recruitment of macrophages in the bones. After a single rhM-CSF injection, we found Mac-2 positive mononuclear cells in the femora of op/op mice. The time course of the appearance of Mac-2-positive cells was very similar to that of tartrate-resistant acid phosphatase (TRAP)-positive cells. In bone sections prepared from the mutant mice that received rhM-CSF 3 days earlier, 91% of the TRAP-positive mononuclear cells were also positive for Mac-2 antigen. These results demonstrate the expression of Mac-2 antigen in preosteoclasts. The antigen was detected on the plasma membrane of preosteoclasts, as well as in their cytoplasm and nucleus, and in the extracellular matrix in the space between the cells and bone. Since Mac-2 is a galactose-specific lectin, a potential role of the lectin in cell-cell and cell-matrix adhesion during osteoclast differentiation is suggested.
Relationship of the plasminogen activator/plasmin cascade to osteoclast invasion and mineral resorption in explanted fetal metatarsal bones
Authors: Leloup Gaetane , Delaisse Jean-Marie , DR. Vaes Gilbert
Published: February 2010

An attempt was made to establish whether the activation of plasminogen into plasmin is necessary either for the preparatory phases to bone resorption, involving the recruitment of osteoclast precursors, their migration toward mineralized surfaces, and their final differentiation, or for the subsequent osteoclastic resorption phase. 45Ca-labeled fetal (17 day) mouse metatarsals were cultured under conditions in which they pursue their modeling for a few days. In this model, the resorption phase, monitored by the release of 45Ca into the medium, is entirely dependent on the preparatory phases affecting osteoclast precursors. It was, as expected, stimulated by parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 and inhibited by calcitonin. PTH also enhanced the activity of tissue-type plasminogen activator (PA) in extracts of metatarsals but not that of urokinase (which is, however, the main PA present in the mouse fetal metatarsal culture model). The resorption processes were not dependent on the presence of plasminogen in the media, even when the rudiments were precultured with tranexamic acid to remove their endogenous plasminogen. Moreover, they were not influenced by inhibitors of plasmin, either the plasma inhibitors α2-antiplasmin, α2-macroglobulin, and α1-antitrypsin, or aprotinin, which was tested under a variety of conditions. Aprotinin also did not influence the resorption (loss of calcium and hydroxyproline) of 19 day fetal mouse calvariae cultured with PTH in a medium devoid of plasminogen. It is concluded that the various steps implicated in the bone resorption processes that occur in the metatarsals and in the calvariae culture models are not dependent on the activity of plasmin. The function of PAs in bone, however, could be exerted through direct proteolysis of extracellular proteins other than plasminogen or be mediated by a molecular structural domain distinct from their catalytic domain.
Growth hormone-dependent insulin-like growth factor binding protein is a major determinant of bone mineral density in healthy men
Authors: Johansson Anna G. , Forslund Anders , Hambraeus Leif , Blum Werner F. , Ljunghall Sverker
Published: February 2010

To establish the major determinants of bone mass, we assessed relationships between bone mineral density (BMD) and height, weight, body mass index (BMI), muscle strength, physical capacity (Vo2max), body composition, serum concentrations of insulin-like growth factor I (IGF-I), growth hormone (GH), the GH-dependent IGF binding protein (IGFBP-3), testosterone, sex hormone binding globulin (SHBG), osteocalcin, and parathyroid hormone (PTH) in 38 healthy men between 25 and 59 years of age. Values of BMD at all sites (total body, lumbar spine, and hip) were strongly correlated with IGFBP-3 (r = 0.51–0.64, p < 0.001 at all sites), and total-body BMD was also significantly correlated with IGF-I (r = 0.43, p = 0.01). BMD measurements of the total body and of the different sites of the hip were negatively correlated with age and positively with weight, BMI, muscle strength, Vo2max, and fat-free weight. IGF-I and IGFBP-3 were both positively related to muscle strength and Vo2max. In a stepwise forward multiple-regression analysis, the best model was obtained for the femoral neck, where IGFBP-3, GH, PTH, age, IGF-I, and BMI explained 77% of the variation in BMD. The partial regression coefficients of IGFBP-3, PTH, and BMI were all positive, whereas age, GH, and IGF-I were negatively correlated with BMD. In summary, IGFBP-3 correlated better with BMD than any other study parameter. The findings indicate that GH is of importance for bone mass and suggest that IGFBP-3 not only reflects the integrated GH secretion but also has a direct role in the endocrine regulation of bone metabolism.
Mutation analysis of coding sequences for type I procollagen in individuals with low bone density
Authors: Spotila Loretta D. , Colige Alain , Sereda Larisa , Constantinou-Deltas Constantinos D. , Whyte Michael P. , Riggs Lawrence B. , Shaker Joseph L. , Spector Timothy D. , Hume Eric , Olsen Nancy , Attie Maurice , Tenenhouse Alan , Shane Elizabeth , Briney Walter , Prockop Darwin J.
Published: February 2010

Mutations in one of the two genes encoding type I procollagen (COL1A1 and COL1A2) are frequently the cause of osteogenesis imperfecta (OI), a disorder characterized by brittle bones. Here we tested whether patients with low bone density also have mutations in these genes. The 26 patients studied had no apparent metabolic bone disease, but most had a positive family history of osteopenia or osteoporosis. Although a diagnosis of OI was considered by the clinician in some cases, the clinical criteria for OI were not satisfied. Our strategy for mutation analysis consisted of PCR amplification of cDNA made to fibroblast mRNA using primers specific for the coding regions of COL1A1 and COL1A2. The PCR products were then sequenced directly with primers located within each PCR product. We found that 3 of 26 patients had mutations that altered the encoded amino acid. One mutation, at position α2(I)-661 has been reported (Spotila et al. 1991 Proc Natl Acad Sci USA PNAS 88: 5423). The other 2 patients, who were not related to each other, had a mutation that altered the proline codon at α1(I)-27 to alanine. This mutation was not found in 81 normal individuals or in 37 additional osteopenic individuals. However, its effect on the biologic function of type I collagen, as well as its role in osteopenia, is uncertain. In addition to the two mutations, we found a polymorphism in codon α2(I)-459. Although this polymorphism involved an amino acid substitution, it was present with equal frequency in the patient and the normal population. By analyzing this and previously reported neutral sequence variants in the COL1A2 gene, we determined that all patients expressed both alleles of the COL1A2 gene. The 12 patients who were heterozygous for a COL1A1 neutral sequence variant also expressed both alleles. Here we present all PCR primer and sequencing primer information. The results suggest that surveying a larger group of similarly selected individuals may reveal additional mutations in the COL1A1 or COL1A2 genes.
Stimulation of signal transduction pathways in osteoblasts by mechanical strain potentiated by parathyroid hormone
Authors: Carvalho Roberto S. , Scott J. Elliot , Suga Dolores M. , DR. Yen Edwin H. K.
Published: February 2010

Second-messenger systems have been implicated to transmit mechanical stimulation into cellular signals; however, there is no information on how mechanical stimulation is affected by such systemic factors as parathyroid hormone (PTH). Regulation of adenylyl cyclase and phosphatidylinositol pathways in rat dentoalveolar bone cells by mechanical strain and PTH was investigated. Two different cell populations were isolated after sequential enzyme digestions from dentoalveolar bone (group I and group II) to study potential differences in response. Mechanical strain was applied with 20 kPa of vacuum intermittently at 0.05 Hz for periods of 0.5, 1, 5, 10, and 30 minutes and 1, 3, and 7 days using the Flexercell system. Levels of cAMP, measured by RIA, and levels of inositol 1,4,5-triphosphate (IP3) and protein kinase C activity (PKC), measured by assay systems, increased with mechanical strain. When PTH was added to the cells, there was a significant increase in levels of all the intracellular signals, which appeared to potentiate the response to mechanical strain. IP3 levels (0.5 minute) peaked before those of PKC activity (5 minutes), which in turn peaked before those of cAMP (10 minutes). Group II cells showed higher levels of cAMP and IP3 than the group I cells. This suggests that the former may ultimately play the predominant roles in skeletal remodeling in response to strain. Immunolocalization of the cytoskeleton proteins vimentin and α-actinin, focal contact protein vinculin, and PKC showed a marked difference between strained and nonstrained cells. However, the addition of PTH did not cause any significant effect in cytoskeleton reorganization. Staining of PKC and vimentin, α-actinin, and vinculin suggests that PKC participates actively in the transduction of mechanical signals to the cell through focal adhesions and the cytoskeleton, although only PKC seemed to change with short time periods of strain. In conclusion, dentoalveolar osteoblasts responded to mechanical strain initially through increases in levels of IP3, PKC activity, and later cAMP, and this response was potentiated when PTH was applied together with mechanical strain.
Integrins and osteoclastic resorption in three bone organ cultures: Differential sensitivity to synthetic arg-gly-asp peptides during osteoclast formation
Authors: van der Pluijm Gabri , Mouthaan Henk , Baas Corafien , de Groot Henny , Papapoulos Socrates , Löwik Clemens
Published: February 2010

We investigated possible inhibitory effects of five synthetic Arg-Gly-Asp (RGD)-containing peptides on osteoclastic resorption in three distinct in vitro resorption assays (17-day-old fetal mouse bone organ cultures) that differ in stages of osteoclast differentiation. RGD peptides, which can bind the adhesion receptors called integrins, inhibited osteoclastic resorption (45Ca release) in fetal mouse bone explants in which osteoclast precursors have yet to adhere to the mineralized matrix and develop into mature osteoclasts (metacarpals and coculture system). Treatment of metacarpals with RGD peptides inhibited the formation of multinucleated TRAP+ osteoclasts in the mineralized matrix because their mononuclear TRAP+ osteoclast precursors remained localized in the periosteum. In particular, echistatin, a viper venom protein with known affinity for αvβ3 integrin, and GdRGDSP inhibited osteoclastic resorption dose dependently in these systems (ED50 10−9 and 10−4 M, respectively) but did not alter the activity of mature resorbing osteoclasts in radii. In addition, 45Ca release was significantly inhibited by the cyclic peptide GPenGRGDSPCA, which has a relatively higher affinity for the vitronectin than fibronectin receptor(s). In contrast, GRDGdSP, which has a much higher affinity for the fibronectin receptor (than the vitronectin receptors), had no effect on resorption at similar concentrations in any resorption system used. In summary, the data presented in this paper show that peptides with RGD motifs are capable of inhibiting osteoclastic resorption in bone organ cultures. Our studies not only support the hypothesis concerning the importance of αvβ3 in osteoclastic resorption but also suggest an important role of integrin(s) in events preceding the actual resorption of calcified matrix by osteoclasts.
Functional properties of a synthetic chicken parathyroid hormone-related protein 1–36 fragment
Authors: Schermer D.T. , Bradley M.S. , Bambino T.H. , Nissenson R.A. , Strewler G.J.
Published: February 2010

The biologic activities of human parathyroid hormone-related protein [hPTHrP(1–34] and bovine PTH [bPTH(1-34)] are remarkably similar despite marked sequence divergence in their primary binding domain, residues 25–34. Chicken PTHrP (cPTHrP) is identical to hPTHrP through residue 21. However, in the 25–34 region, cPTHrP displays three fewer basic residues than hPTHrP and contains five residues not present in any other member of the PTH/PTHrP family. To assess the biologic consequences of these structural differences, we compared the activities of synthetic [36Tyr]cPTHrP(1–36)NH2 and hPTHrP(1–34)NH2 with those of bPTH(1–34) in avian systems (chicken renal plasma membranes and 19 day chick embryonic bone cells) and mammalian systems [canine renal plasma membranes and rat osteosarcoma cells (UMR-106-H5)]. In both avian and mammalian systems the binding affinity of [36Tyr]cPTHrP(1–36)NH2 (0.8–3.4 nM) was approximately one-half that of hPTHrP(1–34)NH2 (0.4–1.1 nM). The potencies of [36Tyr]cPTHrP(1–36)NH2 and hPTHrP(1–34)NH2 for activation of adenylate cyclase were similar in canine renal membranes (5.2 and 6.7 nM) and chick bone cells (1.0 nM). In UMR-106 cells and chicken renal membranes the potency of [36Tyr[cPTHrP(1–36)NH2 for activation of adenylate cyclase was about one-half that of [36Tyr]hPTHrP(1–36)NH2. Binding of 125I-[36Tyr]cPTHrP(1–36)NH2 to chick bone cells and chicken renal membranes was completely displaced by bPTH(1–34) and hPTHrP(1–34)NH2: thus there was no evidence for a distinct chicken PTHrP receptor. In general, [36Tyr]cPTHrP(1–36)NH2 and hPTHrP(1–34)NH2 activated adenylate cyclase similarly despite their sequence differences in the 25–32 region. This suggests that basic residues in the 25–32 region are not required for the peptide to assume a biologically active conformation at the receptor. In cross-linking studies, both 125I-hPTHrP(1–34)NH2 and 125I-[36Tyr]cPTHrP(1–36)NH2 labeled a major 85 kD PTH/PTHrP receptor component in canine renal plasma membranes. 125I-hPTHrP(1–34)NH2 also labeled a ≤14 kD receptor fragment, whereas 125I-[36Tyr]cPTHrP(1–36)NH2 did not. The present results suggest that retained sequence features in the 25–32 region may be critical determinants of receptor binding and that sequence differences in this region alter the sites of interaction of PTHrP peptides with the PTH/PTHrP receptor.
Automated evaluation of hip axis length for predicting hip fracture
Authors: Faulkner Kenneth G. , Mcclung Michael , Cummings Steven R.
Published: February 2010

The hip axis length has been shown in previous studies to be predictive of hip fracture independent of age and femoral bone density. The first studies of hip axis length were performed by manual measurement of dual x-ray absorptiometry (DXA) scan printouts. In this study, an automated analysis procedure is defined using software tools provided by the DXA manufacturer. Manual and automatic hip axis length measurements in 198 women were highly correlated (r = 0.98). Because of scaling factors of the printout, the automatic measurement was 58% longer than the manual value. Precision of the automatic measurement, based upon triplicate DXA scans of 33 women, was 0.07 cm or 0.68%. To define normative data, the hip axis length was measured from femoral DXA scans of 471 female volunteers aged 40–92 scanned on 14 different Hologic QDR-1000 systems. Mean hip axis length was 10.5 cm, with a standard deviation of 0.62 cm. No significant relationship between hip axis length and age was found (r = 0.07, P = 0.15). Based on previously reported odds ratios corrected for femoral bone density, age, height, and weight, an automatic hip axis length measurement of 11.0 cm is associated with a twofold increase in hip fracture risk compared with a woman with an average hip axis length. A hip axis length value of 11.6 cm increases hip fracture risk by a factor of 4 compared with a woman with a normal hip dimension. We conclude that the hip axis length can be easily incorporated into existing DXA hip analysis software in combination with a bone density measurement. With further research, the hip axis length may become a clinically useful tool for the evaluation of hip fracture risk.
Carbonic anhydrase II mRNA expression in individual osteoclasts under “resorbing” and “nonresorbing” conditions
Authors: Asotra S. , Gupta A.K. , Sodek J. , Aubin J.E. , Heersche J. N. M.
Published: February 2010

Rabbit osteoclasts can be transformed from a nonresorbing state to a resorbing state by transferring them from culture medium at pH 7.5 to one at pH 6.5. We evaluated whether expression of mRNA for carbonic anhydrase (CA-II) could be used as an indicator of the state of activity of individual osteoclasts. A cDNA probe to rabbit carbonic anhydrase II (CA-II) was prepared and used for in situ hybridization analysis of osteoclasts isolated from neonatal rabbit long bones. Quantitation by grain counting revealed heterogeneity within the osteoclast population: osteoclasts with a “compact” (rounded, less spread) morphology expressed higher levels of CA-II mRNA than “spread” osteoclasts with similar numbers of nuclei. When maintained at pH 6.5 for 6 h, the level of CA-II mRNA was increased significantly in osteoclasts of both morphologies compared with those in parallel cultures maintained at pH 7.5. These results were confirmed by quantitating CA-II mRNA using the polymerase chain reaction (PCR). Oligonucleotide primers specific for rabbit CA-II were synthesized and used to amplify CA-II cDNA transcribed from mRNA prepared from single or small numbers (one to eight cells) of osteoclasts that were collected with a micromanipulator. This generated a ∼510 bp PCR product, corresponding to the predicted size of the CA-II fragment encompassed by the primers. For quantitation, CA-II mRNA levels were compared with the levels of a ∼900 bp actin fragment that was coamplified in the same reaction mixture or amplified separately in a duplicate sample of the reaction mixture. The ratio of CA-II mRNA expression to actin mRNA expression was significantly increased in osteoclasts cultured at pH 6.5 for 6 h compared with osteoclasts maintained at pH 7.5 (1.89 + 0.12 versus 0.98 + 0.06, n = 39, mean + SEM, of all assays combined; P < 0.001). Our results demonstrate that CA-II mRNA expression is upregulated in osteoclasts in the resorptive state. The methods used provide a novel molecular approach for analyzing osteoclast activity with assays that are applicable to single cells and obviate the problem of osteoclast impurity, allowing investigation of osteoclast heterogeneity.
Periosteal bone formation stimulated by externally induced bending strains
Authors: Raab-Cullen D.M. , Akhter M.P. , Kimmel D.B. , Recker R.R.
Published: February 2010

The rat tibia four-point bending model is a new mechanical loading model in which force is applied through external pads to the rat lower limb. The advantages of the model are controlled force application to a well-defined bone, noninvasive external loading, and the addition of loads to normal daily activity. A disadvantage of the model is that the pads create local pressure on the leg at the contact sites. This study examined the differences in tibial response to bending strains and to local pressure under the pads. A total of 30 adult Sprague-Dawley rats were randomized into three external loading groups: bending, cyclic pressure, and static pressure. The right leg of each rat was externally loaded to create either bending or local pressure without bending; the left leg served as a control. Strains on the lateral surface averaged 1200 μϵ in compression during bending load application and <200 μϵ in compression during pressure loading. Histomorphometric data were collected from three regions: the maximal bending region, under the loading pads, and outside the maximal bending region. In the maximal bending region, bending loads created greater mineral apposition rate (MAR) on the lateral surface and greater MAR and formation surface on the medial surface of loaded than control tibiae. The region under the bending pad was exposed to similar bending strains and showed the same pattern of increased MAR as sections from the maximal bending region. Cyclic pressure had no effect on periosteal MAR or formation surface. Static pressure increased MAR only on the lateral tibial surface. Bending stimulates bone formation in regions with the highest bending strains. Similar forces applied only in the form of pressure loading do not stimulate tibial formation either at the contact site or between loading pads. These results suggest that externally applied forces of moderate magnitude stimulate bone formation primarily as a result of increased bending strains, not local pressure at the contact site.
Tartrate-resistant acid phosphatase accumulated in the matrix of developing medullary bone induced by estrogen treatment of male Japanese quail
Authors: DR. Yamamoto Toshio , Nagai Hiroshi
Published: February 2010

Tartrate-resistant acid phosphatase (TRAP) in medullary bone matrix and bone cells was histochemically examined employing estrogen-induced medullary bone of male Japanese quail. Within 2 days after estrogen administration, medullary bone matrix was identified and the matrix and osteoblasts were negative for TRAP activity. Osteoclasts showing TRAP activity were occasionally seen. By 4 days, the projection of medullary bone trabeculae was seen and TRAP became positive in the deeper part of the bone matrix of trabeculae as well as the matrix located near cortical bone, but the surface areas and extending margins of medullary bone trabeculae were negative in activity. Osteoblasts were also negative for TRAP activity. By 7 days, TRAP-positive bone matrix conspicuously increased accompanying increment of bone volume, but TRAP-negative bone matrix was still seen at the surface areas or extending areas of the medullary bone trabeculae. Osteoblasts showed no reaction product. Osteoclasts showing TRAP activity increased in number. The results demonstrate that TRAP accumulates in medullary bone matrix with bone formation. Circumstantial evidence suggests that medullary bone osteoblasts may be involved in TRAP accumulation in the matrix despite their negative activity.
Influence of osteoclasts and osteoclast-like cells on osteoblast alkaline phosphatase activity and collagen synthesis
Authors: Galvin Rachelle J. Sells , Cullison James W. , Avioli Louis V. , Osdoby Philip A.
Published: February 2010

Osteoblasts have been shown to modulate osteoclast activity, but the reverse process has not been investigated. In the current study conditioned medium (CM) was collected from osteoclasts and osteoclast-like cells and its effects on osteoblast alkaline phosphatase (ALPase) activity and collagen synthesis ([3H]proline hydroxylation) were determined. In primary chick osteoblasts, cultured chick embryo frontal bones, and UMR-106-01 cells, collagen synthesis and ALPase activity, but not [3H]thymidine incorporation, were inhibited by CM from chick marrow-derived giant cells, which possess some of the phenotypic characteristics of osteoclasts. However, collagen synthesis in chick embryo fibroblasts was not affected by giant cell CM. CM collected from cultures of chicken osteoclasts and human osteoclastoma cells and marrow-derived giant cells inhibited collagen synthesis in UMR-106-01 cells, but the effects on ALPase activity varied with the cell type. In contrast, mononuclear cell and fibroblast CM did not alter collagen synthesis. Initial characterization studies demonstrate that the inhibitor is a heat-labile factor with a molecular weight greater than 3500. In summary, authentic osteoclasts, tumor osteoclast-like cells, and chicken and human multinucleated giant cells produce a soluble factor that alters osteoblast collagen synthesis, suggesting that osteoclasts play a role in the modulation of osteoblast activity.
A number of osteocalcin antisera recognize epitopes on proteins other than osteocalcin in cultured skin fibroblasts: Implications for the identification of cells of the osteoblastic lineage in vitro
Authors: Bradbeer J.N. , Virdi A.S. , Serre C.M. , Beresford J.N. , Delmas P.D. , Reeve J. , Triffitt J.T.
Published: February 2010

Rabbit antisera to bovine osteocalcin were produced independently in two laboratories and their specificities established by western blot analysis. By immunohistochemistry each of the five polyclonal antisera produced an intense cytoplasmic staining in human bone-derived cells. Staining intensity was strongly attenuated by preabsorption of the antisera with osteocalcin. No staining was observed using nonimmune rabbit serum. However, the choice of skin cells as negative controls for osteocalcin synthesis yielded an unexpected positive staining pattern similar to that seen with the bone-derived cells over a range of antiserum dilutions. This was not caused by the uptake of exogenous osteocalcin from the culture medium because a similar pattern of staining was observed when medium was supplemented with osteocalcin-depleted fetal calf serum. Treatment with 1,25-dihydroxyvitamin D3 induced osteocalcin mRNA expression and osteocalcin secretion in cultures of bone-derived cells but not in skin fibroblasts. The results demonstrate that these polyclonal antisera also recognize epitopes shared with other proteins synthesized in culture by skin fibroblasts. Furthermore, three mouse monoclonal antibodies to distinct regions of the osteocalcin molecule show differential staining of human bone-derived cells, skin cells, and osteosarcoma cells (MG63). These observations indicate that the shared epitope resides in the central region of osteocalcin and are consistent with the specific synthesis of osteocalcin by bone cells alone. The observed nonspecificity of many osteocalcin antisera may compromise immunocytochemical studies of the osteoblast phenotype in studies in vitro when based solely on reactivity with inadequately characterized osteocalcin antisera.
Regulation of collagen type I and biglycan mRNA levels by hormones and growth factors in normal and immortalized osteoblastic cell lines
Authors: Thiébaud D. , Guenther H.L. , Porret A. , Burckhardt P. , Fleisch H. , Hofstetter W.
Published: February 2010

Growth factors, such as transforming growth factor β (TGF-β) and insulin-like growth factors (IGF) I and II, have been shown to exert anabolic effects on bone cells in vitro. Hormones, such as PTH and probably insulin and growth hormone, were recently shown to stimulate bone formation in vivo as well. The aim of the present study was to assess by northern blots, which were quantitated by densitometry, the effects of these anabolic growth factors and hormones in two osteogenic cell populations: CRP 10/30 cells, a clonal cell population derived from primary rat calvarial cells, and IRC 10/30-myc cells, which were established from CRP 10/30 by immortalization. Transcripts for α1(I) collagen, biglycan, osteonectin, osteopontin, and osteocalcin were detected in both cell populations, which is consistent with the phenotype expressed by mature osteoblasts. There were no difference in the basal expression of bone matrix mRNAs between the two cell populations. PTH increased α1(I) collagen mRNA levels in both osteoblastic cells but had no effect on the biglycan transcripts. Neither insulin nor growth hormone affected mRNA levels of either matrix protein after 24 h exposure. All three growth factors, TGF-β, IGF-I, and IGF-II, increased α1(I) collagen transcripts in a time- and dose-dependent manner in both cell populations. Biglycan mRNA levels were enhanced in both osteoblastic lines only by IGF-I and IGF-II, but not TGF-β. The transcriptional effect was ascertained by nuclear run-on assays or actinomycin inhibition. When the effects of TGF-β, IGF-I, and IGF-II on the expression of α1(I) collagen and biglycan mRNAs were compared in the two cell populations, there was a similar pattern of response. The only significant difference between the immortalized cell line and the parent cell population was a stronger stimulation of α1(I) collagen mRNA by TGF-β and a delayed increase in biglycan mRNA levels in IRC 10/30-myc. The results show that the immortalized cell line IRC 10/30-myc is useful to study the modulation of two osteoblastic markers, α1(I) collagen and biglycan mRNA, in response to exogenous factors. Biglycan is little modulated and only by IGFs, whereas PTH, IGF-I and II, and TGF-β strongly increase α1(I) collagen transcripts, confirming their anabolic effects on bone cells in vitro.
Normal osteoclastic and osteoblastic responses to exogenous growth hormone in patients with postmenopausal spinal osteoporosis
Authors: Kassem M. , DR. Brixen K. , Blum W.F. , Mosekilde L. , Eriksen E.F.
Published: February 2010

The cause of bone loss in patients with osteoporosis is not known, but both increased bone resorption and decreased bone formation have been reported. Theoretically, these effects may result from either increased activity of osteoclasts or decreased activity of osteoblasts, or both. In vivo, growth hormone (GH) administration leads to activation of osteoclasts and osteoblasts as evidenced by increased biochemical markers of bone resorption and bone formation. To test for disturbances in responsiveness of bone cells to exogenous hormonal stimuli in osteoporosis, we compared 15 patients with postmenopausal osteoporosis with 15 healthy age-matched postmenopausal women before and during a 3 day stimulation test with GH (0.2 IU/kg/day). Serum insulin-like growth factor I increased in both groups (p < 0.001). GH treatment increased biochemical markers of bone resorption (serum carboxyl-terminal telopeptide of type I collagen [ICTP] [p < 0.001] and, to a lesser extent, 24 h urinary hydroxyproline/creatinine) in the two groups. Similarly, biochemical markers for bone formation increased in both groups [osteocalcin (p < 0.01) and procollagen type I C-terminal propeptide, PICP (p < 0.001)]. GH treatment reduced alkaline phosphatase (ALP, p < 0.05) and its bone-specific isoenzyme (bone ALP, p < 0.01) in both groups. The maximal response, the area under the curve (AUC) of response curves for IGF-I, bone resorption markers, and bone formation markers were not different between groups. Our data do not support the hypothesis that osteoporotic patients display major disturbances in responsiveness to GH.
Single-cell analysis of cyclic AMP response to parathyroid hormone in osteoblastic cells
Authors: Civitelli Roberto , Bacskai Brian J. , Mahaut-Smith Martyn P. , Adams Stephen R. , Avioli Louis V. , Tsien Roger Y.
Published: February 2010

We previously demonstrated that the [Ca2+], response to PTH is heterogeneous in single UMR-106-01 osteogenic sarcoma cells. To verify whether response heterogeneity is a universal feature of PTH signal transduction, cAMP production was monitored in monolayer cultures of UMR-106-01 cells and human trabecular bone osteoblasts (HOB) using the cAMP-sensitive fluorescent indicator FICRhR. FICRhR was microinjected into single cells, and the 500-530/>560 nm fluorescence ratio was monitored by confocal laserscanning video imaging as a measure of cAMP concentration ([cAMP]). Virtually all UMR-106-01 cells exposed to bovine PTH(1-34) (10−7 M) exhibited an increase in intracellular [cAMP], with an average fluorescence ratio change of 145 + 17% of baseline (n = 15), corresponding to nearly maximal dissociation of protein kinase A. In the continued presence of the hormone (10−7 M), [cAMP] remained elevated for at least 30 minutes. This effect was accompanied by a slow translocation of the fluorescein-labeled catalytic subunit of protein kinase A from the cytoplasm to the nucleus. In contrast, PTH(1-34) caused no detectable increase in [cAMP] in HOB cells, although PGE2 (3 x 10−6 M) stimulation was able to increase the FICRhR ratio (154 + 27%, n = 10). The truncated fragment PTH (2-34) was only 67% as potent at PTH(1-34), but deletion of the first two amino acids at the N terminus abolished the hormone's ability to stimulate cAMP production in UMR-106-01 cells. Brief exposure to 10−7 M of either PTH(3-34) or PTH(7-34) did not affect the amplitude of the fluorescence ratio change induced by equimolar doses of PTH(1-34). Thus, in osteoblast-like cells stimulated with PTH, the [cAMP] response is much more homogeneous from cell to cell than the [Ca2+]i response.
Correlates of bone mineral density in the postmenopausal estrogen/progestin interventions trial
Authors: Marcus Robert , Greendale Gail , Blunt Barbara A. , Bush Trudy L. , Sherman Sherry , Sherwin Roger , Wahner Heinz , Wells Bradley
Published: February 2010

We assessed the cross-sectional relationship of age, menopausal years, body mass, previous estrogen use, and ethnic background to bone mineral status in a sample of 875 healthy postmenopausal women at the time they were recruited from the community to participate in a multicenter clinical trial. The women were 1-10 years postmenopause, 45-64 years of age, and had not received estrogen replacement therapy within 3 months of enrollment. Of the participants, 89% were white, 69% had a spontaneous menopause, and 53% had a history of previous estrogen replacement therapy. Bone mineral density (BMD) of the lumbar spine (L2-4) and proximal femur was measured by dual-energy x-ray absorptiometry. Results were consistent with a significant negative linear regression of BMD on age or years from menopause. Body mass index (BMI) correlated significantly with BMD at all sites (L 2-4 r = 0.28; femoral neck r = 0.34, p < 0.0001). BMD adjusted for age and BMI were higher at both sites in women who had taken estrogen versus those who had not (L2-4 0.976 + 0.009 versus 0.932 + 0.01; femoral neck 0.740 + 0.006 versus 0.708 + 0.008, p < 0.05). Adjusted BMD also increased with duration of ERT. Parity was negatively associated with L2-4 BMD (p = 0.03) but did not correlate significantly with BMD at the femoral neck. Black women had the highest L2-4 BMD, and Hispanic women had the highest femoral neck BMD, even when results were adjusted for age and BMI. When data were corrected for differences in bone size, these interethnic differences were no longer significant. We conclude that increased body mass is positively correlated with BMD, and this may confer a degree of skeletal protection to heavier postmenopausal women. Exposure for 5 years to exogenous estrogen is associated with significantly increased age- and BMI-adjusted BMD.
The bone-remodeling transient: Implications for the interpretation of clinical studies of bone mass change
Authors: Heaney Robert P.
Published: February 2010

A computer simulation of the bone-remodeling transient is described, in which the focus is explicitly on changes in clinically measurable bone mass (or density). Based upon quantitative remodeling data accumulated by histomorphometry and calcium tracer kinetics, the simulation shows that much of the apparent gain in bone produced by several agents currently employed to treat osteoporosis can be explained as a remodeling transient rather than as a fundamental alteration of remodeling balance. Even gains as large as 30% or more can be produced by nothing more than the remodeling transient under certain plausible combinations of basal remodeling rate, remodeling period, and degree of bone loss. The simulation further highlights the importance, in evaluating bone-active agents, of separating the response across the first remodeling period from bone changes that may ensue thereafter.
Low bone mass in idiopathic renal stone formers: Magnitude and significance
Authors: Jaeger Philippe , Lippuner Kurt , Casez Jean-Paul , Hess Bernhard , Ackermann Daniel , Hug Christian
Published: February 2010

To assess bone mineral density (BMD) in idiopathic calcium nephrolithiasis, dual-energy x-ray absorptiometry was performed at lumbar spine, upper femur (femoral neck, Ward's triangle, and total area), distal tibial diaphysis, and distal tibial epiphysis in 110 male idiopathic calcium stone formers (ICSF); 49 with and 61 without hypercalciuria on free-choice diet). Results were compared with those obtained in 234 healthy male controls, using (1) noncorrected BMD, (2) BMD corrected for age, height, and BMI, and (3) a skeletal score based on a tercile distribution of BMD values at following four sites: lumbar spine, Ward's triangle, tibial diaphysis, and tibial epiphysis. After correction, BMD—and therefore also skeletal score—tended to be lower in the stone formers than in controls at five of the six measurement sites, that is, lumbar spine, upper femur, Ward's triangle, tibial diaphysis, and tibial epiphysis, limit of significance being reached for the last two sites without difference between hypercalciuric (HCSF) and normocalciuric stone formers (NCSF). Estimated current daily calcium intake was significantly lower in patients (616 + 499 mg/24 h, mean + SEM) than in controls (773 + 532, p = 0.02). Of 17 patients who in the past had received a low-calcium diet for at least 1 year, 10 had a low skeletal score (4-6) whereas only 1 had a high score (10-12; p = 0.037). Of the 12 stone formers in the study with skeletal score 4 (i.e., the lowest), 8 had experienced in the past one or more fractures of any kind versus only 19 of the remaining 77 patients with skeletal score 5-12 (p = 0.01). Significant correlations were found between corrected BMD at various sites and 24 h sulfate, uric acid and sodium excretion, and urinary pH, as well as serum uric acid concentration. BMD at tibial diaphysis was negatively correlated with pyridinoline/ creatinine concentration ratio in 24 h urine samples, and skeletal score was negatively correlated with fasting hydroxyprolinuria. In summary, there is a slight decrease in skeletal mineral content in idiopathic calcium stone formers at the tibial site that appears to be related at least in part to dietary habits (low-calcium diet and animal protein and sodium intake) experienced by these patients.
Interleukin-4 inhibits spontaneous and parathyroid hormone-related protein-stimulated osteoclast formation in mice
Authors: Nakano Yoichiro , Watanabe Kenichi , Morimoto Isao , Okada Yosuke , Ura Kazumi , Sato Kanji , Kasono Keizo , Nakamura Toshitaka , Eto Sumiya
Published: February 2010

We examined the in vivo effects of recombinant murine IL-4 (rmIL-4) on spontaneous and stimulated mouse osteoclast formation. EC-GI cells, which produce PThrP and IL-1α, were explanted in nude mice. These EC-GI cell-bearing nude mice developed hypercalcemia (4.90 + 0.68 mM), and the calcium levels were decreased to near normal (3.48 + 0.73 mM, p < 0.05) at day 3 by continuous infusion of rmIL-4 at a dose of 7 μg/day. When infused with 0.6 nmol/day of PTHrP(1-34) in ICR mice, rmIL-4 at a dose of 1 or 5 μg/day for 3 days caused a marked inhibitory effect on hypercalcemia induced by PTHrP(1-34) (3.73 + 0.56-2.54 + 0.14 mM, p < 0.01). However, rmIL-4 alone did not change the serum calcium in mice. Histomorphometric analysis revealed that rmIL-4 inhibits both spontaneous and PTHrP(1-34)-stimulated osteoclast formation in mice, with a decrease in osteoclastic surface and in the number of osteoclasts per mm bone surface, respectively. We conclude that IL-4 inhibits spontaneous and stimulated bone resorption resulting from inhibition of osteoclast formation and modulates the development of humoral hypercalcemia of malignancy.
Abnormal bone mineralization after fluoride treatment in osteoporosis: A small-angle x-ray-scattering study
Authors: Dr. Fratzl P. , Roschger P. , Eschberger J. , Abendroth B. , Klaushofer K.
Published: February 2010

Sodium fluoride treatment of osteoporosis is known to stimulate bone formation and to increase bone mass, but recent clinical trials failed to prove its antifracture effectiveness. The formation of bone with abnormal structure and, therefore, increased fragility is discussed as a possible explanation. Until now, however, exact information on the mineral structure of osteoporotic bone after fluoride treatment has been lacking. Bone biopsies were taken from three patients with postmenopausal osteoporosis before and after fluoride treatment (60 mg NaF/day for 1-2 years), from one patient with iatrogenic fluorosis, as well as from three normal controls. The mineral in these samples was investigated by a combination of backscattered electron imaging and small-angle x-ray scattering. Depending on the total dose of fluoride, an increasing amount of new bone is laid down on the surface of preexisting trabeculae. Its mineral structure is identical to that of heavy fluorosis and is characterized by the presence of additional large crystals, presumably located outside the collagen fibrils. These large crystals, which are not present in the controls or in osteoporotic bone before fluoride treatment, contribute to increase the mineral density without significantly improving the biomechanical properties of the bone. The possible success of fluoride treatment depends not only on the amount of newly formed bone but also on the rate of bone turnover. Indeed, as soon as significant amounts of fluoride are present, bone turnover leads to the replacement of old (normal) bone by new (pathologically mineralized) bone. In particular, in the case of high turnover rates we expect fluoride therapy even to lead to a deterioration in the overall mechanical stability of the skeleton.
Exercise can provide protection against bone loss and prevent the decrease in mechanical strength of femoral neck in ovariectomized rats
Authors: Peng Zhiqi , Tuukkanen Juha , Väänänen Kalervo H.
Published: February 2010

The effect of treadmill exercise on bone loss in ovariectomized (OVX) rats was studied in two different sets of experiments. In the first experiment rats were either ovariectomized (n = 38) or sham operated (n = 18) at the age of 12 weeks. Half the OVX rats were trained twice a day for 30 minutes by running at 10 m/minute for 7 or 17 weeks. In the second experiment 40 female rats, aged 12 weeks, were divided into five groups (n = 8). One group of rats was sacrificed on day 0 for the baseline data. Other rats were sham operated or ovariectomized for 9 weeks. Half of both groups were trained using the same training program as in the first experiment. OVX reduced trabecular bone volume (TBV) in the distal femur to 42.7 and 48.3% in 8 and 18 weeks, respectively. Exercise opposed this effect significantly but could not prevent it totally. Exercise did not have any significant effect on sham-operated animals. OVX induced a 17.7 and 30.7% decrease in maximal failure load of femoral neck in 8 and 18 weeks, respectively. A corresponding decrease was also observed in the torque capacity of tibia. Exercise was able to prevent almost totally the decrease in bone strength of femoral neck, tibia, and humerus. In conclusion, our results suggest that the measurement of bone strength in aging female rat femoral neck can be used as a useful indicator of the deleterious effect of OVX in bone. These results further indicate that exercise can overcome a significant part of the decrease in trabecular bone volume and maintain the mechanical strength of femoral neck and tibial shaft in the OVX rats.
Identification of osteocalcin mrna in nonosteoid tissue of rats and humans by reverse transcription—polymerase chain reaction
Authors: Fleet James C. , Hock Janet M.
Published: February 2010

Diseased or necrotic tissue can become calcified in a way that resembles bone. We examined soft tissues for the presence and regulation of the mRNA for the bone-associated protein, osteocalcin (OC). RNA was isolated from liver, kidney, lung, brain, muscle, and bone of young (2 months) male SD rats and analyzed for β-actin, IGF-I, metallothionein IIa, α1 collagen, calbindin-D9k (CaBP), and OC mRNA by reverse transcription-polymerase chain reaction (RT-PCR). All PCR products but CaBP were found in bone; CaBP was present only in duodenum, kidney, and lung. OC product was detected in all tissues; the identity of the PCR product was confirmed by sequencing. Bone OC mRNA levels were calculated to be 1000-fold higher than duodenal levels. Rats fed a 0.8% strontium diet for 7 days to drive down serum 1,25-dihydroxyvitamin D3 levels [1,25(OH)2D3] and then injected with 300 ng 1,25(OH)2D3/100 body weight had increased duodenal CaBP (2.5-fold) and femur OC mRNA (2.2-fold) 24 h after treatment. Duodenal OC mRNA was unchanged. OC mRNA was found in nondiseased human aortae, and the amount of message was elevated in calcified aorta and calcified aortic plaques. These results demonstrate that (1) tissues other than bone have low basal expression of OC mRNA, (2) OC mRNA is not regulated by vitamin D in nonosteoid tissue, and (3) expression of OC mRNA in atherosclerotic aorta reflects a role for bone-forming cells in ectopic bone formation observed in certain disease conditions.
Bafilomycin A1 inhibits bone resorption and tooth eruption in vivo
Authors: Sundquist Kai T. , Marks Sandy C.
Published: February 2010

It has been shown that a specific inhibitor of vacuolar H+-ATPases, bafilomycin A1, inhibits bone resorption by isolated chicken osteoclasts by blocking the proton pump in the ruffled border membrane. We report here the effects of bafilomycin A1 on bone resorption in vivo. Using a cannulated osmotic minipump delivery system, we infused bafilomycin locally to the eruption pathway of permanent premolars of beagle dogs. We used pit formation by osteoclasts in vitro to estimate the concentrations and heat stability of bafilomycin to be used in vivo. In this model, osteoclasts were cultured on thin bone slices, in which they form pits indicative of resorption. After 2 weeks preincubation at 37°C, bafilomycin concentrations of 10−6 and 10−7 M but not 10−8 M completely inhibited the resorptive activity of cultured osteoclasts, and the two larger doses were chosen for use in vivo. Local delivery of 10−6 M bafilomycin to the eruption pathway of the fourth permanent mandibular premolar during mideruption inhibited tooth eruption by blocking bone resorption as assayed by radiography, light microscopy, and scanning electron microscopy. Bafilomycin at 10−7 M had similar but less intensive effects. Moreover, osteoclasts in the alveolar bone of crypts treated with 10−7 M bafilomycin A1 stained very weakly for tartrate-resistant acid phosphatase. The effect of bafilomycin on bone resorption was shown to be very local, and no side effects of treatment with bafilomycin were observed in adjacent teeth or the behavior of dogs. We report here, for the first time, inhibition of tooth eruption caused by inhibited bone resorption using bafilomycin A1 in vivo.
Bone sialoprotein is localized to the root surface during cementogenesis
Authors: Macneil R.L. , Sheng N. , Strayhorn C. , Fisher L.W. , Somerman M.J.
Published: February 2010

Bone sialoprotein (BSP), an RGD-containing protein with cell attachment properties, is believed to play a regulatory role in the biomineralization of various connective tissues. To determine its possible role in tooth root formation, murine dentoalveolar tissues at sequential phases of development were analyzed immunohistochemically for the presence of BSP. BSP was localized to alveolar bone and cementum at time points associated with initial mineralization of these tissues. In addition, northern blot analyses of dental follicle tissue at day 27 of tooth development indicated that BSP mRNA is expressed by dental follicle cells at a time point coincident with the initiation of cementogenesis on the peripheral tooth root surface. Collectively, these findings indicate that BSP may play an important role in the formation and mineralization of cementum.