bluuebunny/crossref_metadata_2025 huggingface dataset:
Aluminum potentiates the effect of fluoride on tyrosine phosphorylation and osteoblast replication in vitro and bone mass in vivo
Authors: Dr. Caverzasio Joseph , Imai Toshio , Ammann Patrick , Burgener Dominique , Bonjour Jean-Philippe
Published: February 2010

Osteosclerosis in workers exposed to fluoride (F) and aluminum (Al) (industrial fluorosis) led to the use of F as a treatment to increase bone mass in osteoporosis patients. Because the influence of traces of Al on the effects of F on bone formation is heretofore unknown, we have investigated this issue both in vitro and in vivo. We have found that minute amounts of Al (≤10−5 M) potentiate the effects of F in vitro such that osteoblast proliferation increased by 15 ± 2.7% at 50 μM (p < 0.001) and by 117.6 ± 5.1% at 750 μM (p < 0.001), concentrations of F with no mitogenic effect alone. F + Al time-dependently modulated a growth factor signaling pathway(s) associated with enhanced tyrosine phosphorylation (TyrP) of several proteins (p90 [2.9x], p77 [4.9x], p68 [9.6x], and mitogen activated protein kinases [3x]). TyrP was only slightly or not at all changed by F and Al alone, respectively. The effects of F + Al on TyrP and cell proliferation were markedly reduced by 100 μM ryrphostin-51, a tyrosine kinase inhibitor. Protein kinase A (PKA) and protein kinase C (PKC) pathways were not involved in this response. In vivo, F + Al administered for 8 months, at doses that had no effect when the minerals were administered individually, significantly enhanced proximal tibia bone mineral density (BMD) by 6.3 ± 1% compared with initial values and by 2-fold compared with control ovariectomized rats (p < 0.0001). These effects are consistent with a crucial role of Al in osteosclerosis observed in industrial fluorosis. The results suggest that the combination of F + Al modulates a growth factor-dependent TyrP pathway enhancing mitogen-activated protein kinase and osteoblastic proliferation and bone mass.
Parathyroid hormone-induced resorption in fetal rat limb bones is associated with production of the metalloproteinases collagenase and gelatinase B
Authors: Witty Jean P. , Foster Shirley A. , Stricklin George P. , Matrisian Lynn M. , Stern Paula H.
Published: February 2010

The role of matrix metalloproteinases in parathyroid hormone (PTH)-induced bone resorption was assayed using a fetal rat limb bone culture system. Cotreatment of bones with PTH and recombinant inhibitor of metalloproteinases, TIMP-1, in vitro, inhibited the PTH-stimulated 45Ca release from the limb bones without affecting β-glucuronidase release. TIMP-1 was fully effective when added during only the final 24 h of a 72 h culture with PTH but was ineffective when added for only the first 24 h of the 72 h culture. In contrast, calcitonin (CT) was effective when added for either the first 24 or the final 24 h of the culture. Using in situ hybridization, the mRNA for collagenase was detected in mononuclear cells of cultured bone. Treatment of the bones with PTH resulted in an increase in the number of cells producing collagenase mRNA, some of which had osteoclastic morphology. PTH also caused a dramatic induction of the mRNA for the 92-kD gelatinase B metalloproteinase in both mononuclear and osteoclastic cells. There was no detectable mRNA for the metalloproteinases stromelysin-1, stromelysin-2, or matrilysin in PTH-treated or control cultures. These results suggest that PTH-induced bone resorption is mediated, at least in part, by the induction of collagenase and gelatinase B mRNA in bone cells.
Expression of osteocalcin in the patella of experimentally immobilized and remobilized rats
Authors: Kannus Pekka , Jozsa Laszlo , Kvist Martti , Järvinen Teppo L.N. , Maunu Vesa-Matti , Hurme Timo , Järvinen Markku
Published: February 2010

The exact mechanisms by which mechanical loading-unloading affects bone tissue are mostly unknown. Recently, osteocalcin, a direct product of osteoblasts, has been shown to reflect the activity of the mineralization phase of the newly formed bone matrix, and therefore, the in situ detection of osteocalcin could be used for studying the effects of physical activity-inactivity on the osteoblast function or bone formation in the target bone. In this study, the effect of various loading states (immobilization and three forms of subsequent remobilization) on the in situ expression of osteocalcin in the rat patellas and their osteotendinous junctions was studied immunohistochemically using a polyclonal rat antiosteocalcin as the primary antibody. Following immobilization for 3 weeks, the immunoreactivity of osteocalcin was markedly decreased or was completely absent in all the patellar areas which normally show intense reaction as a sign of mineralization of the newly formed bone, that is, in the subperiosteal and subchondral regions, in the osteoid tissue that lies on the surface of the trabecular bone, and around the cortical lacunae. The same was true in the mineralized fibrocartilage zone of the osteotendinous junction of the quadriceps tendon. Free remobilization for 8 weeks (free cage activity) could not improve the situation, but after intensified remobilization of the same duration (low and especially high intensity treadmill running) high osteocalcin expression was observed in the above-mentioned anatomic sites. These findings indicate that formation of new bone tissue is rapidly regulated by the loading states of the bone. Higher than normal activity seems to be needed to restore the bone formation from the disuseinduced depression to normal.
Interleukin-6 and soluble interleukin-6 receptors in the synovial fluids from rheumatoid arthritis patients are responsible for osteoclast-like cell formation
Authors: Kotake Shigeru , Sato Kazuto , Kim Kang Jung , Takahashi Naoyuki , Udagawa Nobuyuki , Nakamura Ichiro , Yamaguchi Akira , Kishimoto Tadamitsu , Suda Tatsuo , Kashiwazaki Sadao
Published: February 2010

Chronic immune responses and inflammatory reactions in rheumatoid arthritis (RA) often cause severe destruction of cartilage and bone, but its mechanism is still a matter of controversy. We reported that interleukin-6 (IL-6) alone does not induce osteoclast formation, but soluble interleukin-6 receptors (sIL-6R) triggered the formation in the presence of IL-6 in cocultures of murine osteoblastic cells and bone marrow cells. In this study, we examined the involvement of sIL-6R and IL-6 in joint destruction in patients with RA. Although the frequency of patients having osteoclast-like multinucleated cells in synovium derived from the knee joint was not significantly different between RA (65%) and osteoarthritis (OA) patients (43%), the number of osteoclast-like cells found in the synovium was greater in the former than in the latter. Multinucleated cells obtained from RA synovium expressed the osteoclast-specific phenotype such as tartrate-resistant acid phosphatase, carbonic anhydrase II, vacuolar proton-ATPase and vitronectin receptors at similar levels to those from a human giant cell tumor of bone. The concentration of both IL-6 and sIL-6R was significantly higher in the synovial fluids from patients with RA than with OA. The concentration of IL-6 and sIL-6R correlated well with the roentgenologic grades of joint destruction. Dose-response curves for human IL-6 and human sIL-6R in inducing osteoclast-like cell formation in cocultures indicated that the RA synovial fluids contained sufficient IL-6 and sIL-6R to induce osteoclastogenesis. When synovial fluids from RA and OA patients were added to the cocultures, some of the RA synovial fluids containing high levels of IL-6 and sIL-6R stimulated osteoclast-like cell formation, which was strikingly inhibited by adding anti-IL-6R antibody simultaneously. These results suggest that IL-6 in the RA synovial fluids is at least in part responsible for joint destruction in the presence of sIL-6R through osteoclastogenesis.
Evidence for preservation of cortical bone mineral density in patients on continuous ambulatory peritoneal dialysis
Authors: Mottet Jean-J. , Horber Fritz F. , Casez Jean-P. , Descoeudres Claude , Jaeger Philippe
Published: February 2010

It is still unclear whether dialysis modality, i.e., continuous ambulatory peritoneal dialysis (CAPD) versus hemodialysis (HD) specifically affects bone mineral density (BMD). To answer this question, 34 patients on HD and 25 on CAPD were matched for age, sex, height, and body weight with 125 normal subjects. BMD was measured using dual-energy X-ray absorptiometry (DXA; Hologic QDR 1000/W) at the lumbar spine (trabecular bone), the femoral neck (mixed cortical and trabecular bone), the distal tibial diaphysis (cortical bone), and the epiphysis (trabecular bone) in all subjects. No significant difference for blood hemoglobin, albumin, total and ionized calcium, intact parathyroid hormone (PTH) or phosphorus concentrations, as well as for alkaline phosphatase activity, failed renal allograft, prior steroid therapy, prior parathyroidectomy, duration of uremia, or of dialysis was found between patients on HD and those on CAPD. However, the residual daily urine volume and renal function at the time of the absorptiometry were higher in CAPD than in HD patients (p < 0.05) as well as the mean dialysate calcium concentration during dialysis, the blood bicarbonate concentration, and the residual renal function at the initiation of dialysis (p, 0.01, p < 0.05, and p < 0.005, respectively). In contrast, the total dose of calcium carbonate was lower in CAPD than in HD patients (p < 0.01). Results of BMD were expressed as Z scores (the number of standard deviations from the appropriate mean of BMD of 623 healthy subjects adjusted for age and sex). At the lumbar spine, no significant difference with respect to BMD was observed between the three groups. At the femoral neck and tibial epiphysis, HD patients had lower BMD (p < 0.001) than normal controls, whereas no difference was observed between HD and CAPD patients. At tibial diaphysis, patients on HD had lower BMD (p < 0.001) than patients on CAPD and than normal controls, with the values being similar in patients on CAPD and in normal controls. The results remained identical after exact matching of HD (n = 25) and CAPD (n = 25) patients for dialysis duration (1.9 ± 03 and 1.7 ± 03 years, respectively). Multiple regression analysis revealed significant negative correlations between Z scores at the lumbar spine (p < 0.05), femoral neck (p < 0.02), tibial diaphysis (p < 0.005), and tibial epiphysis (p < 0,05) on the one hand and plasma alkaline phosphatase activity on the other. The Z score at tibial diaphysis was also correlated with residual renal function at the initiation of dialysis (p < 0.05). In conclusion, this study provides evidence for the preservation of cortical bone with CAPD as opposed to HD. The higher residual renal function observed in the former treatment modality might account, at least in part, for this finding.
Juvenile paget disease: Life-long features of a mildly affected young woman
Authors: Golob Deborah S. , McAlister William H. , Mills Barbara G. , Fedde Kenton N. , Reinus William R. , Teitelbaum Steven L. , Beeki Suganthi , Dr. Whyte Michael P.
Published: February 2010

Unusually mild Juvenile Paget Disease (JPD) was extensively investigated in a mentally retarded 21-year-old white woman. Progressive bowing deformitity of her lower limbs began at age 1 1/2 years. Nontraumatic fractures of both femora and both tibias occurred between ages 9 and 14 years. During adulthood, cortical thickening, osteosclerosis, and bowing affected these bones. Serum alkaline phosphatase (ALP) activity was persistently elevated. We found her serum osteocalcin and urinary hydroxyproline and pyridinoline/deoxypyridinoline to also be increased. The iliac crest histology, at ages 14 and 21 years, showed wide cortices and enhanced skeletal remodeling yet the bone was exclusively lamellar. Features of classic Paget Bone Disease (PBD)-such as hypermultinucleated osteoclasts, peritrabecular fibrosis, and mosaic or woven bone-were absent. Electron microscopy revealed no cytoplasmic or nuclear inclusions. Her dermal fibroblasts in culture synthesized unremarkable levels of ALP with proper membrane topography and lipid anchoring; ALP released into the medium also appeared normal. Six months of synthetic human calcitonin therapy daily appeared to reduce her lower limb pain and warmth, but the radiographs, biochemical parameters of skeletal turnover, and bone scintigraphy were unaltered. Lamellar bone has been reported in JPD but accompanied by excessive amounts of woven bone. Our patient reveals that lamellar bone without features of PBD can characterize the skeletal histopathology of the especially rare case of mild JPD.
Comparison of three intravenous regimens of clodronate in paget disease of bone
Authors: Khan Sohail A. , Mccloskey Eugene V. , Eyres Keith S. , Nakatsuka Kiyoshi , Sirtori Paulo , Orgee Jane , Coombes Gillian , Prof. Kanis John A.
Published: February 2010

We compared the effects of three different regimens of intravenous clodronate in a retrospective study of 60 patients with Paget disease. A total dose of 1500 mg of clodronate was given as 300 mg for 5 consecutive days (n = 20), 1500 mg as a single infusion (n = 20), or 300 mg as a single infusion for 5 consecutive months (n = 20). The response to treatment and the duration of the effect were assessed from sequential changes in the activity of serum alkaline phosphatase. Treatment with dodronate induced a significant response in 85% of patients. The response rate was comparable in patients treated with 5 daily infusions (90%), with a single infusion (75%), and with 5 monthly infusions (90%). The median duration of response from the start of treatment was 11 months for those treated with five daily infusions and 12 months for the other two regimens. At one year, 22, 40, and 44% of patients had maintained their response in the daily, single, and monthly infusion regimen, respectively (NS). Six patients (32%) treated with 5 daily infusions achieved a remission (complete response) compared with 3 patients treated with a single infusion and 5 monthly infusions, respectively (16 and 15% respectively, NS). Patients attaining a complete response had a significantly longer duration of response compared with partial responders (median time 15.0 versus 11.5 months, respectively, p < 0.05). We conclude that intravenous clodronate (total dose 1500 mg) suppresses disease activity in the majority of patients with Paget disease of bone. The degree and duration of response were similar for the three regimens. Thus, in the treatment of Paget disease, the choice of regimen is a matter of convenience.
The effect of systemically administered PDGF-BB on the rodent skeleton
Authors: Mitlak Bruce H. , Finkelman Richard D. , Hill Esther L. , Li Jun , Martin Bruce , Smith Tait , D'Andrea Mark , Antoniades Harry N. , Lynch Samuel E.
Published: February 2010

Platelet-derived growth factor (PDGF), an osteoblast mitogen, has been demonstrated to accelerate fracture healing and periodontal bone repair when applied locally in vivo. To explore whether PDGF could stimulate bone formation in intact bone, we administered it systemically to rats rendered acutely estrogen-deficient. Because PDGF may stimulate bone resorption in vitro, PDGF was administered with and without an antiresorptive agent (alendronate). All treatments were given by intravenous injection 3 times a week for 6 weeks. Spinal bone mineral density (BMD) decreased by 5% in the vehicle-treated ovariectomized (OVX) rats by the end of the study as determined by DXA. Treatment with PDGF prevented this bone loss and significantly (p < 0.05) increased the bone density in the spine (9%) and whole skeleton (5.8%). Combined treatment with PDGF and alendronate resulted in a greater increase at the spine (18%) and whole skeleton (12.8%) than either agent alone. Histomor-phometric analysis demonstrated that treatment with PDGF increased the osteoblast number and osteoblast perimeter without consistent changes in osteoclast estimates. Biomechanical testing demonstrated that PDGF administration increased the vertebral body compressive strength and femoral shaft torsional stiffness and resulted in a trend for enhanced femoral head shearing strength. Coadministration of alendronate further increased these indices of bone strength. PDGF administration also caused premature closure of the growth plate, decreased body fat, and resulted in extraskeletal collagen deposition. We therefore demonstrate, for the first time, that systemic administration of PDGF can increase bone density and strength throughout the skeleton.
A multicenter comparison of dual-energy X-ray absorptiometers: In vivo and in vitro measurements of bone mineral content and density
Authors: Economos Christina D. , Nelson Miriam E. , Fiatarone Maria A. , Dallal Gerard E. , Heymsfield Steven B. , Wang Jack , Russell-Aulet Mary , Yasumura Seiichi , Ma Ruimei , Vaswani Ashok N. , Pierson Richard N.
Published: February 2010

As part of a multicenter research project, we compared dual-energy X-ray absorptiometry (I)XA) instruments at three research centers (sites 1, 2, and 3) to determine both intra- and intersite variability of bone mineral content (BMC), bone mineral density (BMD), and bone area (BA). Scans of the total body and lumbar spine were performed in duplicate on five humans (in vivo), and scans of the total body were performed on two whole body phantoms with artificial skeletons and thickness overlays (in vitro) at all sites over 15 days. The average intrasite variability in two consecutive total body BMD measurements, expressed as a percent difference, was significantly higher in vitro, 1.74 + 1.97%, than in vivo, 0.71 + 0.38% (p < 0.05). Average intrasite variability of the in vivo lumbar spine BMD was 1.08 + 1.12%. The intersite coefficients of variation for all BMD, BMC, and BA measurements were <2.0%. The total body BMD from site 2 was systematically lower than at sites 1 and 3 both in vivo and in vitro (p < 0.05) with no differences in BMC and BA. Although significant, the total body BMD differences between sites were small (< 1.2%) in vivo compared with in vitro (<2.6%) and are encouraging for the comparison and pooling of human data from multicenter trials, provided that appropriate standardized cross-calibration and analysis procedures are applied.
Increased bone formation and osteoblastic cell phenotype in premature cranial suture ossification (craniosynostosis)
Authors: de Pollack Cinderella , Renier Dominique , Hott Monique , Dr. Marie Pierre J.
Published: February 2010

Craniosynostosis is a heterogeneous disorder characterized by premature fusion of the skull bone sutures. To evaluate the pathogenesis of premature cranial suture ossification in craniosynostosis, we have evaluated the histologic indices of bone formation and the characteristics of osteoblastic cells derived from normal and affected cranial sutures in 47 infants and children, aged 3–18 months, with nonsyndromic craniosynostosis. The histomorphometric analysis of normal and fused sutures showed an age-related decline in the extent of endosteal bone surface covered with osteoid and osteoblasts during postnatal suture ossification. Bone formation was 20–50% higher at 3–6 months of age in fused sutures compared with normal sutures in the same patients. Cells derived from normal and fused sutures displayed characteristics of the osteoblast phenotype in culture. Analysis of [3H]thymidine incorporation into DNA from 1–14 days of culture showed an age-related decrease in osteoblastic cell growth in both normal and affected sutures. The proliferation of osteoblastic cells isolated from fused sutures was similar at all ages to that of cells isolated from normal sutures in the same patients. In contrast, alkaline phosphatase activity and osteocalcin production by osteoblastic cells cultured in basal conditions and after stimulation with 1,25-dihydroxyvitamin D (1,25[OH]2D3), were 53–74% higher in fused sutures compared with cells isolated from normal sutures in the same patients. The results indicate that bone formation activity at the suture site is locally increased in craniosynostosis, and this disorder is associated with increased in vitro parameters of osteoblastic cell differentiation, suggesting that an increased maturation of osteoblastic cells at the site of the suture leads to the premature ossification in nonsyndromic craniosynostosis.
Sequential effects of chronic human PTH (1-84) treatment of estrogen-deficiency osteopenia in the rat
Authors: Mitlak Bruce H. , Burdette-Miller Pamela , Schoenfeld David , Neer Robert M.
Published: February 2010

Although daily injections of parathyroid hormone (PTH) can rapidly reverse estrogen-deficiency bone loss in rats, PTH treatment of osteoporotic humans has to date produced more modest increases in bone mass. To explore the reasons for this important difference, we evaluated the dose- and time-dependence of human PTH 1–84 treatment effects on bone mass and biochemical markers of bone metabolism in rats with estrogen-deficiency bone loss. The highest doses of PTH increased spinal, femoral, and total skeletal mass to supra-normal levels and stimulated cortical endosteal bone formation. Spine and whole skeleton mass and density increased rapidly at first, but then increased more slowly; the rate of change decreased significantly (p < 0.01) during continued treatment with the highest doses of PTH. The effects of PTH treatment on biochemical markers also were both dose-dependent and time-dependent. Serum osteocalcin, a marker of osteoblast function, increased with the highest doses of PTH (p < 0.001), but reached an early plateau and later returned toward baseline. Urinary excretion of pyridinolines, a marker of osteoclast function, increased in a time-dependent fashion throughout treatment (p < 0.001). Serum 1,25(OH)2 vitamin D levels increased in a dose-related fashion, but then decreased toward control levels despite continued treatment. We demonstrate that both osteoblast and osteoclast function are increased during daily PTH therapy in the rat. The pattern of response depends on both the dose of PTH and the duration of therapy. These dose- and time-related effects should be taken into account when designing experimental PTH treatments for osteoporosis, and they deserve intensive study.
Increase of vascular endothelial growth factor mRNA expression by 1,25-dihydroxyvitamin D3 in human osteoblast-like cells
Authors: Wang Da Shen , Yamazaki Kazuko , Nohtomi Kyoko , Shizume Kazuo , Ohsumi Kazuoki , Shibuya Masabumi , Demura Hiroshi , Dr. Sato Kanji
Published: February 2010

Vascular endothelial growth factor (VEGF), a secreted endothelial cell-specific mitogen, is produced in endocrine organs and regulated by trophic hormones. Because angiogenesis and osteogenesis are closely regulated, we studied whether human osteoblast-like cells produce VEGF, and if so, what factors regulate VEGF mRNA expression. Human osteoblast-like cells (HObLC) derived from trabecular bone explants were cultured in α-MEM supplemented with 10% fetal calf serum. Northern blot analysis revealed that HObLC expressed VEGF mRNA, as did several human osteosarcoma cells. 1,25-(OH)2D3 increased the steady-state levels of VEGF mRNA in a time- and concentration-dependent manner in HObLC and one of the osteosarcoma cell lines, SaOS-2, accompanied by an increase in the concentration of immunoreactive VEGF in the conditioned medium. PTH and IGF-I also increased the level of VEGF mRNA in HObLC and SaOS-2 cells. Furthermore, 12-O-tetradecanoylphorbol ester stimulated VEGF mRNA in a time-and concentration-dependent manner. The VEGF mRNA expression induced by 1,25-(OH)2D3 was completely inhibited by H-7, but only partially by staurosporine. We have demonstrated that PTH, IGF-I, and most potently 1,25-(OH)2D3 stimulate the mRNA expression and secretion of VEGF in human osteoblast-like cells, suggesting that one of the anabolic effects of 1,25-(OH)2D3 on skeletal tissue may be mediated by VEGF produced by osteoblasts.
Mechanical loading and sex hormone interactions in organ cultures of rat ulna
Authors: Cheng Ming Zhao , Zaman Gul , Rawlinson Simon C. F. , Suswillo Rosemary F. L. , Lanyon Professor Lance E.
Published: February 2010

The separate and combined effects of loading and 17β-estradiol (E2) or 5α-dihydrotestosterone (DHT) on [3H]thymidine and [3H]proline incorporation were investigated in cultured ulna shafts from male and female rats. Ulnae were cultured and loaded to produce physiological strains in the presence or absence of 10−8 M E2 or DHT. Loading engendered similar increases in incorporation of [3H]thymidine and [3H]proline in male and female bones. E2 engendered greater increases in incorporation in females than in males, and DHT greater increases in males than in females. In males E2 with loading produced increases in both [3H]thymidine and [3H]proline incorporation, which approximated to the arithmetic addition of the increases due to E2 and loading separately. In females E2 with loading produced increases greater than those in males, and substantially greater than the addition of the effects of E2 and loading separately. Loading with DHT in males also showed additional [3H]thymidine and [3H]proline incorporation. In females there was additional incorporation of [3H]proline, but not [3H]thymidine. The location of incorporation of [3H]thymidine and [3H]proline was consistent with their level of incorporation reflecting periostea! osteogenesis, in which case the early osteogenic effects of sex hormones are gender-specific when acting alone and in combination with loading. In males the effects of estrogen and testosterone add to, but do not enhance, the osteogenic responses to loading. In females testosterone with loading produces an additional effect on [3H]proline incorporation but no greater effect than loading alone on that of [3H]thymidine. In contrast, estrogen and loading together produce a greater effect than the sum of the two influences separately. Because premenopausal bone mass will have been achieved under the influence of loading and estrogen acting together, these findings suggest that the bone loss which follows estrogen withdrawal may result, at least in part, from reduction in the effectiveness of the loading-related stimulus on bone cell activity. This stimulus is normally responsible for maintaining bone mass and architecture.
Constitutive expression of non-bone/liver/kidney alkaline phosphatase in human osteosarcoma cell lines
Authors: Dr. Ali Nadire N. , Rowe Janice , Teich Natalie M.
Published: February 2010

Alkaline phosphatase (ALP) plays an important role in bone mineralization; high levels in differentiated osteoblasts allows their identification easily in vitro. It is generally assumed that the activity of ALP in osteosarcomaderived cell lines commonly used in studies of bone cell biology is exclusively due to the bone/liver/kidney (BLK) isoenzyme. However, we noted that two human osteosarcoma cell lines, U-2 OS and U-393 OS, predominantly expressed a truncated 1.8 kb mRNA for BLK-ALP. This observation stimulated further investigation upon the ability of ALP to form functional protein. We found that, unlike the BLK-ALP of the Saos-2 osteosarcoma cell line, the activity of U-2 OS ALP was thermostable, unaffected by L-homoarginine and levamisole, but inhibited by L-phenylalanine; these properties are characteristic of the placental and/or placental-like (PL-/PL-like ALP) isoenzymes which are 98% homologous at the amino acid level. In the U-393 OS cell line, which expresses the normal-sized 2.5 kb mRNA in substantially higher levels than that produced by U-2 OS cells, the ALP activity had kinetic properties very similar to that produced by the Saos-2 line for all criteria tested. The HOS osteosarcoma cell line (also known as TE-85), which expresses the normal-sized 2.5 kb BLK-ALP mRNA only, exhibited ALP activity with kinetic properties of both the BLK and PL-/PL-like isoenzymes. The three test lines, U-2 OS, U-393 OS and HOS, produced PL-/PL-like ALP mRNA and protein constitutively, and levels of these increased in cells treated with 1 μM dexamethasone. However, dexamethasone treatment of cells did not alter the types of ALP isoenzyme expressed. Thus our results show that, like Saos-2 cells, U-393 OS cells produce active BLK-ALP exclusively, whereas U-2 OS cells produce PL-/PL-like ALP only, and the HOS cell line produces both. Our findings have important implications for phenotypic characterization of various human osteosarcoma cell lines, and suggest that the production of PL-/PL-like ALP may be a more common occurrence in osteosarcomas than was originally thought.
Establishment of bone morphogenetic protein 2 responsive chondrogenic cell line
Authors: Aikawa Tomonao , Shirasuna Kanemitsu , Iwamoto Masahiro , Watatani Kazuya , Nakamura Takashi , Okura Masaya , Yoshioka Hideo , Matsuya Tokuzo
Published: February 2010

A clonal cell line named RMD-1 was established from the skeletal muscle of a 20-day fetal rat. RMD-1 represents a morphologically homogeneous population of undifferentiated mesenchymal cells, expressing α-smooth muscle actin and type I collagen, but no cartilage-associated genes. When cultured in agarose gel containing 100 ng/ml of recombinant human bone morphogenetic protein 2 (rhBMP-2; BMP-2), RMD-1 cells formed colonies and showed chondrocyte-like features as assessed by their ultrastructure, metachromatic staining with toluidine blue, and the production of large hydrodynamic-size proteoglycans. RMD-1 cells also differentiated into chondrocytes when the cells were plated at high density (over 2.5 × 105 cells/cm2) on type I collagen and incubated in medium containing 0.5% fetal bovine serum and 100 ng/ml of BMP-2. This chondrogenic differentiation was evidenced by a distinct morphological change into spherical cells, an increase in the levels of sulfated glycosaminoglycans, a decrease in type I collagen mRNA and the expression of cartilage-associated genes, including type II collagen, type IX collagen, aggrecan and alkaline phosphatase. In the presence of ascorbic acid and 10% serum, RMD-1 cells increased in size and expressed type X collagen as well as high alkaline phosphatase activity, then induced matrix mineralization. Thus, RMD-1 is a unique cell line that can differentiate from undifferentiated mesenchymal cells into hypertrophic chondrocytes.
Doubling skeletal mass during adult life: The syndrome of diffuse osteosclerosis after intravenous drug abuse
Authors: Dr. Whyte Michael P. , Teitelbaum Steven L. , Reinus William R.
Published: February 2010

A former intravenous substance abuser, seropositive for hepatitis C virus infection, was referred for diffuse osteosclerosis. There was no history of fracture or skeletal deformity. Cortical and trabecular bone density was approximately twice the mean value for controls. Skeletal histology revealed dense lamellar bone. Recognized causes of acquired generalized osteosclerosis or hyperostosis were excluded. This patient verifies the syndrome of painful diffuse osteosclerosis after intravenous drug abuse and shows that skeletal mass can be markedly increased with histologically normal, structurally sound bone during adult life. Elucidation of the etiology and pathogenesis could offer an effective treatment for osteoporosis.
Phosphorylation of the cytoplasmic tail of the PTH/PTHrP receptor
Authors: Blind Eberhard , Bambino Tom , Huang Zhengmin , Bliziotes Michael , Nissenson Robert A.
Published: February 2010

Activation of the G protein–coupled receptor for parathyroid hormone (PTH)/PTH-related protein (PTHrP) produces homologous desensitization of receptor signaling. We have shown recently that the opossum PTH/PTHrP receptor stably expressed in human embryonic kidney (HEK) 293 cells is phosphorylated upon agonist binding and upon activation of serine/threonine protein kinases (PKA and PKC), an event which for some G protein-coupled receptors has been linked to desensitization. To locate the sites of phosphorylation, mutated forms of the opossum PTH/PTHrP receptor were stably expressed in HEK 293 cells, and ligand-stimulated receptor phosphorylation was evaluated. The five serine and threonine residues of the third cytoplasmic loop of the receptor were not required for receptor phosphorylation. Basal and ligand-induced phosphorylation were, however, completely abolished upon deletion of all but the 16 juxtamembrane residues of the cytoplasmic C-terminal tail of the receptor, even though this truncated receptor resembled the wild-type receptor in its level of expression based on Western blotting and radioligand binding. To identify further the phosphorylation sites, the 129 amino acid C-terminal tail of the rat PTH/PTHrP receptor was expressed in E. coli as a recombinant glutathione S-transferase fusion protein. Elimination of a single PKA consensus site in the tail (serine 491) resulted in ≥90% loss of PKA-mediated phosphorylation, identifying this as the preferential site for PKA, with two other sites (serine 473 and/or 475) being minor sites. Phosphorylation by PKC occurred largely in the proximal portion of the tail, whereas β-adrenergic receptor kinase 1 (βARK1) phosphorylated more distally in the tail. The ability of these kinases to phosphorylate the PTH/PTHrP receptor at distinct sites on the cytoplasmic tail may allow differential regulation of receptor signaling and trafficking.
Administration of the bisphosphonate ibandronate (BM 21.0955) by intravenous bolus injection
Authors: Pecherstorfer Martin , Ludwig Heinz , Schlosser Karl , Buck Signe , Huss Hermann-Josef , Body Jean-Jacques
Published: February 2010

Bisphosphonates (BPs) are used for the treatment of both benign and malignant diseases characterized by increased bone resorption. Because of their potential nephrotoxicity, currently available BPs have to be administered by slow intravenous infusion, with conventional doses requiring an infusion time of at least 2 h. In the present investigation, we evaluated the safety and efficacy of the new BP ibandronate as administered by intravenous bolus injection. On day 0, 15 normocalcemic breast cancer patients with bone metastases were treated with 3 mg of ibandronate injected intravenously over 60–120 s. Ibandronate treatment led to significant decreases in serum levels of calcium (p < 0.0001) and phosphate (p < 0.0001) and to subsequent increases in serum concentrations of parathyroid hormone (p < 0.0001) and calcitriol (p < 0.0001). Moreover, there was a significant reduction in the urinary excretion of calcium (p < 0.0001), pyridinoline (p < 0.001), and deoxypyridinoline (p < 0.0001). Three serious adverse events were observed: vomiting (WHO grade 3), pulmonary infection (WHO grade 2), and deterioration of a pre-existing impaired glucose tolerance (WHO grade 3). Only vomiting appeared to be related to administration of the drug. The most frequent nonserious adverse events were 10 cases of transient clinically asymptomatic hypocalcemia and 8 cases of asymptomatic hypophosphatemia. Serum levels of creatinine and urea nitrogen did not increase, nor did creatinine clearance deteriorate. When tested with the dipstick method, proteinuria was present in five (33%) patients prior to ibandronate treatment (median protein concentration, 30 mg/dl). Following the BP injection, seven (47%) patients showed slight (highest protein concentration, 30 mg/dl) transient proteinuria at at least one time point, of which six cases appeared in conjunction with leucocyturia and three with microhematuria. Side effects specific to aminosubstituted BPs (fever, reduction in white blood cell counts, and lymphocyte counts) were not seen in these 15 patients. In conclusion, a single intravenous injection of 3 mg of ibandronate significantly inhibited osteoclast activity as reflected by the decrease in serum calcium and in urinary parameters of bone resorption. Serum creatinine levels and estimates of creatinine clearance were not affected by therapy. However, before repeated bolus injections of ibandronate at this dosage can be recommended for further clinical trials, whether a relationship exists between the transient pathological urinary findings and injected ibandronate needs to be determined.
Dual X-ray absorptiometry quality control: Comparison of visual examination and process-control charts
Authors: Lu Ying , Mathur Ashwini K. , Blunt Barbara A. , Glüer Claus C. , Will A. Steve , Fuerst Thomas P. , Jergas Michael D. , Andriano Kim N. , Cummings Steven R. , Genant Harry K.
Published: February 2010

Dual X-ray absorptiometry (DXA) is widely used to monitor treatment efficacy in reducing the rate of bone mineral loss. In order to assure the validity of these measurements, instrument quality control of the DXA scanners becomes very important. This paper compares five quality control procedures (visual inspection, Shewhart chart with sensitizing rules, Shewhart chart with sensitizing rules and a filter for clinically insignificant mean changes, moving average chart and standard deviation, and cumulative sum chart [CUSUM]) in their ability to identify scanner malfunction by means of (1) an analysis of five longitudinal phantom data sets that had been collected during a clinical trial and (2) an analysis of simulated data sets. The visual inspection method is relatively subjective and depends on the operator's experience and attention. The regular Shewhart chart with sensitizing rules has a high false alarm rate. The Shewhart chart with sensitizing rules and an additional filter for clinically insignificant mean changes has the lowest false alarm rate but a relatively low sensitivity. The CUSUM method has good sensitivity and a low false alarm rate. In addition, this method provides an estimate of the date a change in the DXA scanner performance might have occurred. The method combining a moving average chart and a moving standard deviation chart came closest to the performance of the CUSUM method. Comparing the advantages and disadvantages of all methods, we propose the use of the CUSUM method as a quality control procedure for monitoring DXA scanner performance. For clinical trials use of the more intuitive Shewhart charts may be acceptable at the individual sites provided their scanner performance is followed up by CUSUM analysis at a central quality assurance center.
Dual-energy X-ray absorptiometry for histologic bone sections
Authors: Dr. Denissen Harry , De Blieck Jolanda , Verhey Hans , Klein Christel , Van Lingen Arthur
Published: February 2010

In fundamental osteoporosis research precise and accurate assessment of the mineral quantity in histological bone sections is of particular importance when studying the local effects of implants releasing bone modulating agents. A potentially useful technique to estimate the bone mineral density (BMD) is dual-energy X-ray absorptiometry (DXA). A highly collimated (0.13 mm) Hologic 2000 with a line spacing and point resolution of 0.13 mm was used. The mineral content was measured in regions of 3.1 mm2. A ceramic hydroxyapatite (CHA) phantom was developed as a reference standard. The phantom was made of a single-phase hydroxyapatite starting powder by compressing and sintering at 1000°C. The true density was 3.14 + 0.001 g/cm3. The calcium/phosphorus ratio was close to the theoretical one of 1.67. The mean precision error expressed as the coefficient of variation (CV) of the mineral density (MD) measurements of the phantoms with thicknesses of 1, 2, and 3 mm was 0.2%. Embedded undecalcified alveolar bone sections of dogs (0.0015-1 mm in thickness) were scanned simultaneously with a phantom 1 mm in thickness. The precision error (CV) of the BMD measurements calculated by DXA for sections ≥0.1 mm and with a BMD ≥0.14 g/cm2 was 0.81%. There was a linear relationship between the BMD calculated by DXA and the estimated BMD in the histological bone sections by means of the true density of the phantom. It is concluded that DXA using a standard CHA phantom is a precise and accurate method to measure MD changes as small as 1% in histological bone areas of 3.1 mm2 provided that the loss or gain in BMD is ≥0.14 g/cm2.
Bone sialoprotein expression in primary human breast cancer is associated with bone metastases development
Authors: Bellahcène A. , Kroll M. , Liebens F. , Castronovo V.
Published: February 2010

Breast cancer metastasizes to bone more frequently than to any other organ, and over 80% of advanced breast cancer patients develop bone metastases. Our recent demonstration that human breast cancer cells express bone sialoprotein (BSP), a bone matrix protein, provides a possible clue for the selective affinity of breast cancer cells for bone. We tested the hypothesis that detection of BSP in primary human breast cancer could be a potential indicator of the ability of breast cancer cells to metastasize to bone. BSP expression was evaluated in the primary breast cancers of 39 patients using immunoperoxidase and two specific anti-BSP antibodies. None of these patients presented clinically or scintigraphically detectable bone metastases at the time of surgery. In the course of their disease, 22 patients developed clinically diagnosed bone metastases. Expression of BSP in breast cancer cells from patients who developed bone metastases was significantly higher (p = 0.008, according to the Mann-Whitney test) than in patients with no bone involvement. No association was found between BSP expression in the primary breast lesions and axillary lymph node metastases. BSP expression was significantly increased in infiltrating ductal carcinoma compared with infiltrating lobular carcinoma (p = 0.0023). No correlation was found between immunoreactivity to BSP antibodies and estrogen receptor (ER) status, progesterone receptor (PR) status, or age. Our data suggest that BSP could help to identify which women will develop bone metastases and provide new bases for the understanding of the molecular mechanism(s) responsible for breast cancer cells osteotropism.
Osteoclastic superoxide production and bone resorption: Stimulation and inhibition by modulators of NADPH oxidase
Authors: Darden Alix G. , Ries William L. , Wolf William C. , Rodriguiz Ramona Marie , Key L. Lyndon
Published: February 2010

Production of superoxide radicals by osteoclasts is necessary for normal bone degradation. White blood cell superoxide, needed for bacterial killing, is produced by activated NADPH oxidase. Since osteoclasts and white blood cells share a common hematopoietic origin, we initiated experiments to test the hypothesis that superoxide radicals at the osteoclast-bone interface are produced by NADPH oxidase. Diphenyl iodonium (IDP), an inhibitor of NADPH oxidase, blocked superoxide generation and decreased osteoclastic bone resorption in cultures of calvarial explants from normal mice. Interferon (IFN) gamma, a stimulant of NADPH oxidase activity, increased superoxide production and bone resorption in cultures of calvarial explants from osteopetrotic (microphthalmic) mice. IDP blocked the stimulatory effects of IFN in this bone resorption model. These data suggest that osteoclastic superoxide is produced by NADPH oxidase.
Unilateral hip replacement causes bilateral changes in tibial bone mineral content in a canine model
Authors: Smith Alberta M. , Turner Thomas M. , Sumner Dale R.
Published: February 2010

The presence of asymmetry in tibial bone mineral content (BMC) of the operated and control limbs at the end of the experimental period following unilateral hip replacement surgery is used as a marker of limb function. The goal of the present study was to determine the contribution of ipsilateral and contralateral bone gain and loss to control-treated side differences in BMC of the tibia in dogs following unilateral hip replacement surgery. Seven animals were followed longitudinally with single beam photon absorptiometry for 6 months after unilateral hip hemiarthroplasty. Bone loss, compared with preoperative baseline values, was observed in both limbs, with recovery in bone mass beginning 1 month after surgery in the contralateral tibia and 3 months after surgery in the ipsilateral tibia. Thus, the asymmetry in tibial BMC frequently seen after unilateral experimental hip replacement in the canine appears to be caused by differential timing of recovery of bone mass following a transient loss in both limbs. The mechanism defined in this study is in contrast to an alternative mechanism involving bone loss in the treated limb coupled with bone gain in the control limb.
Translational regulation of parathyroid hormone gene expression and RNA: Protein interactions
Authors: Vadher Shilpa , Hawa Nigar S. , O'Riordan Jeffrey L. H. , Dr. Farrow Sheelagh M.
Published: February 2010

The aim of this study was to investigate the mechanism by which translation of parathyroid hormone (PTH) mRNA is regulated with regard to the subcellular distribution of PTH mRNA and RNA:protein interactions. Sucrose density ultracentrifugation of RNA from bovine parathyroid cells indicated that there was no evidence for a pool of nonribosomal PTH mRNA, and the extracellular calcium concentration had no effect on polysome size. UV cross-linking studies revealed two proteins in parathyroid cell cytosol which bound specifically to the 5′-untranslated region (UTR) of PTH mRNA with molecular masses of 66 and 68 kD while proteins with apparent molecular masses of 48 and 70 kD bound to the 3′-UTR. In vitro translation assays indicated that parathyroid cell cytosol contains factors that inhibit translation of PTH mRNA. Fractionation of cytosol revealed that this effect was associated with proteins within the molecular mass range 30–90 kD. To determine which sequences in PTH mRNA mediate translational regulation, RNA was synthesized from luciferase gene constructs containing the 5′-and/or 3′-UTR of PTH mRNA, and translated in vitro. Addition of parathyroid cell cytosol reduced the translation of RNA containing the 5′- and 3′-UTR of PTH mRNA by 44 + 7% but had no effect on the translation of RNA containing only the luciferase coding region. Translation of RNA containing only the 5′-UTR of PTH mRNA was unchanged; however, cytosol reduced the translation of RNA containing the 3′-UTR by 31 + 9%. These data demonstrate a role for RNA:protein interactions in the regulation of PTH synthesis and that translational control is mediated primarily through interactions with the 3′-UTR of PTH mRNA.
Regulation of NFIL-6 and IL-6 expression by basic fibroblast growth factor in osteoblasts
Authors: Hurley Marja M. , Abreu Christine , Marcello Kristina , Kawaguchi Hiroshi , Lorenzo Joseph , Kalinowski Judith , Ray Anuradha , Gronowicz Gloria
Published: February 2010

We determined whether basic fibroblast growth factor (bFGF) regulated the expression of IL-6 and NFIL-6 in osteoblasts. In mouse osteoblastic MC3T3-E1 cells, bFGF (10−8 M) increased NFIL-6 mRNA 2-fold at 30 minutes and 3-fold at 2 h. IL-6 mRNA was increased by bFGF 10−8 M after 1 h. IL-6 protein was detectable in control cultures but was significantly increased by bFGF (10−8 M) at 4 h. Immunofluorescence analysis of MC3T3-E1 cells showed primarily cytoplasmic and perinuclear NFIL-6 staining in control cultures while bFGF-treated cells showed increased NFIL-6 staining at 2 and 4 h. Western blotting revealed that bFGF increased NFIL-6 protein at 2 h. In calvarial mouse osteoblasts, bFGF 10−8 M induced IL-6 mRNA as early as 1 h and significantly increased IL-6 protein levels as early as 2 h. In conclusion, bFGF stimulates IL-6 and NFIL-6 mRNA in osteoblasts. The increase in NFIL-6 mRNA was associated with increased NFIL-6 protein. The increase in IL-6 mRNA was also associated with increased IL-6 protein. We propose that activations of NFIL-6 and IL-6 may be important mediators of the effects of bFGF on bone cells.