bluuebunny/crossref_metadata_2025 huggingface dataset:
Recombinant human transforming growth factor β1 modulates bone remodeling in a mineralizing bone organ culture
Authors: Chen Theresa L. , Bates Rebecca L.
Published: February 2010

Tgf-β1 Increases Cell Proliferation And Collagen Synthesis In Osteoblast-Like Cells And Bone Organ Cultures. However, The Effects Of Tgf-β1 On Bone Resorption Remain Contradictory. Therefore, The Exact Role That This Growth Factor Plays In The Process Of Bone Remodeling Is Still Not Clear. We Studied The Effects Of Recombinant Human Tgf-β1 (Rhtgf-β1) On Bone Formation And Resorption In A Mineralizing Bone Organ Culture System. Parietal Bones From 20-Day-Old Fetal Rat Calvariae Were Cultured Up To 7 Days In Serum-Free BgjB Medium. They Responded To A 1 Day Pulse Or Continuous Treatment Of Rhtgf-β1 With Dose-Dependent Increases In Dry Weight, [3H]thymidine ([3H]TdR) incorporation, and collagen synthesis. In contrast, rhTGF-β, reduced the calcium content of the bones. This is not due to increased bone resorption but rather to failure of calcium deposition. The following responses occurred at 1 nM rhTGF-β1. Dry weight was increased 25–50% after 6 days in culture. DNA synthesis was increased to a maximum at day 1, reaching twofold of the control level. Adding hydroxyurea at day 0 reduced [3H]TdR incorporation in rhTGF-β, treated bones to 20% of the control and indomethacin abrogated the increase in [3H]TdR stimulated by rhTGF-β1 to the control level. Both treatments completely blocked the increase in dry weight induced by rhTGF-β1 at day 6. rhTGF-β1 stimulated collagen synthesis to reach its maximum at day 2, with a twofold increase in [3H]proline incorporation. Basal alkaline phosphatase activity fell continuously in culture, reaching 35% of day 0 level at day 6. Enzyme activity was not altered by rhTGF-β1. Morphologic observations by light and electron microscopy confirmed these findings. In summary, rhTGF-β1 altered bone remodeling by increasing organic components and decreasing calcification in a mineralizing bone organ culture system.
Bone formation periods studied with triple tetracycline labels in women with postmenopausal osteoporosis
Authors: Ott Susan M.
Published: February 2010

In Adult Cancellous Bone, Cyclic Remodeling Occurs In Discrete Locations (Bmus, Or Basic Multicellular Units). Some Aspects Of These Remodeling Cycles Are Still Not Clear. Only About Half The Osteoid Surface Normally Has Tetracycline Labels; The Remainder Is Thought To Be Immature Or In A Resting Phase. This Study Was Designed To Test The Assumption That Pauses Occur During The Bone Formation Periods. Iliac Crest Bone Biopsies Were Performed In 84 Women With Postmenopausal Osteoporosis. The Subjects Were Given Three Tetracycline Labels; The Middle Label Had A Distinctive Color. The Osteoid Surface Was 10 s± 6% Of The Bone Surface; The Range Was 0.6–24.4%. The Mineralizing Surface Was 42 ± 26% Of The Osteoid Surface. Immature Osteoid Was 7.6 ± 5.3% Of The Osteoid Surface. There Were No Bmus With The First And Third Tetracycline Label But Without The Middle Demeclocycline Label. The Expected Length Of Double 1–3 Label, Based On An Assumption That Unlabeled Mature Osteoid Was Temporarily In A Resting Phase, Was Significantly Higher Than The Observed Length (68 ± 77 μm/Mm2 tissue area versus 0, p < 0.0001). The mean active formation period was 77 ± 16 days calculated by standard methods but was only 55 ± 26 days when calculated by label escape (p < 0.001). The correlation coefficient between these two methods was only modest (r = 0.27, p < 0.01). The failure to find any double 1–3 labels despite abundant triple labels provides strong evidence against the presence of pauses in bone mineralization between 3 and 34 days. Adjusting the formation period or the mineral apposition rate by the mineralizing surface/osteoid surface ratio may not be valid in women with osteoporosis.
Bone mineral density and parameters of bone metabolism in patients with acromegaly
Authors: Kotzmann Harald , Bernecker Peter , Hübsch Peter , Pietschmann Peter , Woloszczuk Wolfgang , Svoboda Thomas , Geyer Georg , Luger Anton
Published: February 2010

To Assess The Effect Of Chronically Elevated Plasma Growth Hormone (Gh) Levels On Bone Metabolism And Bone Mineral Density (Bmd), 16 Patients (10 Females And 6 Males) With A Mean Age Of 49.1 ± 13.2 Years (Range 33–68) With Active Acromegaly Were Studied And Compared To A Control Group Of 16 Sex- And Age-Matched Subjects. Bmd Of The Lumbar Spine And Two Different Sites Of The Proximal Femur Were Measured By Dual-Energy X-Ray Absorptiometry (Norland Xr-26). In The Acromegalic Patients The Mean Plasma Gh Concentration Was 30.1 ± 11.1 μg/Liter, And The Mean Plasma Somatomedin C (Smc) Concentration Was 6.5 ± 1.5 U/Liter. Mean Serum Osteocalcin (Oc) Levels (14.3 ± 1.1 Versus 7.2 ± 0.4 Ng/Ml, P < 0.001) As Well As The Urinary Hydroxyproline Excretion (Ohp; 8.8 ± 1.4 Versus 2.7 ± 0.3 Mg, P < 0.0001) Were Significantly Higher In The Acromegalic Patients Than In The Control Subjects. In The Acromegalic Patients Bmd Was Significantly Elevated In The Two Examined Regions Of The Proximal Femur, That Is, The Femoral Neck (1.06 ± 0.05 Versus 0.86 ± 0.03 G/Cm2, p < 0.05) and Ward's triangle (0.92 ± 0.06 versus 0.76 ± 0.03 g/cm2, p < 0.02), whereas the BMD of the lumbar spine was not significantly different from that of control subjects. Among the patients with acromegaly a significant positive correlation between serum OC concentrations, on the one hand, and urinary OHP excretion (r = 0.7, p < 0.004) as well as BMD in the proximal femur (r = 0.64, p < 0.007), on the other hand, could be observed. We therefore conclude that in acromegaly bone turnover (formation and resorption) is increased and, moreover, that BMD is significantly enhanced in the proximal femur but not in the lumbar spine.
Regulation of expression of the chondrocytic phenotype in a skeletal cell line (CFK2) in vitro
Authors: Bernier Suzanne M. , Dr. Goltzman David
Published: February 2010

We Have Examined In Vitro The Spontaneous And Regulated Expression Of Phenotypic Characteristics Associated With Differentiated Chondrocytes In An Established Skeletal Cell Line (Cfk2) Derived From Fetal Rat Calvariae. Extended Culture Of Cfk2 Cells Resulted In The Appearance Of Glycosaminoglycans And Type Ii Collagen In The Cell Layer In Association With The Formation Of Focal Nodes Of Cells. In Addition, Induction Of Mrna-En-Coding Link Protein, Cartilage-Specific Proteoglycan Core Protein, And Thrombospondin Was Observed In The Differentiated Population (Dcfk2 Cells). The Expression Of These Mrnas Was Present For At Least Two Passages After Subculturing The Dcfk2 Cells. The Dcfk2 Cells Also Demonstrated Enhanced Parathyroid Hormone (Pth)-Stimulated Adenylate Cyclase Activity. Proliferation Of Cfk2 Cells Was Stimulated By The Peptide Regulatory Factors Egf And Pth And Inhibited By The Steroidal Agents Dexamethasone And Retinoic Acid. Egf And Retinoic Acid Inhibited The Formation Of Cell Foci And Glycosaminoglycan Deposition And The Expression Of Mrna-Encoding Link Protein. In Contrast, Pth And Dexamethasone Enhanced The Formation Of Focal Cellular Nodes And Augmented Matrix Deposition And Link Protein Mrna Expression. These Studies Therefore Show That The Cfk2 Cell Line Can Serve As A Nontransformed Model Of Rat Chondrocytic Cells In Which Both Induction And Regulation Of The Expression Of Cartilaginous Matrix Components Can Be Observed. This Line Thereby Provides A Unique Renewable Source Of Chondrocytic Precursor Cells And An Excellent In Vitro Model For Evaluating Temporal And Environmental Control Of Chondrocyte Differentiation And Cartilage Matrix Production.
Integrin expression in human bone
Authors: Hughes D.E. , Salter D.M. , Dedhar S. , Simpson R.
Published: February 2010

Integrins are a family of heterodimeric transmembrane glycoproteins that are known to mediate cell-cell and cell-matrix interactions. Members of the VLA (very late activation) family, which consists of β1 integrin in association with the VLA α chains (α1–6), mediate adhesion of a wide range of cells to matrix proteins, such as fibronectin, collagen, and laminin, and may therefore be important for cell-matrix interactions in bone. Integrin expression in human bone was studied immunohistochemically using cryostat sections of fracture callus, tumor-associated reactive bone, and neonatal costochondral junctions, with a panel of well-characterized antibodies against β1–4 integrins, α1–6 and αv integrins, and the αvβ3 dimer (the classic vitronectin receptor). All cell types present in bone expressed β1 and α5 integrins; a subpopulation of osteoblastic cells expressed α4. The αv was uniformly expressed by osteoblasts but was heterogeneously expressed by osteocytes. Osteoclasts also expressed α2, αv, and αvβ3. These results demonstrate differential expression of a restricted range of integrins in bone. This supports the possibility that integrins may mediate the differing interactions of cells of the osteoblast and osteoclast lineages with the matrix of bone.
Bone mineral density and mass in a cross-sectional study of white and asian women
Authors: Russell-Aulet Mary , Wang Jack , Thornton John C. , Colt Edward W.D. , Pierson Richard N.
Published: February 2010

The literature suggests that Asians have lower bone mineral density and mass than whites. It has been proposed that these differences may be due to differences in height, weight, and factors other than ethnicity, but no study has made the appropriate direct comparisons. We compared total-body bone mineral density and mass between Asian and white women while controlling for factors known to be associated with bone mineral density and mass. Measurements were made in 129 Asian (primarily of Chinese ancestry) and 274 white women. A subgroup was formed of women who did not have a history of alcoholism, premenopausal amenorrhea, kidney disease, estrogen use, birth control pill use, thyroid disease, steroid use, hysterectomy, or smoking. In both the main group and the subgroups, bone mineral mass was significantly lower in Asian than in white women, but after analysis of covariance with body weight, height, and age (or years since menopause) as covariates, the differences between ethnic groups disappeared, except in the large group of premenopausal women, in whom average bone mineral density in Asians actually exceeded (p < 0.04) that in whites. The data set was also searched for Asian-white pairs who matched on 17 characteristics related to bone mineral density and mass. In the resulting 16 matched pairs, bone mineral density and mass were not different between ethnic groups. Although Asian women have lower bone mineral mass than white women, when weight, height, and other factors are controlled, bone mineral density and mass do not differ between Asian and white women.
Prostaglandin E2 administered by subcutaneous pellets causes local inflammation and systemic bone loss: A model for inflammation-induced bone disease
Authors: Desimone Daniel P. , Greene Virginia S. , Hannon Kathleen S. , Turner Russell T. , Bell Norman H.
Published: February 2010

The effects of prostaglandin E2 (PGE2) given in controlled-release pellets or by daily sc injection for 21 days on mineral homeostasis and bone histomorphometry were compared in 7-week-old female rats. Sham operation and ovariectomy were performed at the beginning of the studies. In experiment 1, 7.5 mg PGE2 or drug-free, controlled-release pellets were implanted sc at the back of the neck on day 7. In experiment 2, 3 mg/kg body weight of PGE2 or vehicle was injected sc daily beginning on day 7. The animals were sacrificed on day 28 of the two experiments, and the tibiae were removed for histomorphometric analysis of the diaphysis and metaphysis. When administered by pellets in experiment 1, PGE2 lowered serum 1,25-dihydroxyvitamin D and did not influence weight gain, serum calcium, phosphorus, or magnesium, cross-sectional or medullary areas, periosteal bone formation and apposition rates, endosteal bone formation and apposition rates, or endosteal tetracycline-labeled perimeter. PGE2 lowered cancellous bone area and cancellous bone perimeter in both the sham-operated and ovariectomized rats. In contrast, when administered by sc injection in experiment 2, PGE2 reduced weight gain, increased serum magnesium, increased cortical area, and reduced medullary area without changing cross-sectional area, increased periosteal bone formation and apposition rates and endosteal bone and apposition rates, did not alter endosteal tetracycline-labeled perimeter, and increased cancellous bone area and cancellous bone perimeter in both sham-operated and ovariectomized animals. PGE2 produced local inflammation when given by pellets, and the serum concentration of 13,14-dihydro-15-ketoprostaglandin E2, the major metabolite of PGE2, increased when PGE2 was given by sc injection but not when administered by pellets. Thus, PGE2 given sc by controlled-release pellets (1) produces local inflammation and systemic bone loss without increasing PGE2 systemically and (2) provides a model for inflammation-induced loss of cancellous bone. The results also indicate that the pellet is not a valid means for the delivery of PGE2 to the general circulation.
Bone morphometric changes in adjuvant-induced polyarthritic osteopenia in rats: Evidence for an early bone formation defect
Authors: Bonnet Jacqueline , Zerath E. , Picaud N. , Lesur C. , Mattio A. , Tordjman C. , Hott M. , Marie P.J.
Published: February 2010

Adjuvant polyarthritis (AP) in rats is known to result in extensive bone loss. This study investigates the mechanisms responsible for the early trabecular osteopenia evaluated at a single point in time — 2 weeks after adjuvant injection—in the hindpaw of female Lewis rats using biochemical and histomorphometric methods. At this early point in time, the inflammation was generalized (inflammatory score, 20; albumin/globulin, —80% versus control). Histomorphometric analysis of the noninjected femur showed that the trabecular bone volume was significantly decreased (-28% versus control) in both proximal and distal parts, and the femur growth rate was unaffected. The trabecular osteopenia was associated with a 90% decrease in osteoid surface and a concomitant thinning (-19%) of the trabeculae. Both the double-fluorescence-labeled surface and the osteoblast surface were also markedly decreased (-75%). In addition, the mineral apposition rate was reduced (-50%) and the bone formation rate was decreased by as much as 90%. The trabecular bone volume was decreased in relation with the extent of double-fluorescence labeling (r = 0.38, p = 0.03) and bone formation rate (r = 0.42, p = 0.01), suggesting that the generalized osteopenia resulted from the reduced bone formation. This was associated with a 26% reduction in plasma osteocalcin. Neither the osteoclast surface nor the number of osteoclasts was consistently affected. However, urinary hydroxyproline was increased by 100–200%, which likely reflected the cartilage and bone destruction at the site of injection. The present data show that the early extensive osteopenia observed 2 weeks after AP induction in rats results from defective bone formation with unchanged bone resorption. The role of cytokines in such an inhibitory effect on bone formation remains to be determined.
Cross-sectional and longitudinal study of age-related phalangeal bone loss in adult females
Authors: Trouerbach W.T. , Vecht-Hart C.M. , Collette H.J.A. , Slooter G.D. , Zwamborn A.W. , Schmitz P.I.M.
Published: February 2010

To establish a comprehensive model for peripheral phalangeal bone loss, bone mass was studied in 1984 and 1989 using quantitative microdensitometry (QMD) in a total of 330 healthy women (age range 43–78.7 years). Bone mass and changes in bone mass were analyzed in relation to age and menopausal status. Ideal and nonideal populations were distinguished to assess the effect of diseases and medication. Both groups showed a decrease in bone mass, which proved to be more dependent on menopausal status than on age. A substantial loss started in the ideal group in the early postmenopausal period and in the nonideal group in the premenopausal period. Because the nonideal group started to lose bone at an earlier stage, the lifetime risk for osteoporosis is higher than in the ideal group.
Circulating PTHrP concentrations in tumor-induced hypercalcemia: Influence on the response to bisphosphonate and changes after therapy
Authors: Body J.J. , Dumon J.C. , Thirion M. , Cleeren A.
Published: February 2010

We studied the influence of circulating parathyroid hormone-related protein (PTHrP) concentrations on the response of hypercalcemic cancer patients to bisphosphonate therapy. We also examined the changes in circulating PTHrP levels during the normalization of serum Ca to determine if part of the increase in PTHrP concentrations is not secondary to hypercalcemia itself, as suggested by some in vitro data. We sequentially measured in 45 hypercalcemic cancer patients treated by pamidronate the circulating concentrations of PTHrP (by an amino-terminal RIA; normal values <9 pmol/liter), Ca, Ca2+, Pi, intact PTH, and the fasting urinary excretion of Ca (Ca/Cr) and cyclic AMP (cAMP). Mean ± SEM baseline PTHrP levels were 9.5 ± 1.3, with a median (range) value of 6.0 (< 3.4–43) pmol/liter. PTHrP levels were elevated in 18 of 45 patients, more often in epidermoid than in glandular carcinomas (P < 0.05), and they were significantly (P < 0.05) correlated with the concentrations of Pi (r = -0.46), Ca/Cr (r = -0.31), and urinary cAMP (r = 0.47). Mean pretreatment Ca levels were not significantly different between patients with elevated and patients with normal PTHrP levels, 13.3 ± 0.4 versus 12.9 ± 0.4 mg/dl, but the concentrations became significantly different (P < 0.005) 4 days after therapy, 10.2 ± 0.3 versus 9.2 ± 0.1 mg/dl, respectively. The fall in fasting urinary Ca excretion was significantly (P < 0.05 from day 4 to day 14) lower in patients with elevated baseline PTHrP levels: for example, Δ (day 4 - day 0), 0.31 ± 0.11 in patients with elevated PTHrP levels versus 0.64 ± 0.08 mg Ca per mg Cr in patients with normal PTHrP levels. In agreement with a lesser effect on serum Ca, intact PTH levels did not increase significantly in patients with elevated PTHrP levels, in contrast with a clear-cut recovery of PTH secretion in the other group. Last, PTHrP levels did not change significantly after bisphosphonate therapy: for example, at the day of the nadir of Ca, the levels were 8.2 ± 1.2 (6.4, < 3.4–28) pmol/liter. In summary, our data suggest that circulating PTHrP concentrations do not change during correction of tumor-induced hypercalcemia but significantly influence the response to bisphosphonate therapy. Elevated PTHrP concentrations in hypercalcemic cancer patients thus constitute a primary phenomenon of pathogenic importance.
Development and characterization of a polyclonal antiserum-based radioimmunoassay for dog osteocalcin
Authors: Fanti Paolo , Colombo Giovanna , Yao Chinghua , Brown Stephen A. , Vernon Michael W. , Malluche Hartmut H.
Published: February 2010

Determination of the serum concentration of the protein osteocalcin (OC) is useful for the noninvasive evaluation of bone metabolism. Because the dog is an excellent experimental model for the study of bone, we produced and characterized a polyclonal antiserum specific for dog OC and used it to develop a radioimmunoassay (RIA) for the measurement of the concentration of this protein in dog serum. The antiserum expresses higher affinity for Ca2+-bound than for Ca2+-free OC (B50 at 10−5 versus 2 × 10−4 dilution). Also, in the presence of Ca2+ affinity is higher for the carboxylated than for the decarboxylated form of the protein, and under Ca2+-free conditions the affinity is equal for the two forms. The study of peptide fragments of OC demonstrates competitive binding of the peptide comprising amino acids 20–44 but not of other fragments; this suggests that the antigenic epitope of dog OC is located in the midmolecular region of the protein. The RIA displays excellent sensitivity for the measurement of OC in blood (detection limit 0.31 ng/ml), with intraassay and interassay variations of 4.6 and 6.8%, respectively. Analysis of gel chromatography fractions of normal dog serum shows that greater than 90% of the antigenic material coelutes with purified radiolabeled dog OC. Test of parallelism reveals lack of interference of serum constituents with the binding assay. The antiserum displays limited species specificity since it cross-reacts with human OC, but not with the protein from rodents. Consistent with previous observations in other in vivo models, the serum concentration of OC in experimental dogs is decreased significantly 7–10 days after thyroparathyroidectomy and it is unchanged 1 month following ovariohysterectomy.
Calcium retention and hormone levels in black and white women on high- and low-calcium diets
Authors: Dawson-Hughes Bess , Harris Susan , Kramich Carolyn , Dallal Gerard , Rasmussen Helen M.
Published: February 2010

Black-white differences in calcium metabolism are sought because they may reveal why blacks have higher bone mass than whites. Comparative studies of calcium absorption in blacks and whites are not available. In this study, we compare fractional calcium retention, an index of calcium absorption, and calcium regulating hormone levels in black and white women on a high-calcium diet and after adaptation to a low-calcium diet. A total of 30 healthy women (15 black and 15 white) had measurements of fractional 47Ca retention and calcium regulating hormone levels after 8 weeks on a 2000 mg calcium diet and, subsequently, after 1, 2, 4, and 8 weeks on a 300 mg calcium diet. By 2 weeks after the diet change, fractional 47Ca retention, parathyroid hormone (PTH), and 1,25-dihydroxyvitamin D [1,25-(OH)2D] had reached a plateau at higher levels, urine calcium at a low level, and serum calcium at the same level (repeated-measures ANOVA). Fractional 47Ca retention, serum calcium, and PTH were similar on both diets in blacks and whites. Blacks had higher levels of 1,25-(OH)2D on both diets (e.g., 125.1 ± 53.5 SD versus 73.4 ± 19.0 pmol/liter, P = 0.003 on low-calcium diet) and a greater increase in 1,25-(OH)2D after the diet change (33.9 ± 30.1 SD versus 11.8 ± 17.9 pmol/liter, P = 0.021). Serum phosphorus was lower in blacks throughout. For hormone levels and fractional calcium retention to stabilize after a diet change, 2 weeks was needed. Similar levels of fractional retention in black and white women despite higher levels of 1,25-(OH)2D in blacks suggest that blacks may have a gut resistance to the action of 1,25-(OH)2D.
Effects of transforming growth factor β1 and l-ascorbate on synthesis and distribution of proteoglycans in murine osteoblast-like cells
Authors: Takeuchi Yasuhiro , Matsumoto Toshio , Ogata Etsuro , Shishiba Yoshimasa
Published: February 2010

Proteoglycans synthesized by osteoblasts are incorporated into bone matrix and thought to play a role in bone metabolism. Transforming growth factor (TGF) β affects the synthesis of matrix proteins, including proteoglycans, in various stromal cells, and proteoglycans, especially decorin, are associated with matrix collagen. In the present study, the effects of TGF-β1 and L-ascorbate, a factor essential for collagen synthesis, on the synthesis and distribution of proteoglycans were examined using murine osteoblast-like MC3T3-E1 cells. TGF-β1 stimulated the synthesis of proteoglycans in MC3T3-E1 cells. Among various proteoglycans, the synthesis of decorin was preferentially enhanced by TGF-β1 and the effect was more pronounced on secreted decorin compared to that associated with the cell/matrix layer. TGF-β1 also stimulated the initiation and elongation of the dermatan sulfate glycosaminoglycan chain, resulting in a larger molecular size of decorin. TGF-β1 influenced the synthesis of a heparan sulfate proteoglycan only slightly. L-ascorbate had no effect on the synthesis of proteoglycans, but increased those associated with the cell/matrix layer. Furthermore, when L-ascorbate was added to the culture along with TGF-β1, the percentage of proteoglycans associated with the cell/matrix layer increased from 25.8 ± 1.0 to 41.0 ± 0.5%. These data demonstrate that TGF-β1 markedly stimulates the synthesis of proteoglycans, especially decorin, mainly as a secreting form, that the accumulation of decorin into matrix is enhanced by L-ascorbate, and that the effects of TGF-β1 and L-ascorbate are additive. Because L-ascorbate is required for the synthesis of collagen, the effect of L-ascorbate on the distribution of decorin appears to be due to its effect on collagen synthesis.
Effects of high extracellular calcium and strontium on inositol polyphosphates in bovine parathyroid cells
Authors: Shoback Dolores M. , Chen Tsui-Hua , Lattyak Bruce , King Kathleen , Johnson Randolph M.
Published: February 2010

The addition of Ca2+ or a variety of divalent cations increases intracellular Ca2+ in parathyroid cells and suppresses secretion. Since 1,4,5-inositol trisphosphate (IP3) and 1,3,4,5-inositol tetrakisphosphate (IP4) mediate Ca2+ mobilization in other systems, we examined high Ca2+- and Sr2+-induced accumulation of IP3 and IP4 isomers by anion-exchange HPLC and measured 1,4,5-IP3 mass in parathyroid cells. Raising extracellular [Ca2+] from 0.5 to 3.0 mM increased 3H-1,4,5-IP3 within 5 s, which was confirmed by mass measurements. 3H-1,3,4-IP3 rose gradually by 10 s and increased for 60 s after the addition of Ca2+. Although we detected no change in 3H-1,3,4,5-IP4, the increase in 3H-1,3,4-IP3 suggests that 3H-1,3,4,5-IP4 was being formed. The addition of 4 mM SrCl2 produced similar changes in 1,4,5-IP3, which were confirmed by mass assay. 3H-1,3,4,5-IP4 did not change. However, Sr2+ induced a gradual increase in 3H-1,3,4-IP3, which remained above control levels for 5 minutes. Isotopic labeling studies in this system may underestimate changes in 1,4,5-IP3 mass, but both mass and radioisotopic analyses indicate that high extracellular Ca2+ and Sr2+ stimulate substantial increases in 1,4,5-IP3 without significant accumulation of 1,3,4,5-IP4. These studies suggest a role for 1,4,5-IP3 in intracellular Ca2+ mobilization by divalent cations in parathyroid cells.
Linkage of extracellular and intracellular control of cytosolic Ca2+ in rat osteoclasts in the presence of thapsigargin
Authors: Zaidi Mone , Shankar Vijai S. , Bax Christopher M.R. , Bax Bridget E. , Bevis Peter J.R. , Pazianas Michael , Alam A.S.M. Towhidul , Moonga Baljit S. , Huang Christopher L.-H.
Published: February 2010

Cytosolic [Ca2+] was measured in single osteoclasts using fura-2 in experiments investigating the effects of Ca2+ “receptor” activation using thapsigargin as a means of depleting intracellular Ca2+ stores. Application of 4 μM thapsigargin to osteoclasts in Ca2+-free solutions resulted in an elevation of cytosolic [Ca2+]. Under similar conditions, activation of the osteoclast Ca2+ receptor by the substitute divalent cation agonist, Ni2+, resulted in a transient elevation of cytosolic [Ca2+]. In both instances, restoration of extracellular [Ca2+] to 1.25 mM resulted in an “overshoot” of cytosolic [Ca2+]. Prior depletion of intracellular Ca2+ stores by thapsigargin markedly reduced the magnitude of the cytosolic [Ca2+] response to a subsequent application of 5 mM Ni2+. The application of 2 μM thapsigargin to intercept the falling phase of the Ni2+-induced cytosolic Ca2+ signal resulted in a sustained elevation of cytosolic [Ca2+], which was terminated by a second application of the same Ni2+. Furthermore, the sustained elevation of cytosolic [Ca2+] induced by thapsigargin application alone was abolished by late application of Ni2+. We conclude that activation of the surface membrane Ca2+ receptor on the osteoclast results in the cytosolic release of Ca2+ from intracellular storage organelles; the refilling of such stores depends upon a thapsigargin-sensitive Ca2+-ATPase; store depletion induces capacitative Ca2+ influx; and the Ca2+ influx pathway is sensitive to blockade by Ni2+.
Electric fields modulate bone cell function in a density-dependent manner
Authors: Mcleod Kenneth J. , Donahue Henry J. , Levin Paul E. , Fontaine Marie-Anne , Rubin Clinton T.
Published: February 2010

The influence of an extremely low frequency (ELF) electric field stimulus (30 Hz at 6 μV/cm rms), known to promote bone formation in vivo, was evaluated for its ability to affect bone cell function in vitro. To accomplish this, we developed an apparatus for the exposure of monolayer cell systems to electric fields in a manner that provides relatively uniform electric field exposure of multiple cell samples as well as a rigorous sham exposure. We show that field exposure significantly limits the normal increase in osteoblastic cell number and enhances alkaline phosphatase activity compared to sham-exposed samples. Moreover, these alterations are shown to occur in a cell density-dependent manner. Samples plated at 6 × 103 cells/cm2 show no effect of field exposure. In samples plated at 30 × 103 cells/cm2, 72 h of field exposure resulted in 25% fewer cells in the exposed samples, and a doubling of alkaline phosphatase activity in those cells compared to sham exposure. Experiments using a 12 h exposure to preclude significant changes in cell number during the exposure show this density-dependent response to be biphasic. Sparse cultures (>50 × 103 cells/cm2) were not found to be affected by the field exposure, but increases in alkaline phosphatase activity occurred in cultures at densities of 50–200 × 103 and 200–350 × 103 cells/cm2 and no effect on alkaline phosphatase activity was seen in confluent cell cultures of greater than 350 × 103 cells/cm2. This work suggests that the demonstrated osteogenic effect of this specific electric field stimulus on bone tissue may be initiated by an alteration of the differentiated status of the osteoblasts in the tissue rather than by stimulating cell proliferation, as has been previously suggested. As importantly, a more thorough characterization of this density-dependent interaction with electric fields may help to identify the mechanism of transduction by which ELF electric fields interact with cells.
Tumor necrosis factor α decreases 1,25-dihydroxyvitamin D3 receptors in osteoblastic ROS 17/2.8 cells
Authors: Mayur Nitin , Lewis Sharma , Catherwood Bayard D. , Nanes Mark S.
Published: February 2010

Bone remodeling is a complex process regulated by systemic hormones, local cytokines, and growth factors. One cytokine, tumor necrosis factor α (TNF-α), is known to have potent inhibitory effects on osteoblast matrix protein production and to stimulate osteoclast recruitment. We have previously shown that TNF-α inhibits 1,25-(OH)2D3-stimulated synthesis of bone gla protein (BGP), an abundant and osteoblast-specific matrix constituent. We hypothesized that the mechanism of TNF-α action included inhibition of intracellular 1,25-(OH)2D3 receptor (VDR) number or function. To test this, the osteoblastic cell line ROS 17/2.8 was cultured in the presence or absence of TNF-α (100 ng/ml), and binding of [3H]1,25-(OH)2D3 to 0.3 M KCl extracts of cytosol was measured by equilibrium assay. Specific [3H]1,25-(OH)2D3 binding decreased 70%, 25 h after addition of TNF-α. The decrease in [3H]1,25-(OH)2D3 binding was seen by 18 h, was sustained throughout the 72 h culture period, and was greater in low-density cultures. Scatchard analysis confirmed that TNF-α (100 ng/ml for 24 h) caused a decrease in the number of binding sites without change in VDR affinity. Northern analysis with a VDR riboprobe revealed that the decrease in VDR occurred without a change in the 4.4 kb steady-state VDR mRNA [VDR/cyclophilin mRNA signal ratio: control, 2.25; TNF-α, 2.24 (24 h),2.17 (40 h), n = 2 flasks/time point]. These results suggest that TNF-α action on osteoblastic cells includes an inhibitory effect on VDR number at a point distal to the synthesis of VDR mRNA.
Distribution of noncollagenous proteins in the matrix of adult human bone: Evidence of anatomic and functional heterogeneity
Authors: Ingram Ronald T. , Clarke Bart L. , Fisher Larry W. , Fitzpatrick Lorraine A.
Published: February 2010

The microanatomic distribution of several noncollagenous proteins (NCPs) in bone matrix was examined by immunohistochemical analysis of glycol-methyl methacrylate-embedded normal adult human bone biopsies. Osteopontin and bone sialoprotein stained throughout the lamellae of both trabecular and cortical bone. Cement lines (cortical and trabecular) and the mineralized matrix immediately adjacent to each Haversian canal were intensely stained. Osteocalcin was detected in cement lines; however, lamellar staining varied depending on the location within the individual unit of bone. In cortical bone, the inner concentric lamellae of osteons were often unstained but the outer lamellae were heavily stained for osteocalcin. Osteonectin was not detected in cement lines and in most specimens revealed a pattern similar to that of osteocalcin with respect to the absence of immunostaining within the inner concentric lamellae. Decorin was prominent in the perilacunar matrix, the canaliculi of osteocytes, and the matrix immediately adjacent to quiescent Haversian canals. Biglycan appeared evenly distributed throughout cortical and trabecular bone matrix. These results suggest that the incorporation of NCPs into matrix may vary depending on the stage of formation of individual bone units. The specific distribution and spatial relationship of these NCPs may be related to the function of each protein during bone resorption and formation. The distinct patterns of NCP localization in bone support the hypothesis that in addition to their structural and mineral-inducing properties, these proteins may influence the events associated with bone remodeling, such as recruitment, attachment, differentiation, and activity of bone cells.
Post-translational control of collagen fibrillogenesis in mineralizing cultures of chick osteoblasts
Authors: Gerstenfeld Louis C. , Riva Angela , Hodgens Karen , Eyre David R. , Dr. Landis William J.
Published: February 2010

Cultured osteoblasts from chick embryo calvaria were used as a model system to investigate the post-translational extracellular mechanisms controlling the macroassembly of collagen fibrils. The results of these studies demonstrated that cultured osteoblasts secreted a collagenous extracellular matrix that assembled and mineralized in a defined temporal and spatial sequence. The assembly of collagen occurred in a polarized fashion, such that successive orthogonal arrays of fibrils formed between successive cell layers proceeding from the culture surface toward the media. Mineralization followed in the same manner, being observed first in the deepest and oldest fibril layers. Collagen fibrillogenesis, the kinetics of cross-link formation, and collagen stability in the extracellular matrix of the cultures were examined over a 30 day culture period. Between days 8 and 12 in culture, collagen fibril diameters increased from < 30 nm to an average of 30–45 nm. Thereafter, diameters ranged in size from 20 to 200 nm. Quantitation of the collagen cross-linking residues, hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP), showed that these mature cross-links increased from undetectable levels to concentrations found in normal chick bone. Analysis of the kinetics of their formation by pulse-chase labeling the cultures with [3H]lysine showed a doubling time of ˜ 5 days. The relationships between cross-link formation, fibrillogenesis, and collagen stability were examined in cultures treated with β-aminopropionitrile (β-APN), a potent inhibitor of lysyl oxidase and cross-link formation. In β-APN-treated cultures, total collagen synthesis was increased twofold, with no change in mRNA levels for type I collagen, whereas the amount of collagen accumulated in the cell layer was decreased by 50% and mineral deposition was reduced. The rate of collagen retention in the matrix was assessed by pulse-chase analysis of [3H]proline over a 16 day period in control and β-APN-treated cultures. In control cultures, about 20% of the labeled collagen was lost from the cell layers over a 16 day period compared with > 80% in the presence of β-APN. The β-APN-treated cultures also showed a wider diversity of fibril diameters with a median in the > 45–60 nm range. In summary, these data suggest that cross-linking and assembly of collagen fibrils secreted by osteoblasts in vitro occur in a fashion similar to that found in vivo. The rate of cross-link formation is relatively constant and may be correlated with increasing collagen mass. With progressive cross-linking, collagen fibrils become more stable and less susceptible to loss from the cell layer. Thus, collagen deposition is probably controlled at several post-translational stages during fibrillogenesis, including cross-link formation, lateral fibril aggregation, and increasing fibril stability.
Bone resorption activity of particulate-stimulated macrophages
Authors: Glant Tibor T. , Jacobs Joshua J. , Molnár Gyöngyi , Shanbhag Arun S. , Valyon Márta , Galante Jorge O.
Published: February 2010

Particulate wear debris from bone cement or prosthetic components can stimulate macrophages to cause bone resorption in a dose-dependent manner. This bone resorption activity of particulate-stimulated macrophages is associated with increased levels of both prostaglandin E2 (PGE2) and interleukin-1 (IL-1). In this study we compared the effect of particulate size, concentration, and composition on the secretion of IL-1 and PGE2 by peritoneal macrophages and on the bone-resorbing activity of conditioned medium (CM) harvested from particulate-challenged macrophages. Particulates (titanium, Ti; polymethylmethacrylate, PMMA; and polystyrene, PS) only with phagocytosable size stimulated peritoneal macrophages to secrete IL-1 and PGE2 in a dose- and time-dependent manner. Ti particles (1–3 μm) exhibited significantly enhanced bone-resorbing activity measured as 45Ca release. The maximum bone-resorbing response was observed at a concentration of 0.1% Ti (approximately 10–15 Ti particulates per cell), which also corresponded with the highest IL-1 levels measured in particulate-challenged CM. This was measured using either conditioned media from Ti-stimulated macrophages or in cocultures of calvarial bone and macrophages in the presence of Ti. Exogenous PGE2 and recombinant human IL-1 could significantly increase the 45Ca release; indomethacin (IM) significantly reduced both the spontaneous calcium efflux and active 45Ca release from in vivo labeled calvarial bones. However, IM and/or anti-Il-1 antibodies could suppress only partly the macrophage-mediated bone resorption, indicating that, in a macrophage-bone coculture system, factors other than PGE2 and IL-1 also may regulate particulate-induced bone resorption, probably involving multiple cell types.
Regulation of cytokine expression in osteoblasts by parathyroid hormone: Rapid stimulation of interleukin-6 and leukemia inhibitory factor mRNA
Authors: Greenfield Edward M. , Gornik Sandra A. , Horowitz Mark C. , Donahue Henry J. , Shaw Steven M.
Published: February 2010

PTH and other hormones that stimulate resorption affect osteoclasts indirectly by modulating cytokine production by osteoblasts. However, the identity and role of the osteoblast-derived cytokines involved in this process are unclear. To examine which cytokines are regulated by PTH, we assessed cytokine mRNA levels in osteoblasts using the reverse transcription-polymerase chain reaction technique. Of the 16 cytokines we examined, unstimulated MC3T3-E1 osteoblastic cells expressed mRNA for interleukins 5, 6, and 7, macrophage and granulocyte-macrophage colony-stimulating factors, transforming growth factor β1 and leukemia inhibitory factor. PTH specifically increased expression of interleukin-6 (approximately 50-fold) and leukemia inhibitory factor (approximately 10-fold). Levels of both IL-6 and LIF mRNA peaked 30–60 minutes after addition of PTH and returned to baseline by 4–6 h. This rapid and transient mRNA response, which resembles that of immediate early genes, was also observed in primary rat osteoblasts. The transient mRNA response was accompanied by increased secretion of IL-6 protein. Lipopolysaccharide, another stimulator of resorption, increased mRNA levels of a group of cytokines that were not induced by PTH, namely interleukin-1α, tumor necrosis factor α, and granulocyte-macrophage and granulocyte colony-stimulating factors. We conclude that osteoblasts produce complex networks of cytokines that (1) are regulated by bone-resorptive agents and (2) may be involved in controlling bone resorption.
Effect of different amounts of sodium intake for 4 months on calcium metabolism in normal and oophorectomized rats
Authors: Chan Ellen Lai-Ping , Professor Swaminathan R.
Published: February 2010

The effects of different amounts of salt intake on urinary calcium and hydroxyproline (OHP) excretion were studied in oophorectomized (O) and sham-operated (S) rats on normal calcium diet. Groups of O and S rats were given either tap water or 2, 6, or 18 g/liter of NaCl to drink. Urinary excretion of sodium, calcium, and OHP was monitored at 0, 2, and 4 months. Urinary excretion of calcium and OHP increased with increasing salt intake (P < 0.001 by ANOVA) in S and O groups. Oophorectomy did not have any additional effect on calcium or OHP excretion. We conclude that increased salt intake causes increased excretion of calcium and OHP and oophorectomy does not further aggravate the loss.
Simple measurement of femoral geometry predicts hip fracture: The study of osteoporotic fractures
Authors: Faulkner Kenneth G. , Cummings Steven R. , Black Dennis , Palermo Lisa , Glüer Claus-C. , Genant Harry K.
Published: February 2010

Based on engineering principles, geometric measurements of femoral size should be related to femoral strength and the risk for hip fracture. To evaluate whether a simple measurement of femoral geometry is associated with hip fracture risk, we obtained dual x-ray absorptiometry scans of the proximal femur on 8074 white women age 67 or older. During an average of 1.6 years of follow-up, 64 participants suffered hip fractures. In all fracture cases and in a random sample of 134 women who did not subsequently suffer a hip fracture, we measured hip axis length (the distance from greater trochanter to inner pelvic brim), neck width, and the neck/shaft angle on the scan printout, with the observer blinded to subsequent fracture status of the participant. Results were analyzed using multiple logistic models, and odds ratios were determined. After adjustment for age, each standard deviation decrease in femoral neck bone mineral density increased hip fracture risk 2.7-fold (95% confidence interval 1.7, 4.3), and each standard deviation increase in hip axis length nearly doubled the risk of hip fracture (odds ratio = 1.8; 95% CI 1.3, 2.5). The relationship between hip axis length and fracture risk persisted even after adjustment for age, femoral neck density, height, and weight. A longer hip axis length was associated with an increased risk of both femoral neck (OR = 1.9; 95% CI 1.3, 3.0) and trochanteric fractures (1.6; 1.0, 2.4). We found no significant association between the neck width (1.1; 0.8, 1.5) or the neck/shaft angle (1.4; 0.9, 2.2) and risk of hip fracture. In a combined analysis of the control group with an additional population of younger volunteers, no significant relationship was found between the hip axis length and age (r = 0.04, P = 0.60) or femoral neck density (r = 0.01, P = 0.84) in 225 women from 41 to 92 years of age. We conclude that hip axis length predicts hip fractures independently of age and bone mineral density in elderly women. If verified by additional studies, this simple measurement can improve the assessment of hip fracture risk compared to a measurement of femoral neck bone density alone.
Parenteral pamidronate prevents thyroid hormone-induced bone loss in rats
Authors: Rosen Harold N. , Sullivan E. Kelly , Middlebrooks V. Leah , Zeind Adib John , Gundberg Caren , Dresner-Pollak Rivka , Maitland Lauri A. , Hock Janet M. , Moses Alan C. , Greenspan Susan L.
Published: February 2010

Pamidronate (APD) is a bisphosphonate that prevents bone loss from a variety of causes. We studied the role of APD in preventing thyroid hormone-induced bone loss. A total of 32 rats were assigned to one of four treatment groups: (1) −APD/triiodothyronine (−T3), (2) −APD/+T3, (3) +APD/−T3, or (4) +APD/+T3. In the first of two studies, the rats received APD for the first week and T3 for the second week, and then their blood was analyzed for alkaline phosphatase and osteocalcin. Alkaline phosphatase and osteocalcin were significantly higher (p < 0.05) in hyperthyroid rats (−APD/+T3, 3.9 ± 0.25 μcat/liter and 23 ± 1.6 nM, respectively) than in control animals (2.53 ± 0.28 μkat/liter and 18.3 ± 1.4 nM, respectively). Hyperthyroid rats pretreated with APD (+APD/+T3) had levels of alkaline phosphatase and osteocalcin no different from controls. In a second study, rats were divided into the same four groups, except they received APD/placebo and T3/placebo concomitantly for 3 weeks. At the end of the study, bone mineral density (BMD) of the femur, spine, and whole body was measured by dual-energy x-ray absorptiometry, and the calcium content of the femora was measured directly. In hyperthyroid rats (−APD/+T3) BMD was significantly lower than in controls in the spine (0.201 ± 0.004 versus 0.214 ± 0.002 g/cm2, p < 0.05) and femur (0.204 ± 0.003 versus 0.218 ± 0.002, p < 0.05). In hyperthyroid rats pretreated with APD (+APD/+T3) BMD of the spine, femur, and total body was significantly higher than in controls (p < 0.001). Similar differences among groups were seen in femur calcium content. We conclude that hyperthyroid rats have an increased rate of bone turnover and bone loss that can be prevented by coadministration of a bisphosphonate.
Cloning and characterization of the 5′-flanking region of the mouse tartrate-resistant acid phosphatase gene
Authors: Reddy Sakamuri V. , Scarcez Thierry , Windle Jolene J. , Leach Robin J. , Hundley Jeffrey E. , Chirgwin John M. , Chou Janice Y. , Roodman G. David
Published: February 2010

Little information is available on the molecular mechanisms controlling osteoclastic bone resorption. We used tartrate-resistant acid phosphatase (TRAP) to begin to investigate the regulation of bone resorption at the molecular level. TRAP is expressed at high levels in osteoclasts and may play an important role in the bone resorptive process. Therefore, we isolated the murine TRAP gene from a mouse spleen genomic library and characterized its promoter. A restriction map was generated for the 17 kb TRAP insert. A 2 kb Smal fragment, containing the 5′-flanking region, was subcloned and the nucleotide sequence determined. Sequence analysis of the Smal fragment revealed the presence of numerous candidate transcription factor binding sequences, including those for API and H-APF-1. The H-APF-1 site matches the consensus sequence for the IL-6-regulated transcription factor. An intron was identified at −1 to −393 bp relative to the ATG. The presence of an intron was confirmed by PCR analysis of RNA isolated from murine osteoclasts. Primer extension analysis indicated the presence of a transcription initiation site at −552 bp from the ATG. The region from −1846 to 2 bp relative to the ATG initiation codon drove the transient expression of a luciferase reporter gene when transfected into HRE H9 rabbit endometrial cells. PMA treatment of HRE H9 cells enhanced luciferase transcription approximately threefold. These data suggest that the TRAP promoter is complex and contains multiple regulatory elements. The availability of the TRAP promoter may also permit production of transgenic mice, which can be used to develop previously unavailable osteoclast cell lines.