bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical trial of fluoride therapy in postmenopausal osteoporotic women: Extended observations and additional analysis
Authors: Riggs B.L. , O'Fallon W.M. , Lane A. , Hodgson S.F. , Wahner H.W. , Muhs J. , Chao E. , Melton L.J.
Published: February 2010

In a 4 year clinical trial in 202 postmenopausal osteoporotic women receiving NaF at 75 mg/day or placebo (both groups received supplementary calcium at 1500 mg/day), we found (N Engl J Med 322:801, 1990) that NaF increased bone mineral density in the lumbar spine (LS-BMD) substantially but did not decrease vertebral fracture rate (VFR), and it increased the nonvertebral fracture rate. Additional analyses and extended observations are now available on 50 women from the NaF group followed for up to 6 years of treatment. In these women, LS-BMD increased linearly over the 6 years (median rate, 8.7%/year or 0.063 g/cm2/year). Because during the 4 year trial the NaF dosage was decreased (because of side effects) in 54 of the 101 women randomized to NaF, dose-response relationships could be evaluated. For the entire study population, serum F level correlated directly with increase in LS-BMD (r = 0.61, P < 0.001). When individual person-years of observation were grouped by deciles of LS-BMD, VFR (per 100 person-years) decreased to a nadir of 24 as mean LS-BMD for the group increased from 0.6 to 1.2 g/cm2 and then doubled to 52 in the group with mean LS-BMD of 1.6 g/cm2. Multivariate analyses and inspection of three-dimensional plots revealed a complex pattern in which VFR was influenced by interaction of several variables. When the effects of LS-BMD, changes in LS-BMD, and serum F were assessed simultaneously, VFR was seen to decrease with increasing LS-BMD except when the higher LS-BMD was associated with rapid rate of increase in LS-BMD or a large increase from baseline serum F. For some patients (noncompliers or nonresponders), serum F or LS-BMD failed to increase. Thus, it is possible that lower dosages of NaF produce moderate decreases in VFR.
Course of bone mass during and after hormonal replacement therapy with and without addition of nandrolone decanoate
Authors: Erdtsieck R.J. , Pols H.A.P. , van Kuijk C. , Birkenhäger-Frenkel D.H. , Zeelenberg J. , Kooy P.P.M. , Mulder P. , Birkenhäger J.C.
Published: February 2010

A total of 33 women with postmenopausal osteoporosis were matched pairwise by age, years since menopause, and body mass index and randomized to receive either cyclic estrogen-progestagen replacement treatment (group 1) or the same treatment plus nandrolone decanoate (ND; group 2). Both groups were treated during 3 years and subsequently followed for another year off treatment. A year after cessation of the treatment the distal forearm bone mineral content in group 2 was significantly higher than that in group 1. Bone mass measurements in the axial skeleton already showed a significant difference in favor of group 2 after 3 years treatment, which persisted during the year off treatment. The decline in lumbar bone mineral mass and density in the 1 year off treatment was similar in both groups. Correction for body mass did not change these results. Bone turnover parameters did not show significant differences between the two groups after cessation of treatment. A higher muscle mass, induced by ND, could partly explain the differences between the groups since even 1 year after treatment was stopped an increased serum creatinine level was still observed in group 2.
Diagnostic value of estimated volumetric bone mineral density of the lumbar spine in osteoporosis
Authors: Peel N.F.A. , Dr. Eastell R.
Published: February 2010

Bone mineral density (BMD) measurements by dual-energy x-ray absorptiometry (DXA) are planar measurements. By measuring planar BMD in anteroposterior (AP) and lateral projections, it is possible to estimate the volumetric BMD by assuming that the vertebral body is an ellipsoid cylinder. Correction for the third dimension could improve the diagnostic accuracy of spinal BMD measurement in osteoporosis. The aims of this study were (1) to determine the effect of aging on volumetric BMD compared to planar measurements; and (2) to evaluate the diagnostic accuracy of volumetric BMD in comparison to planar measurements. We studied 26 postmenopausal women with osteoporotic vertebral fractures and 114 age- and sex-matched controls without vertebral fractures from a population-based group. AP and lateral decubitus BMD of the lumbar spine were measured by DXA using a Lunar DPX (precision error for AP measurement, 0.8%; for lateral measurement of vertebra L3, 6.4%). Between the ages of 51 and 85 years the decreases in volumetric BMD of L3 (vol L3), lateral BMD of L3 (lat L3), AP BMD of L3 (AP L3), and AP BMD of L2–4 (AP L2–4) were 31, 28, 17, and 17%, respectively. The decrease in BMD compared to controls in the osteoporotics for vol L3, lat L3, AP L3, and AP L2–4 were 31, 34, 23, and 23%; expressed as Z scores, the decreases were −1.36, −1.33, −1.46, and −1.47 standard deviation units. The areas under ROC analysis curves for vol L3, lat L3, AP L3, and AP L2–4 were 85, 86, 87, and 87%, respectively. We conclude that there is a greater decrease in volumetric and lateral BMD of vertebra L3 than the AP measurements, both with age and in postmenopausal osteoporosis compared to age-matched controls. However, the diagnostic accuracy was not improved by correcting BMC for vertebral volume. The greater decreases observed with aging may result from the high proportion of cancellous bone in the vertebral body. The lack of improvement in diagnostic accuracy may be because of the large precision error in lateral decubitus (and hence volumetric) BMD measurements. It is possible that the diagnostic accuracy of estimated volumetric BMD may improve with the use of lateral supine DXA, a measurement with lower precision error.
Bone marrow from mechanically unloaded rat bones expresses reduced osteogenic capacity in vitro
Authors: Keila Senia , Pitaru Sandu , Grosskopf Ayala , Dr. Weinreb Miron
Published: February 2010

Bone formation during mechanical unloading is reduced, mainly as a result of osteoblastic hypofunction. At the same time, the total number of osteoblasts per long bone is also markedly reduced. We tested the hypothesis that the number of osteogenic precursors present in the bone marrow stroma was concomitantly diminished by using an in vitro cell culture system in which femoral adherent bone marrow cells differentiate into active osteoblasts and produce bone-like nodules. Hindlimbs of 32-day-old male rats were either immobilized (unloaded) by sciatic neurectomy (immo) or sham operated (sham) and animals were killed after 11 days. Femora were either ashed to determine bone mass or used to generate bone marrow cultures. Adherent marrow cells were cultured in the presence of ascorbic acid, β-glycerophosphate, and dexamethasone. Bone mass was significantly reduced in unloaded femora (by 16%) and tibiae (by 18%). The number of adherent cells (determined on day 6) was reduced by 50% in the immo group. Reduced cell number did not result from slower proliferation in culture since [3H]thymidine incorporation on days 4 and 6 was similar in the two groups. The osteogenic potential in vitro of marrow from unloaded bones was diminished compared with that from loaded ones as evidenced by (1) lower alkaline phosphatase (ALP) activity per mg protein (by 25–40%, examined on days 6 and 12), and (2) reduced nodule formation (by 70%, expressed as percentage of the dish area stained with Alizarin Red S on day 21). None of these changes occurred in the contralateral limb of operated (immobilized) animals. In an additional experiment, adherent cells from both groups were subcultured on day 6 and seeded at an equal density of 40,000 cells per 35 mm dish. The osteogenic potential in cultures from unloaded bones was reduced as evidenced by lower ALP activity (by 25–40%) and fewer nodules formed (by 40%). These experiments show that unloading for 11 days causes a deficit in bone mass and reduces the number of femoral adherent marrow stromal cells and their osteogenic potential in vitro in subsequent (ex vivo) cultures. They also suggest that unloading specifically reduces the number of osteogenic precursors present within the marrow stroma.
Two-site enzyme immunoassay for measuring intact human osteocalcin in serum
Authors: Parviainen Markku , Kuronen Ilpo , Kokko Harri , Lakaniemi Mari , Savolainen Kari , Mononen Ilkka
Published: February 2010

We developed a sensitive two-site sandwich ELISA for quantitative analysis of human osteocalcin in serum or plasma. Our method is based on two different highly specific antibodies recognizing epitopes at different ends of the protein so that only intact osteocalcin is detected. The method is fast (total analysis time less than 6 h/96 wells), precise (intraassay variation less than 2.3% at four different levels; n = 10, and interassay variation less than 2.5%, n = 5, respectively), and accurate, with a mean recovery of 105%. The detection limit in serum is approximately 0.1 μg/liter. The mean concentration of osteocalcin in normal serum with this assay is 3.3 μg/liter (SD 3.7 μg/liter; range 0.1–13.1 μg/liter; n = 41), and the reference range is 0.28–10.1 μg/liter (10 and 90% confidence limits). The method shows a reasonable positive linear correlation with other osteocalcin assays (Incstar, r = 0.55, p < 0.05, n = 13; Henning Oscatest, r = 0.52, p < 0.005, n = 34). A good correlation (r = 0.70, p < 0.001) between individual osteocalcin and bone-specific alkaline phosphatase serum concentrations was observed in normal subjects. We found a low or undetectable concentration of intact osteocalcin in serum of all four of our patients with acute primary hyperparathyroidism, and in all five patients with hypocalcemic secondary hyperparathyroidism, which suggests that PTH effectively inhibited the synthesis of osteocalcin in osteoblasts. The serum concentration of intact osteocalcin was elevated in two of three patients with chronic primary hyperparathyroidism. In five patients with normocalcemic secondary hyperparathyroidism, the serum concentration of intact osteocalcin was low: three had a normal and two had an elevated value. An age-related decrease in intact osteocalcin in serum of females younger than 50 years and an increase in older women were observed.
Evidence for a diminished maturation of preosteoblasts into osteoblasts during aging in rats: An ultrastructural analysis
Authors: Roholl P.J.M. , Blauw E. , Zurcher C. , Dormans J.A. M. A. , Dr. Theuns H.M.
Published: February 2010

Bone is subject to continuous remodeling throughout life. The age-related loss of (trabecular) bone, leading to senile osteopenia, is mainly due to impaired bone formation. Osteoblasts (OB) and osteoclasts (OC) have been identified as playing a crucial role in the process of bone turnover, but the contribution made by their precursors is not well documented. We analyzed the cells of the osteoblast and osteoclast cell lineage along the trabecular bone of tibiae and the stromal cells in the marrow of aging BN/Bi Rij rats using electron microscopy. It appeared possible to distinguish preosteoblasts (pre-OB), OB, preosteoclasts (pre-OC), OC, and inactive bone-lining cells. Periods of increase, the maximal peak, and the decrease in trabecular bone volume were defined by means of morphometric measurements of trabecular bone volume. We found a decrease of more than 10-fold in the number of OB with age, but the numbers of pre-OB, pre-OC, and OC expressed per unit bone length, although variable, were age independent. The relative bone resorption and formation surface, expressed as a percentage of the total bone surface, decreased 2- and 15-fold, respectively. In 2-year-old animals the total volume of stromal cells, part of which constitutes the stem cell compartment of the osteogenic lineage, was a quarter of that found in 1-month-old animals and a third of that found in 6-month-old animals. The loss of trabecular bone is concomitant with a sharp increase in the ratio of pre-OB/OB, the ratio of OC/OB, and in the ratio of resorption to formation surfaces. There was no relation between the ratio of pre-OC/OC with age. These data lead to the conclusion that the main factor causing bone loss with age is a diminished maturation of pre-OB into OB.
Deposition of cement at reversal lines in rat femoral bone
Authors: Zhou Hong , Chernecky Robert , Dr. Davies John E.
Published: February 2010

Femora from young adult Wistar rats were prepared for both light and electron microscopy. Routinely processed wax sections showed the appearance of cement lines immediately proximal to the resorption surface formed by active osteoclasts and distal to the onset of lamella formation in femoral bone tissue. This early stage of extracellular matrix elaboration at reversal lines was then studied by scanning electron microscopy (SEM) of actively remodeling sites, mainly on trabecular and endosteal surfaces. The resorption surface was shown to comprise a decalcified collagenous mat with individual fibers running either parallel or perpendicular to the surface plane. By examining different, neighboring, areas of resorption lacunae, a temporal sequence of new extracellular matrix production could be established. Before the deposition of new collagen, globular accretions were deposited onto the resorption surface. In areas where individual collagen fibers were oriented perpendicular to the surface plane, this globular matrix was initially deposited on the exposed fiber tips. The globules increased in size and fused laterally to form a continuous cement layer, which not only interdigitated with the collagen mesh of the resorption surface but also provided anchorage for new collagen fibers, which themselves became mineralized. These morphologic results provide a mechanistic explanation of coupling at reversal lines.
Expression of bone morphogenetic protein messenger RNA in prolonged cultures of fetal rat calvarial cells
Authors: Harris S.E. , Sabatini M. , Harris M.A. , Feng J.Q. , Wozney J. , Mundy G.R.
Published: February 2010

In addition to structural proteins of bone, such as type I collagen, bone cells synthesize a number of growth regulatory peptides that are also stored in the bone matrix, presumably as a consequence of local production by osteoblasts. Among the bone growth regulatory peptides found in the bone matrix are the recently described bone morphogenetic proteins (BMPs). These factors were purified from bone matrix by their capacity to stimulate ectopic bone formation, but it is not known whether they are produced by normal bone cells and influence normal bone formation. To determine whether they are expressed by normal osteoblasts during differentiation, we used the technique of prolonged primary culture of fetal rat calvarial osteoblasts. These cultures have been shown to be an informative model for studying expression of bone-related genes by cultured osteoblasts, since specific genes are expressed as the cells undergo proliferation and differentiation. We found that the bone morphogenetic proteins 1, 2, 4, and 6 are expressed by cultures of fetal rat calvarial osteoblasts before they form mineralized bone nodules and as they express alkaline phosphatase, osteocalcin, and osteopontin. This model can be used for study of regulation of expression of bone morphogenetic proteins by osteoblasts.
Long-term exercise of young and adult female rats: Effect on femoral neck biomechanical competence and bone structure
Authors: Søgaard Charlotte H. , Danielsen Carl Christian , Thorling Eivind B. , Mosekilde Lis
Published: February 2010

The present study was designed to examine the effect of exercise on femoral neck bone structure and strength in female rats after 4 and 10 months of exercise. Female Fischer rats aged 2 months were exercised for 4 h a day, 5 days a week on a motor-driven treadmill the speed of which was gradually increased until a daily distance of 2 km was reached. The training level was maintained for 4 months (n = 15) and 10 months (n = 15). Sedentary age-matched rats served as controls (n = 30). At death the proximal third of both femora was obtained from each rat. The left femoral neck was tested in a materials-testing machine, and the right was used for either trabecular bone mass measurement (BV/TV) or scanning electron microscopy (SEM). Biomechanical testing revealed a significant training-induced increase in femoral neck bone strength after 4 months of exercise, which although not accelerated was preserved after 10 months of exercise. Histologic investigation revealed a significant training-induced increase in BV/TV, accounted for by a significantly smaller proportion of marrow space. No difference in cortical area was found. Both histology and SEM revealed a tendency to an earlier closure of the growth line in the exercised animals. However, the exercised animals aged 6 months had a significantly increased total femoral length compared with the sedentary rats. No difference was found in total femoral length between sedentary and exercised rats aged 12 months. On the basis of this study, it is concluded that exercise has a positive effect on femoral neck bone strength. The gain of bone strength may be due partly to an increased bone mass and partly to an earlier maturation of the skeleton. The method presented could form an excellent basis for evaluating potential therapeutic regimens for the treatment of osteoporosis.
Osteoporosis in men with a history of tibial fracture
Authors: Kannus Pekka , Järvinen Markku , Sievänen Harri , Oja Pekka , Vuori Ilkka
Published: February 2010

The long-term effects of bone fractures on bone mineral density (BMD) at various skeletal sites are poorly established, although a serious fracture, such as a tibia fracture, followed by long immobility and disuse may lead to permanently decreased BMD and, through this mechanism, may be a risk factor for osteoporotic fractures in later life. To determine whether such an injury leads to osteoporosis, we measured the areal BMD (g/cm2) from the lumbar spine (L2–4), right distal radius and ulna, and the femoral neck, distal femur, patella, proximal tibia, distal tibia, and calcaneus of both extremities in 14 men with a history of primarily nonunited (finally bone-grafted) shaft fracture of the tibia and 20 men with a history of primary union. For evaluation of the patients' BMD in the spine and distal forearm, 22 age-, weight-, and height-matched normal men were also measured. The average time of immobilization in a long plaster cast was 27 weeks in the former group of patients and 16 weeks in the latter. The measurements were performed an average of 9 years after the fracture using a dual-energy x-ray absorptiometric scanner. Compared with normal men (mean ± SD = 1.116 ± 0.160), the spinal BMDs were significantly lower in men with a history of a primary nonunion (0.979 ± 0.100, −12.3%) and union (1.010 ± 0.124, −9.5%). In distal radius and distal ulna, there were no significant differences between the three groups. In the 14 patients with a primary nonunion, the mean BMD of the injured extremity (compared with the uninjured side) was significantly lower in the distal femur (-10.0%), patella (-11.2%), proximal tibia (-9.2%), distal tibia (-7.9%), and calcaneus (-5.6%). In the 20 patients with a primary union, the side-to-side difference was significant in the femoral neck (-2.4%), distal femur (-4.0%), patella (-3.7%), and proximal tibia (-5.1%). The relative BMD of the injured extremity did not correlate with the fracture type, fracture location, or patients' age but did show significant (r = 0.41–0.79) positive correlation with short immobilization time, low pain assessment, good muscle strength, and high functional scores of the injured extremity. In conclusion, the tibial shaft fracture may be associated with reduced bone density in the lumbar spine and injured extremity. The observed decrease (10–12% in spine and 4–11% in the knee region of the injured limb) is clinically important with respect to age-related bone loss of 1% per year after the age of peak bone mass. Additional follow-up is needed to determine any increased risk of osteoporotic fractures in spine and injured extremity.
Carboxyl-terminal parathyroid hormone peptide (53–84) elevates alkaline phosphatase and osteocalcin mRNA levels in SaOS-2 cells
Authors: Dr. Sutherland M. Kung , Rao L.G. , Wylie J.N. , Gupta A. , Ly H. , Sodek J. , Murray T.M.
Published: February 2010

Previous findings in our laboratory have shown that hPTH-(53–84) stimulates alkaline phosphatase activity in dexamethasone-treated ROS 17/2.8 cells. In the present study, we examined the effects of hPTH-(53–84) and hPTH-(1–34) on the expressions of alkaline phosphatase, osteocalcin, and collagen type I mRNA levels in the human osteosarcoma cell line SaOS-2. The effect of hPTH-(53–84) on alkaline phosphatase and osteocalcin message levels was dose dependent (ANOVA, p < 0.005 and p < 0.001, respectively), with significant stimulation observed at 10 nM. Treatment with 10 nM hPTH-(53–84) for 24 h resulted in significant 2- and 1.4-fold increases in mRNA levels for alkaline phosphatase and osteocalcin, respectively (p < 0.05), but had no effect on collagen type I expression. The increased alkaline phosphatase mRNA levels was associated with a 1.5-fold increase in enzyme activity (p < 0.05). In contrast, under similar incubation conditions, hPTH-(1–34) had no significant effects on alkaline phosphatase or osteocalcin mRNA levels. On the other hand, hPTH-(1–34) had dose-dependent stimulatory effects on collagen type I mRNA levels (ANOVA, p < 0.001), 10 nM hPTH-(1–34) stimulating collagen type I expression 1.6-fold (p < 0.05). The results indicate that carboxyl-terminal hPTH-(53–84) has direct and unique biologic effects in human osteoblast-like cells in culture.
Bone matrix RGD glycoproteins: Immunolocalization and interaction with human primary osteoblastic bone cells in vitro
Authors: Grzesik Wojciech J. , Robey Pamela Gehron
Published: February 2010

The interaction of cells with extracellular matrix is essential for their anchorage, proliferation, migration, and differentiation. In bone matrix there are multiple glycoproteins that contain the integrin-binding RGD sequence: fibronectin (FN), thrombospondin (TSP), osteopontin (OPN), bone sialoprotein (BSP), type I collagen (COLL I), and vitronectin (VN). In this study, the localization of TSP, FN, VN, and several integrins within developing human long bone using immunohistochemical methods was examined, as was the effect of all bone RGD proteins on the adhesion of human osteoblastic cells. Thrombospondin, fibronectin, and vitronectin showed distinct localization patterns within bone tissue. TSP was found mainly in osteoid and the periosteum; VN appeared to be present mainly in mature bone matrix. FN was present in the periosteum as well as within both mature and immature bone matrix. Using a panel of antiintegrin antibodies we found that bone cells in vivo and in vitro express α4, αv, α5β1, αvβ3, and β3/β5 integrins, and these receptors are for the most part expressed on all bone cells at different stages of maturation with quantitative rather than qualitative variations, with the exception of α4, which is expressed mainly by osteoblasts. Cell attachment assays were performed using primary human cells of the osteoblastic lineage under serum-free conditions. COLL I, TSP, VN, FN, OPN, and BSP promoted bone cell attachment in a dose-dependent manner and were equivalent in action when used in equimolar concentrations. In the presence of GRGDS peptide in the medium, the adhesion to BSP, OPN, and VN was almost completely blocked (10, 10, and 15% of control, respectively), and attachment to FN, COLL I, and TSP was only slightly decreased (80, 75, and 55%, respectively). These results suggest that human bone cells may use RGD-independent mechanisms for attachment to the latter glycoproteins.
Carboxyl-terminal parathyroid hormone-related protein inhibits bone resorption by isolated chicken osteoclasts
Authors: Dr. Fenton A.J. , Martin T.J. , Nicholson G.C.
Published: February 2010

Carboxyl-terminal peptides from parathyroid hormone-related protein (PTHrP) have been studied for their effect on bone resorption by osteoclasts isolated from 15 day embryonic chickens. Basal bone resorption by chicken osteoclasts was directly inhibited by chicken and human PTHrP-(107–139) and the pentapeptide PTHrP-(107–111). The chicken and human analogs were equipotent. Both the number of resorption pits and the total area resorbed per bone slice were reduced by PTHrP-(107–139), but it did not alter the size of individual resorption pits. Resorption stimulated by hPTH-(1–34) in cocultures of chicken osteoclasts with osteoblasts was also inhibited by cPTHrP-(107–139) but required a concentration three orders of magnitude greater than that required to inhibit basal resorption in cocultures or cultures of isolated osteoclasts. The finding of resorption inhibitory activity by PTHrP-(107–139) in avian as well as mammalian species strengthens the hypothesis that carboxyl-terminal PTHrP may act as a paracrine regulator of bone cell activity.
Effects of treatment of malignancy-associated hypercalcemia on serum parathyroid hormone-related protein
Authors: Budayr A.A. , Zysset E. , Jenzer A. , Thiébaud D. , Ammann P. , Rizzoli R. , Jaquet-Müller F. , Bonjour J.P. , Gertz B. , Burckhardt P. , Halloran B.P. , Nissenson R.A. , Strewler Gordon J.
Published: February 2010

Parathyroid hormone-related protein (PTHrP) is the primary mediator of hypercalcemia in patients with malignancy-associated hypercalcemia. We conducted this study to examine the effects of treatment with a bisphosphonate on serum PTHrP. We analyzed 41 episodes of hypercalcemia occurring in 38 patients: 22 patients received alendronate, and 16 were treated with pamidronate. At baseline, 29 patients had an increased serum PTHrP (group I) and 9 had low or undetectable levels (group II). The two groups did not differ significantly in baseline hypercalcemia (3.26 versus 3.41 mM) or the response of serum calcium to therapy. Serum calcium was normalized in 88% of group I and 70% of group II patients. Lowering of the mean calcium level was not associated with a change in the level of PTHrP in group I patients (40.2 versus 36.7 pgEq/ml) or group II patients. We also analyzed data on serum PTH and 1,25-(OH)2D in 20 of the patients. Serum PTH rose with treatment in group I patients (9.7–40.2 pg/ml, p < 0.05), as did the serum 1,25-(OH)2D (19.1–32.4 pg/ml, p < 0.001). Similarly, treatment of group II patients was associated with an increase in serum PTH (9.8–37.2 pg/ml) and serum 1,25-(OH)2D (22.9–40.2 pg/ml). The individual increases in 1,25-(OH)2D levels associated with therapy could not be predicted from the level of PTHrP or the changes in levels of serum calcium or PTH. Our data show that effective treatment of malignancy-associated hypercalcemia is not associated with a consistent change in serum levels of PTHrP. Therapy is associated with a variable increase in the serum levels of PTH and 1,25-(OH)2D.
Expression of 92 kD type IV collagenase/gelatinase B in human osteoclasts
Authors: Wucherpfennig Anne L. , Li Yi-Ping , Stetler-Stevenson William G. , Rosenberg Andrew E. , Stashenko Philip
Published: February 2010

The digestion of type I collagen is an essential step in bone resorption. It is well established that osteoclasts solubilize the mineral phase of bone during the resorptive process, but the mechanism by which they degrade type I collagen, the major proteinaceous component of bone, is controversial. Differential screening of a human osteoclastoma cDNA library was performed to characterize genes specifically expressed in osteoclasts. A large number of cDNA clones obtained by this procedure were found to represent 92 kD type IV collagenase (gelatinase B; MMP-9, EC 3.4.24.35), as well as tartrate-resistant acid phosphatase. In situ hybridization localized mRNA for gelatinase B to multinucleated giant cells in human osteoclastomas. Gelatinase B immunoreactivity was demonstrated in giant cells from eight of eight osteoclastomas, osteoclasts in normal bone, and osteoclasts of Paget's disease by use of a polyclonal antiserum raised against a synthetic gelatinase B peptide. In contrast, no immunoreactivity for 72 kD type IV collagenase (gelatinase A; MMP-2, EC 3.4.24.24), which is the product of a separate gene, was detected in osteoclastomas or normal osteoclasts. We propose that the 92 kD type IV collagenase/gelatinase B plays an important role in the resorption of collagen during bone remodeling.
Effects of 1,25-dihydroxyvitamin D3 on bone resorption and natural immunity in osteopetrotic (ia) rats
Authors: Dr. Schneider Gary B. , Relfson Melanie , Langman Craig B.
Published: February 2010

Osteopetrois is an inherited bone disease characterized by an excessive accumulation of bone throughout the skeleton. The disease in the ia (incisors absent) rat is the result of reduced bone resorption caused by defective, although numerous osteoclasts. In addition to the bone defects, ia rats have suppressed natural immunity, even though these animals have excessive numbers of natural killer (NK) cells. The osteopetrotic condition also appears to have an associated abnormality in vitamin D metabolism. Because 1,25-dihydroxyvitamin D3[1,25-(OH)2D3] stimulates bone resorption and has a role in the immunoregulation of NK cells, mutant and normal rats were infused with 1,25-(OH)2D3 for 14 days in an attempt to correct the defects in this mutant. Serum levels of osteocalcin, 25-OHD3, and 1,25-(OH)2D3, as well as NK function and parameters of bone resorption, were evaluated after the infusion period. Serum levels of osteocalcin and 1,25-(OH)2D3 were elevated in both ia and normal rats treated with 1,25-(OH)2D3. Serum 25-OHD3 levels were significantly reduced in the treated animals. The elevated percentage of NK cells normally found in ia rats was reduced to normal in the treated mutants, and NK cell function was elevated to normal levels of lytic activity. The percentage of NK cells and NK function remained unchanged in the treated normal rats. The bone marrow cavity size was significantly increased in the 1,25-(OH)2D3-treated mutants, as was the percentage of osteoclasts exhibiting normal morphology. Radiographically, the mutant bones were less dense. No net change in the bone resorption was noted in the treated normal rats. 1,25-(OH)2D3 appears to correct both the bone resorption and natural immune defects in the ia osteopetrotic mutant.
Lack of involution of hyperplastic parathyroid glands in dogs: Adaptation via a decrease in the calcium stimulation set point and a change in secretion profile
Authors: Cloutier M. , Brossard J.H. , Gascon-Barré M. , D'Amour Pierre
Published: February 2010

This study analyzes the parathyroid function in four dogs before and after 2 years of a low-calcium, high-sodium, vitamin D-deficient diet and the involution of the same function following (1) correction of dietary calcium deficiency and administration of IV 1,25-(OH)2D (0.25 μg twice per day) during 1 month, (2) after an additional month of normal dog chow supplemented with oral vitamin D (25 μg per day), and, finally, (3) after 5 and 17 months of a diet with normal levels of calcium and vitamin D. The parathyroid function was evaluated through IV infusion of CaCl2 and Na2 EDTA with measurement of intact (I) and carboxyl-terminal (C) immunoreactive parathyroid hormone (iPTH). The C-iPTH/I-iPTH ratio was calculated to assess the modulation of molecular forms of iPTH induced by the various treatments. The 2 years of calcium and vitamin D deprivation lowered ionized calcium (1.23 ± 0.04, p < 0.05) and 25-OHD (4.02 ± 2.06 nM, p < 0.005) and tended to decrease 1,25-(OH)2D (80.8 ± 8.6 pM); it increased basal I- and C-iPTH levels approximately eightfold (I-iPTH, 40.2 ± 20.7, p < 0.05; C-iPTH, 185.4 ± 94.9, p < 0.05) and stimulated I-iPTH (60.2 ± 23.0 pM, p < 0.05) and C-iPTH (239.6 ± 80.7 pM, p < 0.05) fivefold. A greater rise in nonsuppressible I-iPTH levels than in C-iPTH levels led to a decreased C-iPTH/I-iPTH ratio in hypercalcemia (12.5 ± 2.8 versus 27.8 ± 6.05 pM, p < 0.005). Dietary deprivation also decreased the I-iPTH calcium stimulation set point (1.25 ± 0.05 pM, p < 0.05). Treatment with dietary calcium and IV 1,25-(OH)2D normalized ionized calcium (1.34 ± 0.02 mM) and basal I-iPTH level (6.09 ± 5.48 pM) more than basal C-iPTH (69.8 ± 48.8 pM), causing the C-iPTH/I-iPTH ratio to increase from 4.80 ± 0.81 to 14.8 ± 6.1 (p < 0.05). Stimulated I-iPTH decreased (46.1 ± 15.3 pM, p < 0.05), but stimulated C-iPTH secretion did not change (282.4 ± 90.9 pM). A greater fall in the nonsuppressible I-iPTH level as opposed to C-iPTH level led the C-iPTH/I-iPTH ratio to increase back to a normal value (23.8 ± 1.40). The I-iPTH calcium stimulation set point remained low at 1.28 mM. Further consecutive treatment modalities over 18 months did not cause additional significant change in basal, stimulated, or nonsuppressible C-iPTH levels, but the same I-iPTH levels tended to decrease further. This explained why all three C-iPTH/I-iPTH ratios tended either to increase (basal) or to increase significantly (stimulated and nonsuppressible, p < 0.05). The I-iPTH calcium stimulation set point again remained low. This study reveals that animals with hyperplastic parathyroid glands can control their I-iPTH level by maintaining a lower set point of I-iPTH stimulation by calcium and by changing their secretion profile with respect to carboxyl-terminal fragments of the PTH molecule.
Bone mineral density measured by dual-energy X-ray absorptiometry and novel markers of bone formation and resorption in patients on antiepileptic drugs
Authors: Dr. Välimäki Matti J. , Tiihonen Mikko , Laitinen Kalevi , Tähtelä Riitta , Kärkkäinen Merja , Lamberg-Allardt Christel , Mäkelä Pekka , Tunninen Riitta
Published: February 2010

In patients on antiepileptic drugs, bone loss has been mainly demonstrated at radial sites using old technology and has been ascribed to drug-induced vitamin D deficiency rather than to any direct effects of the treatment on bone cells. We examined 38 epileptic patients (24 women and 14 men) aged 20–49 years who were using either carbamazepine or phenytoin or both. Bone mineral density (BMD) at the lumbar spine and three femoral sites was measured by dual-energy x-ray absorptiometry (DXA) and serum and urine markers of bone and mineral metabolism were determined. The latter included the C-terminal extension peptide of type I procollagen (PICP), a putative serum marker of bone formation, and the cross-linked carboxyl-terminal telopeptide of human type I collagen (ICTP), a novel serum marker of bone matrix degradation. In female patients on phenytoin, weight- and height-adjusted BMD was reduced at the femoral neck and the Ward's triangle (p < 0.05) but was at the control level in the other patient groups at all four measurement sites. Compared with controls, the serum concentrations of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were reduced by 26% (p < 0.01) and by 27% (p < 0.001) in female patients. These changes were independent of the therapy used. They were not present in male patients. For both genders the serum levels of vitamin D binding protein were normal. Both female and male patients had hypocalcemia, but women only showed hypocalciuria. The serum intact parathyroid hormone was 42% higher (p < 0.01) in women and 26% (p = 0.07) in men. The serum markers of bone formation, bone alkaline phosphatase, osteocalcin, and PICP, were higher for both sexes, the percentage elevations being more striking in men (104%, p < 0.001; 66%, p < 0.001; and 63%; p < 0.001, respectively) than in women (33%, p < 0.01; 13%, p = NS; and 27%, p < 0.05, respectively). Serum ICTP was 46% higher (p < 0.001) in women and 22% (p < 0.05) in men. We conclude that in patients on antiepileptic drugs, bone turnover is accelerated independently of the presence of hypovitaminosis D. Such biochemical alterations do not necessarily lead to reduced bone mass, however.
Lithium inhibits calcitriol-stimulated formation of multinucleated cells in human long-term marrow cultures
Authors: Pepersack Thierry , Corazza Francis , Demulder Anne , Guns Martine , Fondu Pierre , Bergmann Pierre
Published: February 2010

We observed that lithium (3 mM) blocked the 1,25-dihydroxyvitamin D [1,25-(OH)2D3]-stimulated bone resorption in fetal rat long bones in culture. Because this inhibitory effect was not seen when bone resorption was stimulated by parathyroid hormone or interleukin-1, we reasoned that Li specifically inhibited events involved in the 1,25-(OH)D3-stimulated bone resorption. The increased bone resorption induced by vitamin D in culture is associated with differentiation and/or fusion of osteoclast progenitors. In the present work, we studied the effect of Li on the basal and 1,25-(OH)2D3-stimulated generation of multinucleated osteoclast-like cells (MNC) and MNC containing tartrate-resistant acid phosphatase (TRAP+) in long-term human bone marrow cultures. Total MNC and TRAP+ cells were counted after 3 weeks of culture. In the absence of both lithium and 1,25-(OH)2D3, total MNC and TRAP+ cell numbers were 146 ± 22 and 110 ± 18 per well, respectively (mean ± SEM); in the presence of Li, corresponding figures were 79 ± 17 and 59 ± 14. When the generation of MNC and TRAP+ cells was stimulated with 1,25-(OH)2D3, (10−8 M), total MNC and TRAP+ cells were 521 ± 66 and 473 ± 63, respectively, in the absence of Li and 251 ± 44 and 155 ± 27 in the presence of Li (p < 0.05). The inhibitory effect of Li was dose dependent and was not observed when the cultures were exposed to parathyroid hormone instead of 1,25-(OH)2D3. When Li was added to the cells the first week of culture only, we observed the full inhibitory effect; conversely, if Li was added for the last week of culture only, no inhibitory effect was seen. These data show that Li interferes with the recruitment of osteoclast-like cells from their precursors, probably at an early stage of differentiation.
Transient and localized expression of bone morphogenetic protein 4 messenger RNA during fracture healing
Authors: Nakase Takanobu , Nomura Shintaro , Yoshikawa Hideki , Hashimoto Jun , Hirota Seiichi , Kitamura Yukihiko , Oikawa Shinzo , Ono Keiro , Takaoka Kunio
Published: February 2010

Temporal and spatial distribution of a gene encoding murine bone morphogenetic protein 4 (mBMP-4) during fracture repair were investigated in mice by RT-PCR and in situ hybridization. For in situ hybridization, fractured ribs and surrounding tissues were decalcified and hybridized with a mBMP-4-specific complementary RNA probe labeled with digoxigenin-11 UTP. mBMP-4 messenger RNA (mRNA) was not detected in ribs without fracture, whereas it was detected only in the early phase of fracture from 12 to 72 h after the onset of fracture before new cartilage or bone formation. The mBMP-4 mRNAs were present in cells distributed in three distinct regions, namely, the proliferating periosteum, the medullary cavity, and the muscles near the fracture site. These BMP-4-positive cells did not express bone gla protein mRNA, which is a marker of the mature osteogenic cell. RT-PCR also showed a transient increase in the level of BMP-4 mRNA in the early phase of fracture repair. The findings provide us with some new information. (1) The BMP-4 gene is produced by less differentiated osteoprogenitor cells, not by differentiated osteoblasts. (2) The BMP-4 gene is enhanced by the impact of fracture and localized in callus-forming tissue before callus formation. Together with the activities of BMP-4, as was previously described, our results suggest that newly produced BMP-4 gene product is one of the local contributing factors in callus formation in the early phase of fracture healing.
Effects of gallium nitrate in nude mice bearing a canine adenocarcinoma (CAC-8) model of humoral hypercalcemia of malignancy
Authors: Merryman Joyce I. , Capen Charles C. , Dr. Rosol Thomas J.
Published: February 2010

Hypercalcemic nude mice bearing a canine adenocarcinoma (CAC-8) model of humoral hypercalcemia of malignancy (HHM) were treated daily with gallium nitrate (60 mg/kg of elemental gallium subcutaneously (SC) on day 0, followed by 20 mg/kg/day) for 5 days. Gallium nitrate significantly decreased (p < 0.01) serum calcium in tumor-bearing animals on days 2 and 5 of treatment (mean 13.7 ± 0.7 mg/dl on day 0 versus 11.6 ± 0.3 on day 2 and 12.4 ± 0.5 on day 5). Urinary calcium excretion was decreased (p < 0.05) in the gallium-treated, tumor-bearing animals (0.11 ± 0.01 mg calcium/mg creatinine) compared with hypercalcemic tumor-bearing mice (0.30 ± 0.06). Both nontumor control and tumor-bearing mice treated with gallium nitrate lost body weight during the treatment period (p < 0.01). Gallium nitrate had no effect on tumor growth. Histomorphometric evaluation of lumbar vertebrae stained for tartrate-resistant acid phosphatase revealed a significant decrease (p < 0.05) in the number of osteoclasts/mm trabecular bone and perimeter of trabecular bone lined by active osteoblasts (p < 0.01) in the gallium-treated tumor-bearing mice compared with tumor-bearing controls. Osteoclast length (mm) was significantly increased in both the nontumor and tumor-bearing gallium-treated animals (p < 0.05) compared with nontumor and tumor-bearing control mice. Serum tumor necrosis factor α (TNF-α) levels were increased in tumor-bearing animals, but gallium nitrate had no effect on circulating levels (not detectable in nontumor control mice versus 82 ± 21 pg/ml in tumor-bearing mice and 107 ± 12 pg/ml in gallium-treated tumor-bearing mice). Concentrations of gallium in bone were undetectable in untreated control mice (<0.00005 μg/g bone), but markedly elevated concentrations were present in gallium-treated mice (235 ± 6 μg/g bone in nontumor-bearing and 155 ± 18 in tumor-bearing). These results indicate that gallium nitrate decreased osteoclastic bone resorption and serum calcium levels in a nude mouse model of HHM.
Studies on the mechanism of desensitization of the parathyroid hormone-stimulated calcium signal in UMR-106 cells: Reversal of desensitization by alkaline phosphatase but not by protein kinase C downregulation
Authors: Lee Suk Kyeong , Dr. Stern Paula H.
Published: February 2010

The involvement of protein kinase C (PKC), cAMP-dependent protein kinase (PKA), and other phosphorylation mechanisms in the rapid desensitization of the [Ca2+]i response to parathyroid hormone (PTH) stimulation was investigated in osteoblast-like UMR-106 cells. A 5 minute preincubation of the cell suspension with phorbol 12,13-dibutyrate (PDB) decreased the response to PTH in a concentration-dependent manner. 1-Oleoyl-2-acetyl-r-glycerol (OAG) pretreatment likewise decreased the PTH response. Staurosporine, a potent protein kinase inhibitor, completely prevented the desensitization caused by PDB. These PDB and staurosporine effects were also observed in 3 mM EGTA-containing medium ([Ca2+]free < 10−8 M). A 5 minute pretreatment of cells with 1 μM forskolin had no effect on the calcium response to PTH. Homologous and PDB-induced desensitizations differed in several respects. Staurosporine pretreatment resulted in only a slight restoration of the PTH response under conditions of homologous desensitization. Chronic treatment with phorbol ester prevented the desensitization of the PTH response by acute phorbol treatment but not the homologous desensitization. Both homologous and PDB-induced desensitization were relieved by alkaline phosphatase treatment, consistent with the involvement of phosphorylation in the desensitization. This alkaline phosphatase effect on desensitization was inhibited by L-phenylalanine. These results suggest that PTH receptor homologous desensitization involves phosphorylation process(es) other than or in addition to those of PKC.
Estrogen enhances the stimulation of bone collagen synthesis by loading and exogenous prostacyclin, but not prostaglandin E2, in organ cultures of rat ulnae
Authors: DR. Cheng Ming Zhao , Zaman Gul , Lanyon Lance E.
Published: February 2010

The shafts of ulnae from 110 g male rats were cultured, and after a period of 5 h preincubation one of each pair of bones was either loaded cyclically (500 g, 1 Hz, 8 minutes) to produce physiologic strains (-1300 με) or treated with exogenous prostacyclin (PGI2) or prostaglandin E2 (10−6 M, 8 minutes) in the presence or absence of 17β-estradiol (10−8 M). PGI2, PGE2, and loading stimulated almost immediate increases in glucose 6-phosphate dehydrogenase (G6PD) activity in osteocytes and osteoblasts. This increase was uniform throughout the section with exogenous PGs in the medium but was related to local strain magnitude in loading. Elevated G6PD levels in response to loading and PGI2 persisted for 18 h, by which time, ALP activity in surface osteoblasts was elevated and [H]proline incorporation into collagen increased. PGE2 produced similar immediate and sustained increases in G6PD activity and [H]proline incorporation after 18 h but no change in ALP activity. Bones cultured for 18 h with 17β-estradiol increased their [H]proline incorporation, as did those loaded, and treated with PGI2 and PGE2. Loading and PGI2 but not PGE2 produced similar proportional increases in [H]proline incorporation above the increased baseline of estradiol alone. These results suggest that estrogen and loading together produce a greater osteogenic response than either separately. If so, estrogen withdrawal would result in a rapid fall in bone mass to establish a new equilibrium appropriate to the reduced effectiveness of the loading-related stimulus. Such a fall in bone mass is a characteristic feature of estrogen withdrawal at the menopause.
Effects of ascorbic acid on collagen matrix formation and osteoblast differentiation in murine MC3T3-E1 cells
Authors: Franceschi Renny T. , Iyer Bhanumathi S. , Cui Yingqi
Published: February 2010

Treatment of mouse MC3T3-E1 cells with ascorbic acid initiates the formation of a collagenous extracellular matrix and synthesis of several osteoblast-related proteins. We recently showed that ascorbic acid dramatically increases alkaline phosphatase and osteocalcin mRNAs and that this induction is blocked by inhibitors of collagen triple-helix formation (Franceschi and Iyer, J Bone Miner Res 7:235). In the present study, the relationship between collagen matrix formation and osteoblast-specific gene expression is explored in greater detail. Kinetic studies revealed that ascorbic acid increased proline hydroxylation in the intracellular procollagen pool within I h and stimulated the cleavage of type 1 collagen propeptides beginning at 2.5 h. Mature α1(I) and α2(I) collagen components were first detected at 10 h and continued to increase in both cell layer and culture medium for up to 72 h. Ascorbic acid also increased the rate of procollagen secretion from cell layers to culture medium. The secretion of another matrix protein, fibronectin, was only slightly affected. Alkaline phosphatase or its mRNA was first detected 2–3 days after ascorbic acid addition, but osteocalcin mRNA was not seen until day 6. Two inhibitors of collagen triple-helix formation, ethyl-3,4-dihydroxybenzoate and 3,4-dehydroproline, inhibited procollagen hydroxylation and alkaline phosphatase induction. 3,4-Dehydroproline also inhibited the induction of alkaline phosphatase and osteocalcin mRNAs. Surprisingly, induction was not blocked if cells were exposed to ascorbic acid before inhibitor addition. Alkaline phosphatase was also partially inhibited if cells were grown in the presence of purified bacterial collagenase. These results indicate that the induction of osteoblast markers by ascorbic acid does not require the continuous hydroxylation and processing of procollagens and suggest that a stable, possibly matrix-associated signal is generated at early times after ascorbic acid addition that allows subsequent induction of osteoblast-related genes.