bluuebunny/crossref_metadata_2025 huggingface dataset:
Bilateral Tibial Marrow Ablation in Rats Induces a Rapid Hypercalcemia Arising from Extratibial Bone Resorption Inhibitable by Methylprednisolone or Deflazacort
Authors: Magnuson Scott K. , Booth Randal , Porter Susan , Dr. Gorski Jeffrey P.
Published: April 2006

The goals of this study were to quantitate biochemical markers of bone metabolism on days 1–15 after bilateral tibial marrow ablation surgery in young adult rats and to determine the effect of a single dose of methylprednisolone (2 mg/kg) or deflazacort (2.5 mg/kg) given at the time of ablation. Unexpectedly, serum calcium levels rose to a maximum of 15.9 mg/dl on day 7 after marrow ablation and remained above normal through day 15. This increase was blocked by a single intramedullary injection of methylprednisolone or deflazacort immediately following ablation; however, the fact that both drugs produced a characteristic rapid 3- to 10-fold increase in the serum α2-macroglobulin level demonstrates that the drugs rapidly reached the circulation. Both methylprednisolone and deflazacort also inhibited intramedullary deposition of collagen by 40–60% on day 7, a time near which operated control animals achieved maximal accumulation of new bone in this model. Histological comparisons among the three experimental groups were largely consistent with biochemical results. The urinary hydroxyproline/creatinine ratio for the operated control group doubled on day 3 and then returned to presurgical levels on day 7 and later. The timing and size of the hydroxyproline/creatinine peak, as well as the fact that the intratibial osteoclastic response peaks on days 8–10 after ablation, suggests it results from extratibial bone resorption induced by marrow ablation. Consistent with this rationale, urinary calcium excretion in operated controls rose 9-fold from day 0 to day 3 and appeared to plateau over the period from day 3 to day 9, before returning to a near presurgical level on day 15. Elevated excretion of calcium noted on days 9–15 in deflazacort-treated animals, which occurs in the absence of a detectable increase in resorption marker hydroxyproline, may however be due to the known action of glucocorticoids in increasing kidney filtration of calcium. In summary, this is the first report to show that bilateral tibial marrow ablation in rats causes a rapid hypercalcemia and calciuria which is accompanied initially by a peak of bone resorption marker urinary hydroxyproline. We speculate that the source of calcium and hydroxyproline is extratibial osteoclastic bone resorption induced by circulating cytokines whose release from ablated tibias or osteoclastogenic action is inhibitable by methylprednisolone and deflazacort.
Different Responses of Bone Alkaline Phosphatase Isoforms During Recombinant Insulin-like Growth Factor-I (IGF-I) and During Growth Hormone Therapy in Adults with Growth Hormone Deficiency
Authors: Magnusson Per , Degerblad Marie , Sääf Maria , Larsson Lasse , Thorén Marja
Published: April 2006

We studied serum bone alkaline phosphatase (ALP) isoforms and other markers of bone turnover in growth hormone–deficient (GHD) adults (n = 22). The patients were followed during 1 week of insulin-like growth factor-I (IGF-I) administration, 40 μg/kg of body weight/day (n = 6), and during 24 months of growth hormone (GH) therapy, 0.125 IU/kg of body weight/week for the first month, and then 0.250 IU/kg of body weight/week (n = 20). Six ALP isoforms were separated and quantified by high-performance liquid chromatography: one bone/intestinal, two bone (B1, B2), and three liver ALP isoforms. At baseline, the mean levels of B1, B2, and osteocalcin were higher in GHD adults than in healthy adults. After 1 week of IGF-I administration and 1 month of GH therapy, only B1 was decreased. We suggest that the initial decrease of B1 during GH therapy could be an effect of endocrine IGF-I action mediated by GH. After 3 months of GH therapy, both B1 and B2 increased as compared with placebo. Osteocalcin, carboxy-terminal propeptide of type I procollagen (PICP), cross-linked carboxy-terminal telopeptide of type I collagen (ICTP), and urinary pyridinoline cross-links/creatinine ratio increased during GH therapy. PICP increased significantly before bone ALP and osteocalcin, indicating an early stimulation of type I collagen synthesis as previously demonstrated by in vitro models. Different responses of the bone ALP isoforms during IGF-I and during GH therapy suggest different regulations in vivo.
Protein Interactions During Assembly of the Enamel Organic Extracellular Matrix
Authors: Paine Michael L. , Snead Malcolm L.
Published: April 2006

Enamel is the outermost covering of teeth and contains the largest hydroxyapatite crystallites formed in the vertebrate body. Enamel forms extracellularly through the ordered assembly of a protein scaffolding that regulates crystallite dimensions. The two most studied proteins of the enamel extracellular matrix (ECM) are amelogenin and tuftelin. The underlying mechanism for assembly of the proteins within the enamel extracellular matrix and the regulatory role of crystallite-protein interactions have proven elusive. We used the two-hybrid system to identify and define minimal protein domains responsible for supra molecular assembly of the enamel ECM. We show that amelogenin proteins self-assemble, and this self-assembly depends on the amino-terminal 42 residues interacting either directly or indirectly with a 17-residue domain in the carboxyl region. Amelogenin and tuftelin fail to interact with each other. Based upon this data, and advances in the field, a model for amelogenin assemblies that direct enamel biomineralization is presented.
Degradable Bisphosphonate-Alkaline Phosphatase-Complexed Hydroxyapatite Implants In Vitro
Authors: Dr. Denissen Harry , Van Beek Ermond , Van Den Bos Theo , De Blieck Jolanda , Klein Christel , Van Den Hooff Arnold
Published: April 2006

Degradable hydroxyapatite (HA) implants complexed with the resorption inhibiting agent bisphosphonate (PCP) and the mineralizing agent alkaline phosphatase (ALP) can theoretically maintain alveolar bone mass directly after extraction of teeth. The present in vitro study investigated the surface properties of PCP–ALP-complexed HA implants in relation to the requirements of implant behavior and action. Adsorbed PCP (pH 3.49) resulted in a flattening and broadening of the phosphate peaks and the formation of carbonate peaks in the HA pattern of the implant indicating a chemical alteration of the HA surface. Adsorption of ALP onto PCP-altered HA surfaces was 26% lower than onto HA implant blank surfaces. PCP–ALP-complexed HA implants released the PCP and ALP steadily and continuously over observation periods of, respectively, 75 and 14 days. During these observation periods, the ceramic grains of the HA implant became smaller and intergrain boundaries became broader. These morphologic characteristics suggested preconditioning of the HA implant surface for future bonding and degradation in vivo. Individual grains were no longer bonded to other grains and detached from the implant which had become rounded in shape. From in vitro mice experiments we found that PCP concentrations between 10−4 and 10−3 M resulted in45Ca-release from the bone HA. Our calculations showed, however, that only a total concentration of 1.4 × 10−4 M PCP was gradually released over the whole observation period. In another experiment, it appeared that a PCP concentration in solution <10−3 M did not reduce ALP activity. It is concluded that release of PCP by the PCP–ALP-complexed implants is maintained at levels in the range to impair osteoclast bone resorption but not high enough to block osteoblast activity. The amount of ALP released can lead to induction of bone formation onto implant surfaces. pH-induced alterations in the microstructure and chemistry of the HA surface allow for controlled degradation of the HA implants in vitro. A PCP–ALP-complexed HA implant acting as temporary scaffolding for alveolar bone growth enhancement, mineralization, and maintenance seems to be a reasonable concept for preservation of the edentulous alveolus.
Secretory Products of Breast Cancer Cells Specifically Affect Human Osteoblastic Cells: Partial Characterization of Active Factors
Authors: Siwek Brigitte , Lacroix Marc , De Pollak Cindy , Marie Pierre , Body Jean-Jacques
Published: April 2006

The pathogenesis of tumor-induced osteolysis (TIO) following breast cancer metastases in bone remains unclear. We postulated that osteoblasts could be target cells for the secretory products of breast cancer cells. We previously showed that serum-free conditioned medium (CM) of the breast cancer cell line MCF-7 inhibits DNA synthesis by 75% of control values in osteoblast-like cells SaOS-2 and that this effect is only in a minor part due to transforming growth factor β secretion. To establish the specificity of our observations and to look for other biologically active factors, we have tested the effects of medium conditioned by several cancer and noncancer cell lines (breast, colon, placenta, or fibrosarcoma) on the proliferation of osteoblast-like cells (SaOS-2, MG-63), normal human osteoblasts, human fibrosarcoma cells, and normal human fibroblasts. Culture medium (1:2) of the breast cancer cell lines MCF-7, T-47D, MDA-MB-231, and SK-BR-3 inhibited by 25–50% the proliferation of osteoblast-like cells SaOS-2, MG-63, and normal osteoblasts as evaluated by the MTT survival test or [3H]thymidine incorporation. MCF-7 cells completely inhibited the proliferation of normal human osteoblasts in coculture. This inhibitory effect was reversible and not due to cytotoxicity. Moreover, the cyclic adenosine monophosphate (cAMP) response to parathyroid hormone (PTH) of osteoblast-like cells SaOS-2 was also increased by 100–240% by the same CM. Such activities were, however, not detected in medium from the breast noncancer cell line HBL-100 or in the medium conditioned by non–breast cancer cell lines (COLO 320DM, HT-29, JAR, or HT-1080). Medium from the breast cancer cells had no effect on normal human fibroblasts or fibrosarcoma cells (HT-1080), suggesting the specificity of their action on human osteoblasts. After partial purification by ultrafiltration and size-exclusion chromatography, we found that medium of T-47D cells contained at least three nonprostanoid factors of low molecular weights (apparent MW of 700, 1500, and 4000 D) which affected human osteoblast-like cells. These factors were heat stable and could be peptides without disulfide bonds. In summary, our data show that human breast cancer cells release soluble factors that inhibit osteoblast proliferation and increase their cAMP response to PTH, indicating that osteoblasts could be important target cells for breast cancer cells and could be involved in the process of TIO.
Role of Protein Kinase A in Collagenase-1 Gene Regulation by Prostaglandin E1: Studies in a Rabbit Synoviocyte Cell Line, HIG-82
Authors: Suzuki Koji , Rapuano Bruce E. , Bockman Richard S.
Published: April 2006

Gene expression of the matrix-degrading enzyme collagenase-1 in rabbit synoviocytes and human fibroblasts is down-regulated by prostaglandin E1 (PGE1) through a cyclic adenosine monophosphate (cAMP)–dependent pathway. In the current study, we examined the role of protein kinase A (PKA) in the PGE1-mediated effect on collagenase-1 gene expression. Collagenase-1 gene expression was rapidly induced several-fold above control both by a phorbol ester, 12-o-tetradecanoyl phorbol 13 acetate, and interleukin-1β (IL-1β) in HIG-82 synoviocytes. Treatment with PGE1 and forskolin increased PKA activity in the HIG-82 cells within 15 minutes of adding the stimulating agents. Two inhibitors of PKA, the isoquinoline-sulfonamide derivative, H-89 and a cAMP analog, RpcAMP, blocked the ability of PGE1 to down-regulate collagenase-1 gene expression. However, if PGE1 was added from 6 h to 30 minutes before the PKA inhibitor H-89, collagenase-1 gene expression was inhibited. Constitutive PKA activity was increased in HIG-82 synoviocytes stably transfected with an expression vector pCMV.Cα that caused the HIG-82 cells to overexpress an active catalytic subunit of PKA. Cells stably transfected with an inactive, mutated C-α-variant showed no change in PKA activity. Collagenase-1 mRNA levels in TPA-stimulated cells were reduced to baseline levels in the pCMV.Cα but not in the mutated C-α–transfected cells. These data show the importance of PKA in regulating collagenase-1 gene expression in a synoviocyte cell line.
A New Synthetic Steroid, Osaterone Acetate (TZP-4238), Increases Cortical Bone Mass and Strength by Enhancing Bone Formation in Ovariectomized Rats
Authors: Fuse Hiroaki , Fukumoto Seiji , Sone Hideyuki , Miyata Yoshiko , Saito Tomoyuki , Nakayama Konosuke , Takahashi Hiroo , Matsumoto Toshio , Ogata Etsuro
Published: April 2006

A new synthetic steroid, 17α-acetoxy-chloro-2-oxa-4,6-pregnadiene-3,20-dione (osaterone acetate, TZP-4238), has a potent antiandrogenic and gestagenic action with virtually no estrogenic and androgenic activity in their classical target organs. In the present study, the effects of TZP-4238 on the structure, strength, and turnover of the rat long bones were examined. Female Wistar rats at 12 weeks of age were ovariectomized (OVX) and treated with TZP-4238 or 17β-estradiol (E2) every day for 12 weeks. TZP-4238 significantly increased the diameters and maintained bone mineral density (BMD) of the femur of OVX rats. Although the BMD of the total femur was higher in E2-treated rats than that in TZP-treated rats, E2 did not increase the diameters of the femurs. To examine the effects of TZP-4238 and E2 on the BMD of different regions of the femur, the BMD was further analyzed by dividing it into 20 regions of equal longitudinal length. E2 increased the BMD of the distal and proximal metaphysis, regions rich in trabecular bone, but had no effect on the BMD of the femoral diaphysis compared with OVX control rats. In contrast, 2.5 and 12.5 mg/kg TZP-4238 increased the BMD of the femoral diaphysis, regions rich in cortical bone, but did not affect the BMD at the distal metaphysis. In agreement with the changes in the BMD of different regions of the femur, TZP-4238 but not E2 increased the physical strength of the femoral diaphysis assessed by a three-point bending test. Histomorphometric analyses of the cross-sections of the tibia revealed that TZP-4238 increased but E2 reduced the periosteal bone formation rate compared with OVX rats. In addition, TZP-4238 caused an increase in serum bone alkaline phosphatase with only a mild and transient decrease in urinary deoxypyridinoline excretion, while E2 reduced both of these parameters. These results demonstrate that TZP-4238 increases the dimension, BMD, and physical strength of the rat long bones by enhancing cortical bone formation, while estrogen maintains trabecular BMD by inhibiting bone resorption. Because the physical strength of long bones is affected by cortical bone mass and geometry, the effect of TZP-4238 on cortical bone may have a potential for the treatment of osteoporosis with reduced cortical bone formation.
Quantification of Vitamin D Receptor mRNA by Competitive Polymerase Chain Reaction in PBMC: Lack of Correspondence with Common Allelic Variants
Authors: Mocharla Hanna , Butch Anthony W. , Pappas Alex A. , Flick James T. , Weinstein Robert S. , De Togni Pietro , Jilka Robert L. , Roberson Paula K. , Parfitt A. Michael , Manolagas Stavros C.
Published: April 2006

It has been recently claimed that polymorphism for the vitamin D receptor (VDR) influences several aspects of calcium and bone metabolism. To evaluate the physiologic plausibility of these claims, we compared the abundance of the VDR mRNA in peripheral blood mononuclear cells (PBMCs) between different VDR genotypes using a quantitative reverse transcribed polymerase chain reaction–based method. The method is based on the coamplification of VDR cDNA and an internal standard consisting of known concentrations of a human VDR CDNA mutated at a BglII restriction site; the interassay coefficient of variation is 11%. To validate the method, we made use of earlier receptor binding studies indicating that normal human monocytes and activated, but not resting, lymphocytes expressed the VDR. The concentration of the VDR mRNA was 10−8 to 10−7 g/g of total RNA in cell-sorted monocytes and in in vitro activated lymphocytes, but only 10−12 g/g of total mRNA in resting lymphocytes, establishing that the VDR mRNA determined by our method in PBMCs is due to constitutive expression in monocytes. Following an initial genotype screening of 85 normal volunteers by polymerase chain reaction or restriction fragment length polymorphism analysis, 14 individuals with the Bb genotype, 12 with the bb genotype, and 12 with the BB genotype were selected. The concentration of the VDR mRNA, corrected for the number of monocytes, was similar among the three genotype groups, as were the other variables examined: serum calcitriol, serum osteocalcin, and vertebral and hip bone density. We conclude that VDR polymorphism does not affect the abundance of the VDR mRNA.
Plasminogen Activator System in Osteoclasts
Authors: Yang J.-N. , Allan E. H. , Anderson G. I. , Martin T. J. , Minkin C.
Published: April 2006

To determine which genes of the plasminogen activator (PA) system were expressed in osteoclasts, RNA extracted from microisolated mouse osteoclasts was used as template for reverse transcribed polymerase chain reaction (RT-PCR) with gene-specific primer pairs. Using this approach, the expression of RNAs for tissue-type plasminogen activator, urokinase-type plasminogen activator, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, protease nexin, and urokinase receptor isoform 1 (uPAR1) were detected in mouse osteoclasts. The expression of uPAR RNA in osteoclasts was confirmed by in situ hybridization with a uPAR1 probe. RNA encoding the uPAR isoform 2 was not detected in mouse osteoclasts, but a novel unspliced uPAR RNA variant was detected in these cells. The novel uPAR variant and uPAR1 RNA were also detected in mouse calvarial osteoblasts, kidney, muscle, and the mouse macrophage cell line J774A.1 by RT-PCR. The presence of RNAs for most of the components of the PA system in osteoclasts suggests that it may have a functional role in this cell type.
Early Responses to Dynamic Strain Change and Prostaglandins in Bone-Derived Cells in Culture
Authors: Zaman G. , Suswillo R. F. L. , Cheng M. Z. , Tavares I. A. , Lanyon L. E.
Published: April 2006

Mechanical loading of bone explants stimulates prostaglandin E2 (PGE2) and prostacyclin (PGI2) release and increases glucose 6-phosphate dehydrogenase (G6PD) activity. This response is blocked by indomethacin and imitated by exogenous PGs. In the experiments reported here, primary cultures of rat long bone–derived osteoblast-like cells were exposed to a dynamic strain and exogenous PGs in the culture dish. Strain (3400 με, 600 cycles, 1 Hz) caused an immediate release of PGI2 into the culture medium but had no effect on PGE2. Strain also caused an increase in G6PD activity per cell and an increase in the smallest transcript of insulin-like growth factor II (IGF-II) (IGF-II T3) but had no effect on the expression of transforming growth factor-β1 (TGF-β1). Indomethacin inhibited strain-induced release of PGI2 and suppressed strain-induced stimulation of IGF-II T3 transcript. PGI2 (1 μM) increased G6PD activity and mRNA levels of all three transcripts of IGF-II but had no effect on the mRNA levels of IGF-I or TGF-β1. PGE2 (1 μM) stimulated G6PD activity and caused a marked increase in IGF-I and the largest transcript of IGF-II (IGF-II T1) but had no effect on the IGF-II transcripts T2 and T3 or on TGF-β1 mRNA levels. These findings show similarities in response between osteoblast-like cells strained in monolayer culture and bone cells in loaded bone explants in situ. They provide support for a role for IGF-II and PGI2 in the early strain-related response of osteoblasts in loading-related bone modeling/remodeling.
C-Terminal Parathyroid Hormone-Related Protein Inhibits Proliferation and Differentiation of Human Osteoblast-like Cells
Authors: Martínez Maria Eugenia , García-Ocaña Adolfo , Sánchez Maravillas , Medina Sonia , Del Campo Teresa , Valin Alvaro , Sanchez-Cabezudo Maria Jesus , Esbrit Pedro
Published: April 2006

Parathyroid hormone-related protein (PTHrP) is synthesized by osteoblasts, although its local role in bone is not completely understood. The C-terminal (107–111) region of PTHrP seems to be a potent inhibitor of osteoblastic bone resorption. We studied the effect of this PTHrP domain on the proliferation and synthesis of osteoblastic markers in osteoblast-like cells from adult human bone. We found that the human (h)PTHrP(107–139) fragment, between 10 fM and 10 nM, inhibited3H-thymidine incorporation into these cells. The antiproliferative effect of the latter fragment, or that of hPTHrP(107–111), was similar to that induced by [Tyr34]hPTHrP(1–34) amide, bovine PTH(1–34), and hPTHrP(1–141), while hPTHrP(38–64) amide was ineffective. Human PTHrP(7–34) amide, at 10 nM, and 1 μM phorbol-12-myristate-13-acetate also significantly decreased DNA synthesis in human osteoblast-like cells. Neither hPTHrP(7–34) amide nor hPTHrP(107–139), at 10 nM, stimulated protein kinase A (PKA) activity in these cells. Moreover, 100 nM H-89, a PKA inhibitor, did not eliminate the inhibitory effect of hPTHrP(107–139) on these cells' growth. However 100 nM calphostin C, a PKC inhibitor, blunted this effect of PTHrP(107–139). In addition to their antimitogenic effect, hPTHrP(107–139) and hPTHrP(107–111) inhibited basal and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)–stimulated alkaline phosphatase activity in these cells. Both fragments, like 1,25(OH)2D3, decreased C-terminal type I procollagen secretion into the cell-conditioned medium, but osteocalcin secretion by these cells was unaffected by the C-terminal PTHrP fragments. These findings suggest that PTHrP may act as a local regulator of bone formation.
Prevention of Postmenopausal Bone Loss Using Tibolone or Conventional Peroral or Transdermal Hormone Replacement Therapy with 17β-Estradiol and Dydrogesterone
Authors: Lippuner Kurt , Haenggi Willy , Birkhaeuser Martin H. , Casez Jean-Paul , Jaeger Philippe
Published: April 2006

Postmenopausal bone loss can be prevented by continuous or intermittent estradiol (E2) administration. Concomitant progestogen therapy is mandatory in nonhysterectomized women to curtail the risk of endometrial hyperplasia or cancer. However, the recurrence of vaginal bleeding induced by sequential progestogen therapy in addition to continuous estrogen administration is one of the reasons for noncompliance to hormone replacement therapy (HRT). Tibolone, a synthetic steroid with simultaneous weak estrogenic, androgenic, and progestational activity, which does not stimulate endometrial proliferation, has recently been proposed for the treatment of climacteric symptoms. To compare the efficacy of conventional oral and transdermal HRT with that of tibolone in the prevention of postmenopausal bone loss, 140 postmenopausal women (age, 52 ± 0.6 years; median duration of menopause, 3 years) were enrolled in an open 2-year study. Volunteers had been offered a choice between HRT and no therapy (control group, CO). Patients selecting HRT were randomly allocated to one of the following three treatment groups: TIB, tibolone, 2.5 mg/day continuously, orally; PO, peroral E2, 2 mg/day continuously, plus sequential oral dydrogesterone (DYD), 10 mg/day, for 14 days of a 28-day cycle; TTS, transdermal E2 by patch releasing 50 μg/day, plus DYD as above. Bone densitometry of the lumbar spine, upper femur, and whole body was performed using dual-energy X-ray absorptiometry at baseline, and then 6, 12, 18, and 24 months after initiation of therapy. One hundred and fifteen women (82%) completed the 2 years of the study. The dropout rate was similar in each group. Over 2 years, bone preservation was observed in all three treatment groups as compared with controls, without significant differences among treatment regimens. In conclusion, tibolone can be regarded as an alternative to conventional HRT to prevent postmenopausal bone loss.
Regulation of Serum Calcitriol by Serum Ionized Calcium in Rats During Pregnancy and Lactation
Authors: Boass Agna , Garner Sanford C. , Schultz Vijitha L. , Toverud Svein U.
Published: April 2006

Serum calcitriol concentrations in rats follow a biphasic pattern during reproduction, with elevated levels during late pregnancy, a decline after parturition, and a rise to even higher levels during peak lactation. We have previously shown that serum calcitriol in rats at peak lactation correlates significantly with, and appears to be regulated by, serum ionized Ca (Ca2+), with parathyroid hormone (PTH) serving a permissive role. We have extended this study by determining if serum calcitriol also correlates with serum Ca2+ during late pregnancy, when calcitriol levels are clearly elevated, and during early lactation, when only modest increases in serum calcitriol are observed. Analyses of data combined from nonmated, 21-day pregnant (P), and 1-day lactating rats (L) revealed a significant regression (p < 0.001) of calcitriol on Ca2+, but a nonsignificant regression (p = 0.34) of calcitriol on serum PTH. An even stronger correlation (p < 0.001) between calcitriol and Ca2+ was found for the combined data for 5−, 8−, and 14-day L rats. The partial correlation coefficient for calcitriol versus Ca2+, with PTH as the independent variable, was highly significant (p < 0.01) for the data from both combined groups. However, the coefficient for calcitriol versus PTH, with Ca2+ as the independent variable, was not significant (p > 0.05). Fetal weights (uterus and contents) correlated significantly with both maternal calcitriol and Ca2+ concentrations (p < 0.01), but not with maternal PTH levels. Litter weights for 14-day-old pups likewise correlated significantly with maternal calcitriol and Ca2+ (p < 0.001). We conclude that hypocalcemia, induced by the demands for Ca for fetal calcification and milk production, appears to be a controlling factor in serum calcitriol elevation in late pregnancy and throughout lactation, whereas PTH may be important for calcitriol synthesis without playing a direct regulatory role.
Method-Based Differences in the Automated Analysis of the Three-Dimensional Morphology of Trabecular Bone
Authors: Simmons Craig A. , Hipp John A.
Published: April 2006

The three-dimensional (3D) morphology of trabecular bone is frequently quantified using computer programs. However, there are no standardized implementations of morphology programs and many variations are possible. Even though programs may use the same basic method, results can be significantly different because of differences in implementation. Morphology data from different laboratories therefore may not be comparable. The method of directed secants, with parallel plate assumptions, is commonly used to quantify 3D morphology. We examined the effect of several variations in the implementation of this method on measurements of trabecular plate number (Tb.N), trabecular thickness, and trabecular spacing. Three-dimensional micromagnetic resonance images of 10 bovine trabecular bone specimens were analyzed using several variations of the directed secant method. An analysis of covariance with repeated measures suggested that variations in the algorithm used to count test line intersections, variations in the criteria used to classify a test coordinate as bone or marrow, and variations in the number of test grid rotations had significant effects on Tb.N (p < 0.0001). The largest difference in Tb.N (52%) was due to the method used to count test line intersections with the bone–marrow interface. Variations in the classification algorithm and variations in the number of test line grid rotations resulted in a 6% difference in Tb.N. The spacing of the test line grids did not affect Tb.N (p = 0.28), and all differences were independent of volume fraction (p = 0.67). These data show that there can be significant differences in trabecular bone morphology measurements due only to the method used for the measurements. To facilitate comparisons between laboratories, we have made validated computer programs to measure trabecular bone morphology available over the internet.
Resistance to Bone Resorbing Effects of PTH in Black Women
Authors: Cosman Felicia , Morgan Dorcas C. , Nieves Jeri W. , Shen Victor , Luckey Marjorie M. , Dempster David W. , Lindsay Robert , Parisien May
Published: April 2006

Black women have a lower incidence of vertebral and hip fractures than white women, possibly due to differences in skeletal and mineral metabolism. One suggested mechanism is that blacks have decreased skeletal sensitivity to parathyroid hormone (PTH). To test this hypothesis, we infused h(1–34)PTH in healthy premenopausal black (n = 15) and white (n = 18) women over 24 h and measured serum and urine indices of bone turnover and calcium metabolism throughout the infusion. At baseline, the mean 25-hydroxyvitamin D (25(OH)D) concentration was significantly lower in black women (46%). There were also nearly significant trends toward higher PTH and lower urinary calcium and pyridinoline levels in black women. During infusion, there were no racial differences in the mean (1–34)PTH levels achieved or in resultant elevations of serum calcium or 1,25-dihydroxyvitamin D (1,25(OH)2D) levels. Endogenous parathyroid suppression (measured by (1–84)PTH levels) was also similar between blacks and whites. There was an initial decline in urinary calcium/creatinine in both groups with a greater reduction in black women early in the infusion period (p < 0.05 at 8 h). Furthermore, blacks had lower levels of urinary calcium/creatinine throughout the infusion (p < 0.05 group difference). Bone formation markers (carboxy-terminal propeptide of type I procollagen and osteocalcin) decreased within 8 h and continued to decline throughout the infusion with no distinguishable racial differences (p < 0.05 time trend for both). The most dramatic difference between black and white women in response to PTH infusion was represented by the bone resorption markers. Three separate metabolites of bone resorption (cross-linked N-telopeptide of type I collagen, cross-linked C-telopeptide of type I collagen, and free pyridinoline) all showed substantially greater elevations in white (mean peak increments 399, 725, and 43%) compared with black women (mean peak increments 317, 369, and 17%) during the infusion (p < 0.05 group differences for all three variables). These data strongly suggest that blacks have decreased skeletal sensitivity to the acute resorptive effects of increased PTH. This finding indicates that calcium homeostasis may be accomplished in blacks (during times of relative calcium deficiency) by greater conservation of calcium from nonskeletal sources (most likely renal) with relative preservation of skeletal tissue. These differences in calcium economy could account, at least in part, for the increased bone mass and lower incidence of osteoporotic fractures in black women.
Whole Body Bone, Fat, and Lean Mass in Black and White Men
Authors: Barondess David A. , Nelson Dorothy A. , Schlaen Sandra E.
Published: April 2006

This research describes the effects of age, ethnicity, and body size and composition on whole body bone mass and bone density in healthy black and white men. We measured 79 male subjects, 42 white and 37 black, ranging in age from 33 to 64 years. Whole body bone mineral content (WBBMC) and bone mineral density (WBBMD), as well as fat and lean mass, were evaluated with a Hologic 1000W bone densitometer. We explore the utility of different methods of controlling for variations in body size in the two ethnic groups. There are statistically significant ethnic differences only in the bone mass variables. The black men had a 15% higher WBBMC (3111 vs. 2712 g, p < 0.0001) and a 8% higher WBBMD (1.25 vs. 1.16 g/cm2, p = 0.001) than the white men. Dividing WBBMD by height reduced the black/white difference to 6%. WBBMC, WBBMC/height, and WBBMD are strongly and significantly correlated with weight, body mass index (BMI), and body composition; correlations tended to be lower for WBBMD/height. Age is not significantly correlated with any of the variables in either ethnic group (p ≥ 0.10). In multivariate linear regression models for predicting WBBMC or WBBMD, the two best models contained height, weight, and an interaction of ethnicity and weight (model r2 = 0.72 for WBBMC and r2 = 0.47 for WBBMD); and height, lean mass, and an ethnicity–fat interaction (model r2 = 0.69 for WBBMC and r2 = 0.46 for WBBMD). Using analysis of covariance, we found that controlling for lean mass and height reduced the black/white difference in bone mass from 14.7 to 9.8%.
Strain Gradients Correlate with Sites of Periosteal Bone Formation
Authors: Gross Ted S. , Edwards Jonathan L. , Mcleod Kenneth J. , Rubin Clinton T.
Published: April 2006

We examined the hypothesis that peak magnitude strain gradients are spatially correlated with sites of bone formation. Ten adult male turkeys underwent functional isolation of the right radius and a subsequent 4-week exogenous loading regimen. Full field solutions of the engendered strains were obtained for each animal using animal-specific, orthotropic finite element models. Circumferential, radial, and longitudinal gradients of normal strain were calculated from these solutions. Site-specific bone formation within 24 equal angle pie sectors was determined by automated image analysis of microradiographs taken from the mid-diaphysis of the experimental radii. The loading regimen increased mean cortical area (±SE) by 32.3 ± 10.5% (p = 0.01). Across animals, some periosteal bone formation was observed in every sector. The amount of periosteal new bone area contained within each sector was not uniform. Circumferential strain gradients (r2 = 0.36) were most strongly correlated with the observed periosteal bone formation. SED (a scalar measure of stress/strain magnitude with minimal relation to fluid flow) was poorly correlated with periosteal bone formation (r2 = 0.01). The combination of circumferential, radial, and longitudinal strain gradients accounted for over 60% of the periosteal new bone area (r2 = 0.63). These data indicate that strain gradients, which are readily determined given a knowledge of the bone's strain environment and geometry, may be used to predict specific locations of new bone formation stimulated by mechanical loading.
Regional Responsiveness of the Tibia to Intermittent Administration of Parathyroid Hormone as Affected by Skeletal Unloading
Authors: Halloran Bernard P. , Bikle Daniel D. , Harris Jonathan , Tanner Sharon , Curren Tracy , Morey-Holton Emily
Published: April 2006

To determine whether the acute inhibition of bone formation and deficit in bone mineral induced by skeletal unloading can be prevented, we studied the effects of intermittent parathyroid hormone (PTH) administration (8 μg/100 g/day) on growing rats submitted to 8 days of skeletal unloading. Loss of weight bearing decreased periosteal bone formation by 34 and 51% at the tibiofibular junction and tibial midshaft, respectively, and reduced the normal gain in tibial mass by 35%. Treatment with PTH of normally loaded and unloaded animals increased mRNA for osteocalcin (+58 and +148%, respectively), cancellous bone volume in the proximal tibia (+41 and +42%, respectively), and bone formation at the tibiofibular junction (+27 and +27%, respectively). Formation was also stimulated at the midshaft in unloaded (+47%, p < 0.05), but not loaded animals (−3%, NS). Although cancellous bone volume was preserved in PTH-treated, unloaded animals, PTH did not restore periosteal bone formation to normal nor prevent the deficit in overall tibial mass induced by unloading. We conclude that the effects of PTH on bone formation are region specific and load dependent. PTH can prevent the decrease in cancellous bone volume and reduce the decrement in cortical bone formation induced by loss of weight bearing.
Expression of Nitric Oxide Synthase Isoforms in Bone and Bone Cell Cultures
Authors: Helfrich Miep H. , Evans Deborah E. , Grabowski Peter S. , Pollock Jennifer S. , Ohshima Hiroshi , Ralston Stuart H.
Published: April 2006

Recent work has shown that nitric oxide (NO) acts as an important mediator of the effects of proinflammatory cytokines and mechanical strain in bone. Although several bone-derived cells have been shown to produce NO in vitro, less is known about the isoforms of NO synthase (NOS), which are expressed in bone or their cellular distribution. Here we investigated the expression, cellular localization, and regulation of NOS mRNA and protein in cultured bone-derived cells and in bone tissue sections. We failed to detect inducible NOS (iNOS) protein in normal bone using immunohistochemical techniques, even though low levels of iNOS mRNA were detected by sensitive reverse transcribed polymerase chain reaction (RT-PCR) assays in RNA extracted from whole bone samples. Cytokine stimulation of bone-derived cells and bone explant cultures caused dramatic induction of iNOS mRNA and protein in osteoblasts and bone marrow macrophages, but no evidence of iNOS expression was seen in osteoclasts by immunohistochemistry or in situ hybridization. Endothelial NOS (ecNOS) mRNA was also detected by RT-PCR in whole bone, and immunohistochemical studies showed widespread ecNOS expression in bone marrow cells and trabecular lining cells in vivo. Related studies in vitro confirmed that ecNOS was expressed in cultured osteoblasts, stromal cells, and osteoclasts. Neuronal NOS mRNA was detected by RT-PCR in whole bone, but we were unable to detect nNOS protein in bone cells in vivo or in studies of cultured bone-derived cells in vitro. In summary, our data show that mRNAs for all three NOS isoforms are expressed in bone and provide evidence for differential expression and regulation of the enzymes in different cell types. These findings confirm the likely importance of the L-arginine–NO pathway as a physiological mediator of bone cell function and demonstrate that it may be possible to exert differential effects on osteoblast and osteoclast activity in vivo by differential targeting of constitutive and inducible NOS isoforms by selective NOS inhibitors.
Cyclization by a Specific Lactam Increases the Ability of Human Parathyroid Hormone (hPTH)-(1–31)NH2 to Stimulate Bone Growth in Ovariectomized Rats
Authors: Whitfield J. F. , Morley P. , Willick G. , Langille R. , Ross V. , Maclean S. , Barbier J.-R.
Published: April 2006

Human parathyroid hormone (hPTH)-(1–31)NH2 (Ostabolin), which only stimulates adenylyl cyclase (AC) instead of AC and phospholipase-C as do hPTH(1–84) and hPTH(1–34), strongly stimulates femoral cortical and trabecular bone growth in ovariectomized (OVX) rats. Two side-chain lactams have been introduced in the hydrophilic face of the receptor-binding region of the fragment's Ser17-Val31 amphiphilic α-helix in an attempt to develop improved analogs for the treatment of osteoporosis. Replacing the polar Lys27 with an apolar Leu on the hydrophobic face of this α-helix and stabilizing the helix with a lactam between Glu22 and Lys26 produced a fragment, [Leu27]-cyclo(Glu22-Lys26)-hPTH(1–31)NH2, which had six times the AC-stimulating ability of hPTH(1–31)NH2 in ROS 17/2 rat osteosarcoma cells, but the other helix-stabilizing lactam derivative [Leu27]-cyclo(Lys26-Arg30)-hPTH(1–31)NH2 did not have a greater AC-stimulating ability than hPTH(1–31)NH2, to stimulate AC in ROS 17/2 rat osteosarcoma cells. As expected from AC stimulation being responsible for PTH's anabolic action, [Leu27]-cyclo(Glu22-Lys26)-hPTH(1–31)NH2 was, depending on the experimental design, a 1.4 to 2 times better stimulator of trabecular bone growth in the OVX rat model than either hPTH(1–31)NH2 or [Leu27]-cyclo(Lys26-Arg30)-hPTH(1–31)NH2. Thus, there is now a more potently anabolic derivative of hPTH(1–31)NH2, [Leu27]-cyclo(Glu22-Lys26)-hPTH(1–31)NH2, which might ultimately prove to be one of the more effective therapeutics for osteoporosis.
Quantitative Ultrasound Techniques for the Assessment of Osteoporosis: Expert Agreement on Current Status
Authors: Glüer Claus-C. ,   FOR THE INTERNATIONAL QUANTITATIVE ULTRASOUND CONSENSUS GROUP
Published: April 2006

Quantitative ultrasound (QUS) methods have been introduced in recent years for the assessment of skeletal status in osteoporosis. The performance of QUS techniques has been evaluated in a large number of studies. Reviewing existing knowledge, an international expert panel formulated the following consensus regarding the current status of this technology. To date, evidence supports the use of QUS techniques for the assessment of fracture risk in elderly women. This has been best established for water-based calcaneal QUS systems. Future studies should include the predictive validity of other QUS systems. Additional clinical applications of QUS, specifically the assessment of rates of change for monitoring disease progression or response to treatment, require further investigation. Its low cost and portability make QUS an attractive technology for assessing risk of fractures in larger populations than may be suitable or feasible for bone densitometry. Additional investigations that assess innovative QUS techniques in well defined research settings are important to determine and utilize the full potential of this technology for the benefit of early detection and monitoring of osteoporosis.
Binding and Cyclic AMP Stimulation by N-Terminally Deleted Human PTHs (3–84 and 4–84) in a Homologous Ligand Receptor System
Authors: Olstad Ole K. , Reppe Sjur , Loseth Ole P. , Jemtland Rune , Gautvik Kaare M.
Published: April 2006

We have produced in yeast two human parathyroid hormone (hPTH) analogs with amino-terminal deletions, hPTH(3–84) and hPTH(4–84), employing the mating factor α (MFα) expression system. The authenticity of the polypeptides was demonstrated by amino-terminal analysis, amino acid composition, and molecular mass analysis. In cells (LLC-PK1) transfected with the human PTH/parathyroid hormone-related protein (PTHrP) receptor, using [125I-Tyr36]chickenPTHrP(1–36)NH2 as radioligand, binding studies revealed dissociation constants at equilibrium (Kd) for hPTH(3–84) and hPTH(4–84) of 4.7 and 8.0 nM, respectively, only slightly higher than natural recombinant hPTH(1–84) (Kd = 2.3 nM). In comparison, [Nle8,18,Tyr34]bovinePTH(3–34)NH2 and [Tyr36]cPTHrP(1–36)NH2 showed equal Kd's of 1.9 nM. Neither of the N-terminally deleted hPTH analogs showed any detectable stimulation of cAMP production in the cells at concentrations below 20 nM. At supersaturated concentrations (500 nM) with receptor occupancy of more than 95% these hPTH analogs revealed about 15% rest agonism compared with that of hPTH(1–84). hPTH(1–84) and [Tyr36]cPTHrP(1–36)NH2 showed an equal half maximal cyclic adenosine monophosphate (cAMP) stimulation of about 0.8 and 0.7 nM, respectively. The hPTH analogs did not show any ability to antagonize cellular cAMP production induced by either hPTH or [Tyr36]cPTHrP(1–36)NH2. [Nle8,18,Tyr34]bPTH(3–34)NH2 did also not antagonize cAMP stimulation by hPTH, but inhibited [Tyr36]cPTHrP(1–36)NH2-induced cAMP production by 40% when present at a 1000 M excess. These distinct results related to PTH and PTHrP from different species are important to consider in experiments evaluating potential hPTH or PTHrP antagonism, and employment of a hPTH/PTHrP receptor model is a requirement.
Macrophage Colony Stimulating Factor Down-Regulates MCSF-Receptor Expression and Entry of Progenitors into the Osteoclast Lineage
Authors: Fan Xian , Biskobing Diane M. , Fan Dongjie , Hofstetter Willy , Rubin Janet
Published: April 2006

Macrophage colony-stimulating factor (MCSF), although necessary for entry of precursors into the early preosteoclast pathway, inhibits osteoclastogenesis at high doses. To clarify the relationship between MCSF and osteoclast formation, we investigated the effect of exogenous MCSF in murine bone marrow culture. Precursor proliferation and the expression of MCSF-receptor were examined after 4 days of culture in the presence or absence of accessory stromal cells. In both mixed marrow and destromalized cell cultures, exogenous MCSF dose-dependently decreased125I-MCSF binding (by 65 ± 5.0% at 3500 and 87 ± 16.7% at 7000 U/ml, respectively) while enhancing mononuclear cell proliferation after 3 days of exposure (by 2.8- and 6.3-fold, respectively). These effects were maintained 24 h after removal of exogenous MCSF and, as such, likely represented an MCSF-induced change in MCSF receptor-bearing cells. Exposure to exogenous MCSF (3500 U/ml) days 2–4 dose-dependently inhibited tartrate resistant acid phosphatase positive multinuclear cell (TRAP+ MNC) formation counted at the end of day 7, by 64.3 ± 4.1%. This inhibition of TRAP+ MNC formation was preceded by a 92 ± 9% decrease in the expression of carbonic anhydrase II mRNA measurable at 4 days. These results indicate that MCSF promotes proliferation of a population of cells expressing lower cognate receptor sites. Changes in MCSF-receptor expression appear to modulate the final lineage selection of the pluripotent monoblastic progenitor.
Aromatase in Human Bone Tissue
Authors: Sasano Hironobu , Uzuki Miwa , Sawai Takashi , Nagura Hiroshi , Matsunaga Gen , Kashimoto Osamu , Harada Nobuhiro
Published: April 2006

Peripheral aromatization of androgens exert estrogenic actions in many tissues. Recently in situ production of estrogens by aromatase was detected in human bone and cultured osteoblasts and has been proposed to participate in the maintenance of bone mass. We examined aromatase expression by immunohistochemistry and mRNA in situ hybridization in 16 cases of tibia (female 2 male, 14 female, 62 ± 5.2 years old) and quantified the level of aromatase mRNA in 28 cases of rib, femur, and lumbar vertebrae (16 male, 12 female, 58.0 ± 11.3 years old) by reverse transcriptase-polymerase chain reaction (RT-PCR) in order to study whether or not and in which cell types aromatase was expressed in human bone tissues. We also studied alternative use of multiple exons 1 of its gene and immunolocalization of type I 17β-hydroxysteroid dehydrogenase (HSD), which converts estrone produced by aromatase to estradiol. Strong aromatase immunoreactivity and mRNA hybridization as well as type I 17β-HSD immunoreactivity were detected in lining cells, osteoblasts, chondrocytes of articular cartilage, and adipocytes adjacent to bone trabeculae in all the cases examined. Amounts of aromatase mRNA varied greatly among the subjects (11.25 ± 9.77, 0.61–42.84 attomol/ng of total RNA). The amount of aromatase expression was not correlated with age or gender of the subjects but positively correlated with the degree of osteroporotic changes evaluated by radiological findings of lumbar vertebrae. Analysis of multiple exons 1 revealed that 1b or fibroblast type was predominantly (23/26) utilized as a promoter of aromatase gene expression. These results demonstrated that aromatase is expressed widely in human bone tissue and may play important roles in maintenance of human bone tissue.