bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Genistein and Hormone-Replacement Therapy on Bone Loss in Early Postmenopausal Women: A Randomized Double-Blind Placebo-Controlled Study
Authors: Morabito Nunziata , Crisafulli Alessandra , Vergara Caterina , Gaudio Agostino , Lasco Antonino , Frisina Nicola , D'Anna Rosario , Corrado Francesco , Pizzoleo Maria Antonia , Cincotta Maria , Altavilla Domenica , Ientile Riccardo , Squadrito Francesco
Published: April 2006

The natural isoflavone phytoestrogen genistein has been shown to stimulate osteoblastic bone formation, inhibit osteoclastic bone resorption, and prevent bone loss in ovariectomized rats. However, no controlled clinical trial has been performed so far to evaluate the effects of the phytoestrogen on bone loss in postmenopausal women. We performed a randomized double-blind placebo-controlled study to evaluate and compare with hormone-replacement therapy (HRT) the effect of the phytoestrogen genistein on bone metabolism and bone mineral density (BMD) in postmenopausal women. Participants were 90 healthy ambulatory women who were 47–57 years of age, with a BMD at the femoral neck of <0.795 g/cm2. After a 4-week stabilization on a standard fat-reduced diet, participants of the study were randomly assigned to receive continuous HRT for 1 year (n = 30; 1 mg of 17β-estradiol [E2] combined with 0.5 mg of norethisterone acetate), the phytoestrogen genistein (n = 30; 54 mg/day), or placebo (n = 30). Urinary excretion of pyridinoline (PYR) and deoxypyridinoline (DPYR) was not significantly modified by placebo administration either at 6 months or at 12 months. Genistein treatment significantly reduced the excretion of pyridinium cross-links at 6 months (PYR = −54 ± 10%; DPYR = −55 ± 13%; p < 0.001) and 12 months (PYR = −42 ± 12%; DPYR = −44 ± 16%; p < 0.001). A similar and not statistically different decrease in excretion of pyridinium cross-links was also observed in the postmenopausal women randomized to receive HRT. Placebo administration did not change the serum levels of the bone-specific ALP (B-ALP) and osteocalcin (bone Gla protein [BGP]). In contrast, administration of genistein markedly increased serum B-ALP and BGP either at 6 months (B-ALP = 23 ± 4%; BGP = 29 ± 11%; p < 0.005) or at 12 months (B-ALP = 25 ± 7%; BGP = 37 ± 16%; p < 0.05). Postmenopausal women treated with HRT had, in contrast, decreased serum B-ALP and BGP levels either at 6 months (B-ALP = −17 ± 6%; BGP = −20 ± 9%; p < 0.001) or 12 months (B-ALP = −20 ± 5%; BGP = −22 ± 10%; p < 0.001). Furthermore, at the end of the experimental period, genistein and HRT significantly increased BMD in the femur (femoral neck: genistein = 3.6 ± 3%, HRT = 2.4 ± 2%, placebo = −0.65 ± 0.1%, and p < 0.001) and lumbar spine (genistein = 3 ± 2%, HRT = 3.8 ± 2.7%, placebo = −1.6 ± 0.3%, and p < 0.001). This study confirms the genistein-positive effects on bone loss already observed in the experimental models of osteoporosis and indicates that the phytoestrogen reduces bone resorption and increases bone formation in postmenopausal women.
Decreased Bone Density, Elevated Serum Osteoprotegerin, and β-Cross-Laps in Wilson Disease
Authors: Hegedus D. , Ferencz V. , Lakatos P. L. , Meszaros S. , Lakatos P. , Horvath C. , Szalay F.
Published: April 2006

Osteopathia has been reported in Wilson disease (WD), but bone density has not been measured; therefore, we performed bone mineral density (BMD), bone mineral content (BMC), and quantitative bone ultrasound (QUS) assessments, as well as measured the serum levels of osteocalcin (OCN), β-cross-laps (β-CTx's), and the recently discovered osteoprotegerin (OPG) and its ligand RANKL to investigate the underlying mechanism of osseous disorders. Serum OCN, β-CTx, OPG, and RANKL levels were measured by ELISA in 21 WD patients and in 20 age- and gender-matched healthy subjects. BMD, BMC, and QUS parameters were also determined. Osteoporosis was present in 9/21 (43%) WD patients. Abnormal QUS parameters were found in 7 (33%) of the patients. Although serum OCN levels were similar in patients and controls (29.93 ± 24.65 mg/ml vs. 29.84 ± 6.89 mg/ml), β-CTx and OPG levels were significantly increased in WD compared with the healthy controls (625.4 ± 312.3 pg/ml vs. 423.6 ± 144.3 pg/ml and p = 0.022 and 7.2 ± 3.4 pM vs. 3.5 ± 1.0 pM and p < 0.001, respectively). No difference was observed in the RANKL level. There was a positive correlation between OCN and β-CTx (r = 0.55; p = 0.01). We proved high occurrence of osteoporosis in WD. Negative bone remodeling balance is a consequence of increased bone resorption, which is indicated by elevated β-CTx. The novel finding of elevated serum OPG may reflect a compensatory reaction to enhanced osteoclast activity, despite the normal OCN level.
Pleiotrophin/Osteoblast-Stimulating Factor 1: Dissecting Its Diverse Functions in Bone Formation
Authors: Tare Rahul S. , Oreffo Richard O. C. , Clarke Nicholas M. P. , Roach Helmtrud I.
Published: April 2006

OSF-1, more commonly known as pleiotrophin (PTN) or heparin-binding growth-associated molecule (HB-GAM), belongs to a new family of secreted HB proteins, which are structurally unrelated to any other growth factor family. The aims of this study were to dissect the diverse functions of PTN in bone formation. The study showed that PTN was synthesized by osteoblasts at an early stage of osteogenic differentiation and was present at sites of new bone formation, where PTN was stored in the new bone matrix. Low concentrations (10 pg/ml) of PTN stimulated osteogenic differentiation of mouse bone marrow cells and had a modest effect on their proliferation, whereas higher concentrations (ng/ml) had no effect. However, PTN did not have the osteoinductive potential of bone morphogenetic proteins (BMPs) because it failed to convert C2C12 cells, a premyoblastic cell line, to the osteogenic phenotype, whereas recombinant human BMP-2 (rhBMP-2) was able to do so. When PTN was present together with rhBMP-2 during the osteoinductive phase, PTN inhibited the BMP-mediated osteoinduction in C2C12 cells at concentrations between 0.05 pg/ml and 100 ng/ml. However, when added after osteoinduction had been achieved, PTN enhanced further osteogenic differentiation. An unusual effect of PTN (50 ng/ml) was the induction of type I collagen synthesis by chondrocytes in organ cultures of chick nasal cartilage and rat growth plates. Thus, PTN had multiple effects on bone formation and the effects were dependent on the concentration of PTN and the timing of its presence. To explain these multiple effects, we propose that PTN is an accessory signaling molecule, which is involved in a variety of processes in bone formation. PTN enhances or inhibits primary responses depending on the prevailing concentrations, the primary stimulus, and the availability of appropriate receptors.
A Case for Strain-Induced Fluid Flow as a Regulator of BMU-Coupling and Osteonal Alignment
Authors: Smit Theo H. , Burger Elisabeth H. , Huyghe Jacques M.
Published: April 2006

Throughout life, human bone is renewed continuously in a tightly controlled sequence of resorption and formation. This process of bone remodeling is remarkable because it involves cells from different lineages, collaborating in so-called basic multicellular units (BMUs) within small spatial and temporal boundaries. Moreover, the newly formed (secondary) osteons are aligned to the dominant load direction and have a density related to its magnitude, thus creating a globally optimized mechanical structure. Although the existence of BMUs is amply described, the cellular mechanisms driving bone remodeling—particularly the alignment process—are poorly understood. In this study we present a theory that explains bone remodelling as a self-organizing process of mechanical adaptation. Osteocytes thereby act as sensors of strain-induced fluid flow. Physiological loading produces stasis of extracellular fluid in front of the cutting cone of a tunneling osteon, which will lead to osteocytic disuse and (continued) attraction of osteoclasts. However, around the resting zone and the closing cone, enhanced extracellular fluid flow occurs, which will activate osteocytes to recruit osteoblasts. Thus, cellular activity at a bone remodeling site is well related to local fluid flow patterns, which may explain the coordinated progression of a BMU.
The Role of Interstitial Fluid Flow in the Remodeling Response to Fatigue Loading
Authors: Tami A. E. , Nasser P. , Verborgt O. , Schaffler M. B. , Tate M. L. Knothe
Published: April 2006

Load-induced fluid flow enhances molecular transport through bone tissue and relates to areas of bone resorption and apposition. Remodeling activity is highly coordinated and necessitates a means for cellular communication via intracellular and extracellular means. Osteocytes, osteoblasts, and osteoclasts, which reside in disparate locations within the tissue, communicate intracellularly via the cellular syncytium and extracellularly via the pericellular fluid space of the lacunocanalicular system. Both of these communications systems are physically disrupted by microdamage incurred during fatigue loading of bone. The purpose of this study was to develop an analytical model to understand the role of interstitial fluid flow in the remodeling response to fatigue loading. Adequate transport was assumed a prerequisite for maintenance of cell viability in bone. Diffusive and convective transport were simulated through the lacunocanalicular network in a healthy undamaged state as well as in a damaged state after fatigue loading. The model predicts that fatigue damage impedes transport from the blood supply, depleting the concentration of molecular entities in and downstream from areas of damage. Furthermore, the presence of microcracks alters the distribution of molecular entities between individual lacunae. These effects were confirmed by the results of an in vivo pilot study in which fluorescent, flow-visualizing agents pooled within microcracks and were absent from areas surrounding microcracks, corresponding to areas deprived of fluid flow. Loss of osteocyte viability is coupled to targeting and initiation of new remodeling activity. Taken as a whole, these data suggest a link between interstitial fluid flow, mass transport, maintenance of osteocyte viability, and modulation of remodeling activity.
MLO-Y4 Osteocyte-Like Cells Support Osteoclast Formation and Activation
Authors: Zhao S. , Kato Y. , Zhang Y. , Harris S. , Ahuja S. S. , Bonewald L. F.
Published: April 2006

Osteocytes are terminally differentiated cells of the osteoblast lineage that have become embedded in mineralized matrix and may send signals that regulate bone modeling and remodeling. The hypothesis to be tested in this study is that osteocytes can stimulate and support osteoclast formation and activation. To test this hypothesis, an osteocyte-like cell line called MLO-Y4 and primary murine osteocytes were used in coculture with spleen or marrow cells. MLO-Y4 cells support osteoclast formation in the absence of 1,25-dihydroxyvitamin D3 [1,25(OD)2D3] or any other exogenous osteotropic factor. These cells alone stimulate osteoclast formation to the same extent or greater than adding 1,25(OH)2D3. Coaddition of 1,25(OH)2D3 with MLO-Y4 cells synergistically increased osteoclast formation. Optimal osteoclast formation and pit formation on dentine was observed with 200–1000 MLO-Y4 cells per 0.75-cm2 well. No osteoclast formation was observed with 2T3, OCT-1, or MC3T3-E1 osteoblast cells (1000 cells/well). Conditioned media from the MLO-Y4 cells had no effect on osteoclast formation, indicating that cell contact is necessary. Serial digestions of 2-week-old mouse calvaria yielded populations of cells that support osteoclast formation when cocultured with 1,25(OH)2D3 and marrow, but the population that remained in the bone particles supported the greatest number of osteoclasts with or without 1,25(OH)2D3. To examine the mechanism whereby these cells support osteoclast formation, the MLO-Y4 cells were compared with a series of osteoblast and stromal cells for expression of macrophage colony-stimulating factor (M-CSF), RANKL, and osteoprotegerin (OPG). MLO-Y4 cells express and secrete large amounts of M-CSF. MLO-Y4 cells express RANKL on their surface and their dendritic processes. The ratio of RANKL to OPG mRNA is greatest in the MLO-Y4 cells compared with the other cell types. RANK-Fc and OPG-Fc blocked the formation of osteoclasts by MLO-Y4 cells. These studies suggest that both RANKL and OPG may play a role in osteocyte signaling, OPG and M-CSF as soluble factors and RANKL as a surface molecule that is functional in osteocytes or along their exposed dendritic processes.
Evidence From the Aged Orchidectomized Male Rat Model That 17β-Estradiol Is a More Effective Bone-Sparing and Anabolic Agent Than 5α-Dihydrotestosterone
Authors: Vandenput L. , Boonen S. , Van Herck E. , Swinnen J. V. , Bouillon R. , Vanderschueren D.
Published: April 2006

This study was designed to evaluate the impact of estrogen versus androgen action on orchidectomy (ORX)-induced bone loss and associated changes in body composition. During an experimental period of 4 months, aged (12-month-old) ORX rats were treated with 17β-estradiol (E2; 0.75 μg/day) or different doses of the nonaromatizable androgen 5α-dihydrotestosterone (DHT; 45, 75, and 150 μg/day, respectively), via subcutaneous (sc) silastic implants. Low doses of DHT and E2 inhibited the ORX-induced rise of bone turnover markers (serum osteocalcin and urinary deoxypyridinoline [DPD]) to a similar extent. High-dose DHT prevented the ORX-induced decrease of trabecular bone density but had no significant effect on cortical thinning as assessed by peripheral quantitative computed tomography (pQCT). This bone-sparing action of DHT occurred at the expense of hypertrophy of the ventral prostate and seminal vesicles. On the other hand, E2 restored both trabecular bone density and cortical thickness in ORX rats and even prevented age-related bone loss. In contrast to DHT, E2 increased lean body mass and inhibited the ORX-associated increase of fat mass, as measured by DXA. Administration of E2 was associated with increased serum concentrations of insulin-like growth factor (IGF) I and decreased circulating levels of leptin. We conclude that, in the aged ORX rat model, E2 is more effective in preventing ORX-induced bone loss than DHT. Additionally, E2 has anabolic effects on muscle tissue and prevents the ORX-related increase of fat mass. Overall, these data suggest that androgen action on bone and body composition is dependent on stimulation of both androgen receptors (ARs) and estrogen receptors (ERs).
Leptin Inhibits Osteoclast Generation
Authors: Holloway Wayne R. , Collier Fiona McL. , Aitken Cathy J. , Myers Damian E. , Hodge Jason M. , Malakellis Mary , Gough Tamara J. , Collier Gregory R. , Prof. Nicholson Geoffrey C.
Published: April 2006

Originally, leptin was described as a product of adipocytes that acts on the hypothalamus to regulate appetite. However, subsequently, it has been shown that leptin receptors are distributed widely and that leptin has diverse functions, including promotion of hemopoietic and osteoblastic differentiation. It has been recognized for some time that both serum leptin and bone mass are correlated positively to body fat mass and, recently, we have shown a direct positive relationship between serum leptin and bone mass in nonobese women. We now report that leptin inhibits osteoclast generation in cultures of human peripheral blood mononuclear cells (PBMCs) and murine spleen cells incubated on bone in the presence of human macrophage colony-stimulating factor (hM-CSF) and human soluble receptor activator of NF-κB ligand (sRANKL). The half-maximal concentration inhibitory of leptin was approximately 20 nM in the presence of sRANKL at 40 ng/ml but decreased to approximately 2 nM when sRANKL was used at 5 ng/ml. The majority of the inhibitory effect occurred in the first week of the 3-week cultures. Inhibition did not occur when the PBMC cultures were washed vigorously to remove nonadherent cells or when purified CD14+ monocytes were used to generate osteoclasts, indicating an indirect or permissive effect via CD14− PBMC. Leptin increased osteoprotegerin (OPG) messenger RNA (mRNA) and protein expression in PBMC but not in CD14+ cells, suggesting that the inhibitory effect may be mediated by the RANKL/RANK/OPG system. Leptin may act locally to increase bone mass and may contribute to linkage of bone formation and resorption.
Chondrocytes Provide Morphogenic Signals That Selectively Induce Osteogenic Differentiation of Mesenchymal Stem Cells
Authors: Gerstenfeld Louis C. , Cruceta Johanna , Shea Colleen M. , Sampath Kuber , Barnes George L. , Einhorn Thomas A.
Published: April 2006

During endochondral bone development cartilage formation always precedes that of bone, leading to the hypothesis that chondrocytes provide inductive signals for osteogenesis. To test this hypothesis, C3H10T½ mesenchymal stem cells were cocultured in membrane separated trans-well culture chambers with nonhypertrophic chondrocytes, hypertrophic chondrocytes, calvaria osteoblasts, or tendon fibroblasts derived from embryonic chickens to assess if individual cell types would selectively promote osteogenic differentiation. Then, differentiation of C3H10T½ mesenchymal stem cells in coculture were compared with that induced by bone morphogenetic protein 7 or osteogenic protein-1 (BMP-7; OP-1) treatment. Osteogenesis, as determined by the expression of Cbfa1 and osteocalcin (OC) messenger RNAs (mRNAs), was induced strongly in C3H10T½ cells cocultured with both chondrocyte cell populations but was not induced by coculture with either osteoblasts or skin fibroblasts. Interestingly, treatment of C3H10T½ cells with BMP-7 induced both chondrogenesis and osteogenesis, and only osteogenic differentiation was observed in the C3H10T½ cells cocultured with chondrocytes. No alterations in the expression of mRNAs for BMP-1 to -8 were observed in the C3H10T½ cells under any of the coculture conditions. This shows that the induction of endogenous BMPs by coculture does not regulate osteogenesis in an autocrine manner. These results show that chondrocytes express soluble morphogenetic factors that selectively promote osteogenesis, and this selective effect is not mimicked by an exogenously added BMP.
Stimulation of Osteoblastic Cell Differentiation by Notch
Authors: Tezuka Ken-ICHI , Yasuda Masafumi , Watanabe Naoko , Morimura Naoko , Kuroda Kazuki , Miyatani Seiji , Hozumi Nobumichi
Published: April 2006

Notch is a transmembrane protein that plays a critical role in the determination of cellular differentiation pathways. Although its importance in the development of mesenchymal tissues has been suggested, its role in skeletal tissues has not been well investigated. Northern blot experiments showed the expression of Notch1 in MC3T3-E1 osteoblastic cells at early differentiation stages. When a Notch1 cytoplasmic domain (Notch-IC [NIC]) delivered by an adenovirus vector was expressed in osteoblastic MC3T3-E1 cells, a significant increase in calcified nodule formation was observed in long-term cultures. Activation of endogenous Notch in MC3T3-E1 by coculturing them with Delta-like-1 (Dll1)-expressing myeloma cells also resulted in a stimulation of calcified nodule formation. Not only affecting nodule formation, Notch activation also had effects on osteoblastic differentiation of multipotent mesenchymal cells. Osteoblastic differentiation of C3H10T1/2 cells induced by bone morphogenetic protein 2 (BMP-2) was significantly stimulated, whereas adipogenic differentiation was suppressed strongly, resulting in a dominant differentiation of osteoblastic cells. NIC expression in primary human bone marrow mesenchymal stem cells (hMSCs) also induced both spontaneous and stimulated osteoblastic cell differentiation. These observations suggest that osteoblastic cell differentiation is regulated positively by Notch and that Notch could be a unique and interesting target molecule for the treatment of osteoporosis.
Involvement of Phosphodiesterase Isozymes in Osteoblastic Differentiation
Authors: Wakabayashi Shinji , Tsutsumimoto Takahiro , Kawasaki Satoshi , Kinoshita Tetsuya , Horiuchi Hiroshi , Takaoka Kunio
Published: April 2006

The cyclic monophosphate nucleotides (cyclic adenosine monophosphate [cAMP] and cyclic guanosine monophosphate [cGMP]) are found ubiquitously in mammalian cells and act as second messenger transducers to effect the intracellular actions of a variety of hormones, cytokines, and neurotransmitters. In turn, these nucleotides also modulate the signal transduction processes regulated by a range of cytokines and growth factors. Previously, we have reported that pentoxifylline, a nonselective phosphodiesterase (PDE) inhibitor, can promote osteoblastic differentiation by elevating intracellular cAMP levels and, consequently, enhance bone formation in vivo and in vitro. In this study, reverse-transcription polymerase chain reaction (RT-PCR) analysis of the osteoblastic cell lines, MC3T3-E1 and ST2 revealed the presence of PDE1, PDE2, PDE3, PDE4, PDE7, PDE8, and PDE9. We examined the effect of selective inhibitors for a respective PDE isozyme on the capacity of bone morphogenetic protein 4 (BMP-4)-induced alkaline phosphatase (ALP) activity, a cellular differentiation marker, in cells with osteogenetic potential. The results indicate that selective inhibitors for PDE2, PDE3, and PDE4 enhanced the BMP-4-induced ALP activity in a dose-dependent manner in ST2 cells but not in MC3T3-E1 cells. Northern blot analysis also revealed that the selective inhibitors for PDE2, PDE3, and PDE4 enhanced the levels of expression of messenger RNAs (mRNAs) of ALP, osteopontin (OP), and collagen type I in ST2 cells but not in MC3T3-E1 cells except for the treatment with PDE4 inhibitor. Given these data, we conclude that PDE isozymes are involved in the modulation of osteoblastic differentiation mainly at an early stage. Additionally, selective inhibitors for PDE2, PDE3, and PDE4 appear to promote the differentiation of osteogenic precursor cells toward an osteoblastic phenotype.
Fluid Flow Induction of Cyclo-Oxygenase 2 Gene Expression in Osteoblasts Is Dependent on an Extracellular Signal-Regulated Kinase Signaling Pathway
Authors: Wadhwa Sunil , Godwin Stephen L. , Peterson Donald R. , Epstein Mary A. , Raisz Lawrence G. , Pilbeam Carol C.
Published: April 2006

Mechanical loading of bone may be transmitted to osteocytes and osteoblasts via shear stresses at cell surfaces generated by the flow of interstitial fluid. The stimulated production of prostaglandins, which mediates some effects of mechanical loading on bone, is dependent on inducible cyclo-oxygenase 2 (COX-2) in bone cells. We examined the fluid shear stress (FSS) induction of COX-2 gene expression in immortalized MC3T3-E1 osteoblastic cells stably transfected with −371/+70 base pairs (bp) of the COX-2 5′-flanking DNA (Pluc371) and in primary osteoblasts (POBs) from calvaria of mice transgenic for Pluc371. Cells were plated on collagen-coated glass slides and subjected to steady laminar FSS in a parallel plate flow chamber. FSS, from 0.14 to10 dynes/cm2, induced COX-2 messenger RNA (mRNA) and protein. FSS (10 dynes/cm2) induced COX-2 mRNA within 30 minutes, with peak effects at 4 h in MC3T3-E1 cells and at ≥8 h in POBs. An inhibitor of new protein synthesis puromycin blocked the peak induction of COX-2 mRNA by FSS. COX-2 promoter activity, measured as luciferase activity, correlated with COX-2 mRNA expression in both MC3T3-E1 and POB cells. FSS induced phosphorylation of extracellular signal-regulated kinase (ERK) in MC3T3-E1 cells, with peak effects at 5 minutes. Inhibiting ERK phosphorylation with the specific inhibitor PD98059 inhibited FSS induction of COX-2 mRNA by 55-70% and FSS stimulation of luciferase activity by ≥80% in both MC3T3-E1 and POB cells. We conclude that FSS transcriptionally induces COX-2 gene expression in osteoblasts, that the maximum induction requires new protein synthesis, and that induction occurs largely via an ERK signaling pathway.
A Role for the BMP Antagonist Chordin in Endochondral Ossification
Authors: Zhang Donghui , Ferguson Cristin M. , O'Keefe Regis J. , Puzas J. Edward , Rosier Randy N. , Reynolds Paul R.
Published: April 2006

Bone morphogenetic proteins (BMPs) are ubiquitous regulators of cellular growth and differentiation. A variety of processes modulate BMP activity, including negative regulation by several distinct binding proteins. One such BMP antagonist chordin has a role in axis determination and neural induction in the early embryo. In this study, a role for chordin during endochondral ossification has been investigated. During limb development, Chordin expression was detected only at the distal ends of the skeletal elements. In cultured embryonic sternal chondrocytes, Chordin expression was related inversely to the stages of maturation. Further, treating cultured chondrocytes with chordin interfered with maturation induced by treatment with BMP-2. These results suggest that chordin may negatively regulate chondrocyte maturation and limb growth in vivo. To address this hypothesis, chordin protein was expressed ectopically in Hamburger-Hamilton (HH) stage 25-27 embryonic chick limbs. The phenotypic changes and alteration of gene expression in treated limbs revealed that overexpression of chordin protein delayed chondrocyte maturation in developing skeletal elements. In summary, these findings strongly support a role for chordin as a negative regulator of endochondral ossification.
Age-Related Osteoporosis in Biglycan-Deficient Mice Is Related to Defects in Bone Marrow Stromal Cells
Authors: Chen Xiao-Dong , Shi Songtao , Xu Tianshun , Robey Pamela Gehron , Young Marian F.
Published: April 2006

Biglycan (bgn) is an extracellular matrix proteoglycan that is enriched in bone and other skeletal connective tissues. Previously, we generated bgn-deficient mice and showed that they developed age-dependent osteopenia. To identify the cellular events that might contribute to this progressive osteoporosis, we measured the number of osteogenic precursors in the bone marrow of normal and mutant mice. The number of colonies, indicative of the colony-forming unit potential of fibroblasts (CFU-F), gradually decreased with age. By 24 weeks of age, colony formation in the bgn knockout (KO) mice was significantly more reduced than that in the wild type (wt) mice. This age-related reduction was consistent with the extensive osteopenia previously shown by X-ray analysis and histological examination of 24-week-old bgn KO mice. Because bgn has been shown previously to bind and regulate transforming growth factor β (TGF-β) activity, we also asked whether this growth factor would affect colony formation. TGF-β treatment significantly increased the size of the wt colonies. In contrast, TGF-β did not significantly influence the size of the bgn colonies. An increase in apoptosis in bgn-deficient bone marrow stromal cells (BMSCs) was observed also. The combination of decreased proliferation and increased apoptosis, if it occurred in vivo, would lead to a deficiency in the generation of mature osteoblasts and would be sufficient to account for the osteopenia developed in the bgn KO mice. The bgn KO mice also were defective in the synthesis of type I collagen messenger RNA (mRNA) and protein. This result supports the suggestion that the composition of the extracellular matrix may be regulated by specific matrix components including bgn.
Bone Histomorphometric and Biochemical Marker Results of a 2-Year Placebo-Controlled Trial of Raloxifene in Postmenopausal Women
Authors: Ott Susan M. , Oleksik Anna , Lu Yili , Harper Kristine , Lips Paul
Published: April 2006

Raloxifene is a selective estrogen receptor modulator that has been shown to increase bone density. The purpose of this study was to examine the effects of raloxifene on bone tissue by studying bone biopsy specimens before and after 2 years of raloxifene or placebo therapy. The women in this study were participants of the double-blind, placebo-controlled, multicenter study, the Multiple Outcomes of Raloxifene Evaluation (MORE) trial. Subjects from two U.S. sites and two European sites were included if they consented to a bone biopsy. Iliac crest bone biopsies were performed at baseline and after 2 years. Tetracycline labeling preceded each biopsy. A total of 65 paired biopsy specimens were evaluated with 25, 22, and 18 patients in the placebo, raloxifene HCl (60 mg) and raloxifene HCl (120 mg) treatment groups, respectively. They were analyzed using standard histomorphometry. None of the biopsy specimens showed evidence of toxic effects on bone or bone cells or met criteria for osteomalacia. Biopsy specimens in the placebo and raloxifene groups had the appearance of normal bone, with no evidence of marrow fibrosis or increases in the amount of woven bone or numbers of empty osteocyte lacunae. Compared with the baseline, the bone formation rate (BFR) decreased significantly in both raloxifene groups. The change in BFR in the group treated with 120 mg of raloxifene was −62.3%, which was significantly lower than the change in the placebo group of −21.0% (p = 0.03). No change in resorption parameters could be measured by histomorphometry, but there was a decrease in urinary type I collagen excretion. The results from this study suggest that raloxifene has actions on bone tissue that are similar to those observed with estrogen. The depressive effects on bone remodeling are less marked than the effects seen with alendronate.
Quantity and Quality of Trabecular Bone in the Femur Are Enhanced by a Strongly Anabolic, Noninvasive Mechanical Intervention
Authors: Rubin Clinton , Turner A. Simon , Müller Ralph , Mittra Erik , McLeod Kenneth , Lin Wei , Qin Yi-Xian
Published: April 2006

The skeleton's sensitivity to mechanical stimuli represents a critical determinant of bone mass and morphology. We have proposed that the extremely low level (<10 microstrain), high frequency (20-50 Hz) mechanical strains, continually present during even subtle activities such as standing are as important to defining the skeleton as the larger strains typically associated with vigorous activity (>2000 microstrain). If these low-level strains are indeed anabolic, then this sensitivity could serve as the basis for a biomechanically based intervention for osteoporosis. To evaluate this hypothesis, the hindlimbs of adult female sheep were stimulated for 20 minutes/day using a noninvasive 0.3g vertical oscillation sufficient to induce approximately 5 microstrain on the cortex of the tibia. After 1 year of stimulation, the physical properties of 10-mm cubes of trabecular bone from the distal femoral condyle of experimental animals (n = 8) were compared with controls (n = 9), as evaluated using microcomputed tomography (μCT) scanning and materials testing. Bone mineral content (BMC) was 10.6% greater (p < 0.05), and the trabecular number (Tb.N) was 8.3% higher in the experimental animals (p < 0.01), and trabecular spacing decreased by 11.3% (p < 0.01), indicating that bone quantity was increased both by the creation of new trabeculae and the thickening of existing trabeculae. The trabecular bone pattern factor (TBPf) decreased 24.2% (p < 0.03), indicating trabecular morphology adapting from rod shape to plate shape. Significant increases in stiffness and strength were observed in the longitudinal direction (12.1% and 26.7%, respectively; both, p < 0.05), indicating that the adaptation occurred primarily in the plane of weightbearing. These results show that extremely low level mechanical stimuli improve both the quantity and the quality of trabecular bone. That these deformations are several orders of magnitude below those peak strains which arise during vigorous activity indicates that this biomechanically based signal may serve as an effective intervention for osteoporosis.
Two New Single-Nucleotide Polymorphisms in the COL1A1 Upstream Regulatory Region and Their Relationship to Bone Mineral Density
Authors: Garcia-Giralt Natalia , Nogués Xavier , Enjuanes Anna , Puig Jordi , Mellibovsky Leonardo , Bay-Jensen Anne , Carreras Ramon , Balcells Susana , Díez-Pérez Adolfo , Grinberg Daniel
Published: April 2006

Single-nucleotide polymorphisms (SNPs) in regulatory regions of candidate genes may determine variability in bone mineral density (BMD) because they may be responsible for differences in levels of a gene product in response to external signals. Under this hypothesis, we scanned an 800-base pair (bp) region within the COL1A1 promoter, known to harbor cis elements important for in vivo expression, and we found two new polymorphisms: −1663indelT and −1997 G/T. The G to T transversion at −1997 was associated with lumbar spine BMD (p = 0.015) when tested in a cohort of 256 postmenopausal women after adjusting by age, body weight, and years since menopause; a lower degree of association was detected also for femoral neck BMD in a subgroup of 146 women in univariate analysis and after adjusting by age (p = 0.044). The polymorphism −1663indelT, which corresponds to a deletion of a T in a tract of eight T residues (−1670 to −1663), did not show significant association with BMD. Interestingly, −1663indelT is in strong linkage disequilibrium (LD) with the previously described Sp1 polymorphism of intron 1, which in this study did not show association with BMD either. Significant interaction between −1997 G/T and −1663indelT (p = 0.019), and between −1997 G/T and Sp1 (p = 0.045) was observed also. Individuals heterozygous for the three polymorphisms showed the highest mean BMD value. Gel retardation assays showed that oligonucleotides containing either the −1663 or the −1997 polymorphic sites specifically bind primary osteoblast nuclear proteins. We named these binding sites as PCOL1 and PCOL2, respectively. In summary, this study describes two new SNPs in the COL1A1 promoter, which may affect bone mass determination.
The Uptake of Lipoprotein-Borne Phylloquinone (Vitamin K1) by Osteoblasts and Osteoblast-Like Cells: Role of Heparan Sulfate Proteoglycans and Apolipoprotein E
Authors: Newman Paul , Bonello Fiona , Wierzbicki Anthony S. , Lumb Peter , Savidge Geoffrey F. , Shearer Martin J.
Published: April 2006

Vitamin K is essential for the γ-carboxylation of Gla-containing bone proteins such as osteocalcin and a suboptimal vitamin K status has been linked to osteoporosis but nothing is known of how the lipoprotein-borne vitamin accesses the bone matrix. We have studied the mechanism of transport of lipoproteins labeled with [3H]-phylloquinone (vitamin K1 [K1]) into osteoblasts using both tumor-derived cell lines and normal osteoblast-rich cell populations. We also investigated the effect of heparin in this model since long-term heparin treatment causes osteopenia and the anticoagulant is known to impair normal lipoprotein metabolism. Heparinase treatment, which removes heparan sulfate proteoglycans (HSPG), reduced uptake of [3H]-K1 from triglyceride-rich lipoproteins (TRL) and low-density lipoproteins (LDL). The effect of heparin in this model was complex depending on cell type, concentration, and time but, overall, the results were consistent with an inhibition of vitamin K uptake by osteoblasts. Anti-apolipoprotein E (apoE) antiserum reduced uptake of TRL-[3H]-K1 by 55 ± 4% and LDL-[3H]-K1 uptake by 35 ± 2%. Exogenous apoE4 increased uptake of TRL-[3H]-K1 by 90 ± 1% compared with 53 ± 11% for apoE3 and 52 ± 5% for apoE2. Our findings show that HSPG on the cell surface and apoE in the lipoprotein particles contribute to lipoprotein-K1 uptake by osteoblasts as is known for lipoprotein uptake by hepatocytes. This mechanism is significant in view of the epidemiological association of both undercarboxylation of osteocalcin and the presence of an apoϵ4 allele with increased fracture risk and reduced bone mineral density (BMD). The inhibition by heparin of lipoprotein-mediated carriage of vitamin K and possibly other lipids to bone may provide a basis for the future understanding of heparin-induced osteoporosis.
Evidence That Peroxynitrite Affects Human Osteoblast Proliferation and Differentiation
Authors: Da Rocha Francisco Airton Castro , De Brum-Fernandes Artur José
Published: April 2006

Peroxynitrite (PN), a nitric oxide (NO·)-derived anion, has been associated with NO· damage in various cell types. We examined the effects of adding PN to cultured human osteoblast-like (hOB) cells obtained after hip arthroplasty. Exposure to PN (0.1-0.4 mM) decreased both hOB proliferation and differentiation, measured by [3H]thymidine uptake and alkaline phosphatase production, respectively. Incubation with 3-morpholinosydnonimine (SIN-1; 0.25-1 mM), an NO· and O2− donor that leads to PN release, also reduced both hOB proliferation and differentiation. Coincubation with both superoxide dismutase (SOD; 100 U/ml) and catalase (CAT; 50 U/ml), rendering SIN-1 a pure NO· donor, reversed its effects on hOB proliferation and differentiation. However, SIN-1-induced NO· production, measured by nitrite release to the hOB medium, was not altered by cotreatment with SOD and CAT. Expression of nitrotyrosine by hOB, a marker of PN action, was significantly increased after SIN-1 addition, as compared with untreated cells, as revealed by Western blot analysis. Interleukin-1α (IL-1α) and interferon γ (IFN-γ) but not tumor necrosis factor α (TNF-α) also significantly increased nitrotyrosine expression in these cells. These data show that PN is at least partially responsible for osteoblast derangement by NO· and that cytokines released during inflammatory arthropathies can induce PN production in hOB cells.
Nuclear Localization of Type I Parathyroid Hormone/Parathyroid Hormone-Related Protein Receptors in Deer Antler Osteoclasts: Evidence for Parathyroid Hormone-Related Protein and Receptor Activator of NF-κB-Dependent Effects on Osteoclast Formation in Regenerating Mammalian Bone
Authors: Faucheux C. , Horton M. A. , Price J. S.
Published: April 2006

Parathyroid hormone-related protein (PTHrP) is not required for osteoclastogenesis during embryonic development; however, after birth it has been shown to regulate osteoclast formation during tooth eruption. Our study explores the hypothesis that PTHrP also may regulate osteoclast differentiation in the regenerating skeletal tissues of deer antlers, bones capable of complete regeneration. Osteoclast-like multinucleated cells (MNCs) formed spontaneously in micromass cultures derived from antler cartilage and these cells had the phenotypic characteristics of osteoclasts. PTHrP and receptor activator of NF-κB ligand (RANKL) stimulated antler osteoclast formation although the effect of RANKL was less marked than that of PTHrP. The addition of osteoprotegerin (OPG) only partially decreased (by ∼65%) the number of osteoclasts in PTHrP-treated cultures. To determine whether PTHrP also potentially could have direct effects on antler osteoclasts, we studied, by confocal microscopy, the expression of the type I PTH/PTHrP receptor (PTH1R) in MNCs cultured on glass and found the receptor protein to have a nuclear localization. In situ hybridization showed that antler MNCs also expressed PTH1R and PTHrP messenger RNAs (mRNAs). PTHrP was immunolocalized in MNCs cultured on glass but was undetectable in cells resorbing a dentine substrate. In tissue sections of antler cartilage, PTHrP and PTH1R were expressed in vitronectin receptor-positive (VNR+) osteoclast-like cells localized in the perivascular stroma. Thus, these data show that PTHrP plays a role in the regulation of osteoclast differentiation in regenerating skeletal tissues and that PTHrP can have effects on osteoclastogenesis that are independent of RANKL synthesis. Ours is the first study to describe the expression of the type I PTH/PTHrP receptor in mammalian osteoclasts at a protein and mRNA level, which indicates that PTHrP also may have a direct effect on osteoclasts. This also is the first study to show a nuclear localization of the PTHIR in cells of the osteoclast lineage, although the functional significance of this observation has yet to be established.
Incidence and Natural History of Paget's Disease of Bone in England and Wales
Authors: Van Staa T. P. , Selby P. , Leufkens H. G. M. , Lyles K. , Sprafka J. M. , Prof. Cooper C.
Published: April 2006

This study used a large, primary care, record-linkage resource (the General Practice Research Database [GPRD]) to evaluate the incidence, clinical presentation, and natural history of Paget's disease of bone in England and Wales. Between 1988 and 1999, we identified 2465 patients with the recorded diagnosis of Paget's disease of bone, within the five million subjects ≥18 years old who were registered in the GPRD. The validity of diagnostic recording was assessed by questionnaire to individual general practitioners (GPs) in 150 patients; the diagnosis was confirmed in 93.8% of responders. The mean age of patients with Paget's disease was 75 years and 51% were men. The prevalence of the disorder was 0.3% among men and women aged ≥55 years; incidence rates for clinically diagnosed Paget's disease rose steeply with age (men, 5 per 10,000 person-years; women, 3 per 10,000 person-years at the age of 75 years). Over the 11-year period of the study, the age- and sex-adjusted incidence rate of clinically diagnosed Paget's disease declined from 1.1 per 10,000 person-years to 0.7 per 10,000 person-years. Each patient with Paget's disease was matched to three controls matched by age, gender, and general practice. Cases had a greater risk of back pain (relative risk [RR], 2.1; 95% CI, 1.9-2.3), osteoarthritis (OA; RR, 1.7; 95% CI, 1.5-1.9), hip arthroplasty (RR, 3.1; 95% CI, 2.4-4.1), knee arthroplasty (RR, 1.6; 95% CI, 1.0-2.6), fracture (RR, 1.2; 95% CI, 1.0-1.5), and hearing loss (RR, 1.6; 95% CI, 1.3-1.9). Seven patients with Paget's disease developed a malignant bone neoplasm (0.3%). Using life table methodology, the estimated number of people who died within 5 years of follow-up was 32.7% among the patients with Paget's disease and 28.0% among the control patients.
Nuclear Magnetic Resonance Spin-Spin Relaxation of the Crystals of Bone, Dental Enamel, and Synthetic Hydroxyapatites
Authors: Wu Yaotang , Ackerman Jerome L. , Kim Hyun-Man , Rey Christian , Barroug Allal , Glimcher Melvin J.
Published: April 2006

Studies of the apatitic crystals of bone and enamel by a variety of spectroscopic techniques have established clearly that their chemical composition, short-range order, and physical chemical reactivity are distinctly different from those of pure hydroxyapatite. Moreover, these characteristics change with aging and maturation of the bone and enamel crystals. Phosphorus-31 solid state nuclear magnetic resonance (NMR) spin-spin relaxation studies were carried out on bovine bone and dental enamel crystals of different ages and the data were compared with those obtained from pure and carbonated hydroxyapatites. By measuring the31P Hahn spin echo amplitude as a function of echo time, Van Vleck second moments (expansion coefficients describing the homonuclear dipolar line shape) were obtained and analyzed in terms of the number density of phosphorus nuclei.31P magnetization prepared by a 90° pulse or by proton-phosphorus cross-polarization (CP) yielded different second moments and experienced different degrees of proton spin-spin coupling, suggesting that these two preparation methods sample different regions, possibly the interior and the surface, respectively, of bone mineral crystals. Distinct differences were found between the biological apatites and the synthetic hydroxyapatites and as a function of the age and maturity of the biological apatites. The data provide evidence that a significant fraction of the protonated phosphates (HPO4−2) are located on the surfaces of the biological crystals, and the concentration of unprotonated phosphates (PO4−3) within the apatitic lattice is elevated with respect to the surface. The total concentration of the surface HPO4−2 groups is higher in the younger, less mature biological crystals.
A Comparison of Type I Collagen, Fibronectin, and Vitronectin in Supporting Adhesion of Mechanically Strained Osteoblasts
Authors: Lacouture Mario E. , Schaffer Jonathan L. , Klickstein Lloyd B.
Published: April 2006

We used an adhesion assay for cells cultured under high dynamic strain to measure human osteoblast-like HOS cell adherence to immobilized type I collagen, fibronectin, and vitronectin. These conditions were designed to model the increased forces present at unstable fractures or loose joint prostheses. At a constant, low protein-coating density (1000 molecules/μm2) and 20% cyclic strain for 24 h, type I collagen, fibronectin, and vitronectin supported 24.6 ± 2%, 16.7 ± 3%, and 1.1 ± 1% adherence, respectively, which paralleled the relative number of integrin-binding sites in each protein. Thus, when the number of available binding sites was limited, strain resistance was proportional to the number of integrin-ligand interactions. In contrast, at high protein-coating densities (≥2,500 molecules/μm2), vitronectin supported greater adherence (45.7 ± 2%) when compared with type I collagen (37 ± 2%) or fibronectin (34.8 ± 2%) and directed constitutive expression of osteopontin (OPN), which suggested that there exist discrete signals on vitronectin receptor occupancy that promoted cell adherence and survival under strain. Integrin-mediated binding was necessary for resistance to strain, as evidenced by the low levels of strain resistance observed when cells were adherent in a nonintegrin-dependent manner. These findings support the utilization of at least two distinct mechanisms (i.e., tensegrity and integrin-mediated signal transduction) by HOS cells to remain adherent and viable on exposure to mechanical forces.
Noninvasive Loading of the Murine Tibia: An In Vivo Model for the Study of Mechanotransduction
Authors: Gross Ted S. , Srinivasan Sundar , Liu Chung C. , Clemens Thomas L. , Bain Steven D.
Published: April 2006

Transgenic and knockout mice present a unique opportunity to study mechanotransduction pathways in vivo, but the difficulty inherent with applying externally controlled loads to the small mouse skeleton has hampered this approach. We have developed a novel device that enables the noninvasive application of controlled mechanical loads to the murine tibia. Calibration of tissue strains induced by the device indicated that the normal strain environment was repeatable across loading bouts. Two in vivo studies were performed to show the usefulness of the device. Using C57Bl/6J mice, we found that dynamic but not static loading increased cortical bone area. This result is consistent with previous models of bone adaptation, and the lack of adaptation induced by static loading serves as a negative control for the device. In a preliminary study, transgenic mice selectively overexpressing insulin-like growth factor 1 (IGF-1) in osteoblasts underwent a low-magnitude loading regimen. Periosteal bone formation was elevated 5-fold in the IGF-1-overexpressing mice but was not elevated in wild-type littermates, showing the potential for synergism between mechanical loading and selected factors. Based on these data, we anticipate that the murine tibia-loading device will enhance assessment of mechanotransduction pathways in vivo and, as a result, has the potential to facilitate novel gene discovery and optimization of synergies between drug therapies and mechanical loading.