bluuebunny/crossref_metadata_2025_split huggingface dataset:
Establishment of a Novel Chondrocytic Cell Line N1511 Derived From p53-Null Mice
Authors: Kamiya Nobuhiro , Jikko Akitoshi , Kimata Koji , Damsky Caroline , Shimizu Katsuji , Watanabe Hideto 
Published: April 2006

Abstract
We established a clonal chondrocytic cell line N1511 derived from rib cartilage of a p53-null mouse. N1511 cells proliferated in polygonal shape and elicited differentiation at confluence when treated with combination of bone morphogenetic protein (BMP) 2 and insulin or parathyroid hormone (PTH) and dexamethasone. BMP-2/insulin-treated cells became refractile without forming cartilaginous nodules and reached terminal differentiation, became positive for alizarin red staining, and developed considerable ALP activity. In contrast, PTH/dexamethasone-treated cells formed Alcian blue-positive nodules but remained negative for alizarin red staining and ALP activity. Northern blot analysis revealed that BMP-2/insulin-treated cells sequentially expressed type II, IX, and X collagens, whereas PTH/dexamethasone-treated cells slowly expressed type II collagen and then type IX, and they did not exhibit type X collagen expression. These results show that BMP-2/insulin treatment induces full differentiation toward hypertrophy, whereas treatment with PTH/dexamethasone slows and limits differentiation. Recovery of p53 expression in N1511 cells by transient transfection inhibited cell proliferation, suggesting that cell proliferation could be regulated with p53 in this cell line. These results indicate that N1511 is the only cell line with known genetic mutation, which undergoes multiple steps of chondrocyte differentiation toward hypertrophy, and because proliferation could be regulated by expression of p53, N1511 could be an excellent model for studies of chondrogenesis, the function of p53, and genetic engineering of cartilage tissue.
Accelerated Chondrogenesis of the Rabbit Cranial Base Growth Plate by Oscillatory Mechanical Stimuli
Authors: Wang Xin , Mao Jeremy J. 
Published: April 2006

Abstract
How mechanical stimuli modulate chondral growth is not well understood. To test a hypothesis that chondral growth is accelerated by oscillatory mechanical stimuli rather than the peak magnitude of mechanical force, we delivered 2-N tensile forces with static (frequency = 0 Hz) and cyclic (f = 1 Hz) profiles noninvasively to the maxillae of growing New Zealand white rabbits for 20 minutes/day over 12 days. Computerized histomorphometry revealed significantly greater maximum height of the cranial base growth plate (GP) treated with cyclic forces (870 ± 130 μm) than static forces (654 ± 29 μm) and sham controls (566 ± 47 μm). In addition, the average total GP area treated with cyclic forces (2.63 ± 0.17 mm2) was significantly greater than static forces (2.12 ± 0.99 mm2) and sham controls (1.65 ± 0.13 mm2). The proliferating zone of GPs treated with cyclic forces (158 ± 38.5 μm) was significantly longer than the corresponding zones of static forces (117 ± 8.6 μm) and sham controls (54 ± 14.9 μm). The average number of chondrocytes in the proliferating zone treated with cyclic forces (1045 ± 127) was significantly greater than static forces (632 ± 85) and sham controls (632 ± 60) in standardized grids. Like natural GPs, the cartilage matrix treated with cyclic and static tensile forces consisted of abundant aggrecan-like proteoglycans. These findings indicate that oscillatory components of mechanical force rather than its peak magnitude are potent anabolic stimulus for chondral growth. A cascade of oscillatory mechanical stimuli is likely capable of engineering chondral growth beyond naturally occurring chondrogenesis.
Annexin VIII Is Differentially Expressed by Chondrocytes in the Mammalian Growth Plate During Endochondral Ossification and in Osteoarthritic Cartilage
Authors: White Alicia H. , Watson Rachel E. B. , Newman Bill , Freemont Anthony J. , Wallis Gillian A. 
Published: April 2006

Abstract
Endochondral ossification is the developmental process that leads to the formation and coordinated longitudinal growth of the majority of the vertebrate skeleton. Central to this process is chondrocyte differentiation occurring in the growth plate that lies at the junction between the epiphyseal cartilage and the bone. To identify novel factors involved in this differentiation process, suppression subtractive hybridization was performed to amplify preferentially cDNAs uniquely expressed in fetal bovine growth plate chondrocytes as opposed to epiphyseal chondrocytes. The subtracted product was used to screen a fetal bovine chondrocyte cDNA library. One of the cDNA clones identified encoded the bovine orthologue of annexin VIII, a protein not previously described in the growth plate. Northern and Western blotting confirmed that annexin VIII was expressed by growth plate chondrocytes and not by epiphyseal chondrocytes. Immunohistochemistry of the fetal bovine growth plate identified a gradient of increasing annexin VIII protein from the proliferative to the hypertrophic zone. Immunofluorescence localized annexin VIII largely to the chondrocyte cell membrane. In a preliminary study, we examined the distribution of annexin VIII in normal and osteoarthritic (OA) articular cartilage. In OA cartilage, the protein was located in a subset of mid- to deep zone chondrocytes and in the matrix surrounding these cells; no annexin VIII was detected in normal articular cartilage. Thus annexin VIII is a marker for chondrocyte differentiation during normal endochondral ossification and may act as a marker for cells undergoing inappropriate differentiation in OA.
Basic Fibroblast Growth Factor Stimulates Osteoclast Recruitment, Development, and Bone Pit Resorption in Association With Angiogenesis In Vivo on the Chick Chorioallantoic Membrane and Activates Isolated Avian Osteoclast Resorption In Vitro
Authors: Collin-Osdoby Patricia , Rothe Linda , Bekker Simon , Anderson Fred , Huang Yuefang , Osdoby Philip 
Published: April 2006

Abstract
Increased local osteoclast (OC)-mediated bone resorption coincides with angiogenesis in normal bone development and fracture repair, as well as in pathological disorders such as tumor-associated osteolysis and inflammatory-related rheumatoid arthritis or periodontal disease. Angiogenic stimulation causes recruitment, activation, adhesion, transmigration, and differentiation of hematopoietic cells which may therefore enable greater numbers of pre-OC to emigrate from the circulation and develop into bone-resorptive OCs. A chick chorioallantoic membrane (CAM) model, involving coimplantation of a stimulus in an agarose plug directly adjacent to a bone chip was used to investigate if a potent angiogenic stimulator, basic fibroblast growth factor (bFGF), could promote OC recruitment, differentiation, and resorption in vivo. Angiogenesis elicited by bFGF on the CAM was accompanied by increased OC formation and bone pit resorption (both overall and on a per OC basis) on the bone implants in vivo. In complementary in vitro assays, bFGF did not directly stimulate avian OC development from bone marrow mononuclear cell precursors, consistent with their low mRNA expression of the four avian signaling FGF receptors (FGFR)-1, FGFR-2, FGFR-3, and FGFR-like embryonic kinase (FREK). In contrast, bFGF activated isolated avian OC bone pit resorption via mechanisms inhibited by a selective cyclo-oxygenase (COX)-2 prostaglandin inhibitor (NS-398) or p42/p44 MAPK activation inhibitor (PD98059), consistent with a relatively high expression of FGFR-1 by differentiated avian OCs. Thus, bFGF may sensitively regulate local bone resorption and remodeling through direct and indirect mechanisms that promote angiogenesis and OC recruitment, formation, differentiation, and activated bone pit resorption. The potential for bFGF to coinduce angiogenesis and OC bone remodeling may find clinical applications in reconstructive surgery, fracture repair, or the treatment of avascular necrosis. Alternatively, inhibiting such bFGF-dependent processes may aid in the treatment of inflammatory-related or metastatic bone loss.
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

Abstract
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 &lt;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 &lt; 0.001) and 12 months (PYR = −42 ± 12%; DPYR = −44 ± 16%; p &lt; 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 &lt; 0.005) or at 12 months (B-ALP = 25 ± 7%; BGP = 37 ± 16%; p &lt; 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 &lt; 0.001) or 12 months (B-ALP = −20 ± 5%; BGP = −22 ± 10%; p &lt; 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 &lt; 0.001) and lumbar spine (genistein = 3 ± 2%, HRT = 3.8 ± 2.7%, placebo = −1.6 ± 0.3%, and p &lt; 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

AbstractOsteopathia 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 &lt; 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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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).