bluuebunny/crossref_metadata_2025 huggingface dataset:
Histomorphometric Assessment of Bone Mass, Structure, and Remodeling: A Comparison Between Healthy Black and White Premenopausal Women
Authors: Parisien May , Cosman Felicia , Morgan Dorcas , Schnitzer Michele , Liang Xiaoguang , Nieves Jeri , Forese Laura , Luckey Marjorie , Meier Diane , Shen Victor , Lindsay Robert , Dempster David W.
Published: April 2006

While noninvasive studies of bone mass and turnover in blacks and whites abound, histologic evaluations are very rare. We have performed a comparative bone histomorphometric study of iliac biopsies from 55 healthy, premenopausal women including 21 blacks (mean age 33.4 + 1.2 years) and 34 whites (mean age 32.5 + 0.8 years) of comparable age, weight, body composition, education, and lifestyle. Biochemical indices of mineral metabolism: parathyroid hormone, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, serum ionized calcium, serum phosphorus, and urinary calcium/creatinine were measured in the fasting state. Blacks had lower 25-hydroxyvitamin D (31.5 ± 3.36 vs. 63.21 ± 3.79 nmol/l, p = 0.0001). Histomorphometric indices of bone volume, structure, and connectivity were not different between groups. The following indices of bone remodeling were also similar in both groups: eroded perimeter, osteoid width, mineralizing perimeter, tissue-based bone formation rate, osteoid maturation time, active formation period, and activation frequency. However, osteoid perimeter (black [B] = 15.85 ± 1.30 vs. white [W] = 9.49 ± 0.70%, p = 0.0002), osteoid area (B = 2.55 ± 0.32 vs. W = 1.39 ± 0.12%, p = 0.003), single-labeled perimeter (B = 5.46 ± 0.54 vs. W = 4.04 ± 0.33%, p = 0.03), mineralization lag time (B = 38.18 ± 4.04 vs. W = 21.83 ± 1.60 days, p < 0.009), and total formation period (B = 148.15 ± 19.70 vs. W = 84.04 ± 7.62 days, p = 0.0056) were higher in blacks than in whites. The quiescent perimeter (B = 76.91 ± 1.40 vs. W = 84.25 ± 0.91%, p = 0.0001), mineral apposition rate (B = 0.70 ± 0.02 vs. W = 0.75 ± 0.02 μm/day, p = 0.066), mineralizing osteoid perimeter (B = 0.49 ± 0.04 vs. W = 0.75 ± 0.04%, p = 0.0001) and adjusted apposition rate (B = 0.35 ± 0.04 vs. W = 0.58 ± 0.04 μm3/μm2/day, p = 0.0001) were all lower in blacks than in whites. These results indicate that there are no differences in bone volume, microstructure, or turnover between black and white premenopausal women. However, there are significant differences in the mechanism of bone formation between the two groups, with a lower rate of mineralized matrix apposition within each remodeling unit and a longer total formation period in blacks than in whites. The differences appear to be the result of more frequent and/or longer inactive periods in the life span of the bone formation units in blacks. These differences may allow a greater overall deposition of bone mineral in black women and therefore help explain a higher bone mass and perhaps better bone quality in black than white women.
Efferent Targets of Osseous CGRP-Immunoreactive Nerve Fiber Before and After Bone Destruction in Adjuvant Arthritic Rat: An Ultramorphological Study on Their Terminal-Target Relations
Authors: Imai Shinji , Rauvala Heikki , Konttinen Yrjö T. , Tokunaga Toshimitsu , Maeda Toshihir , Hukuda Sinsuke , Santavirta Seppo
Published: April 2006

We report the ultramorphological characterization of the terminal-target relation of sensory peptidergic nerve fibers in healthy and diseased osseous tissues. Bone tissue sections were immunoelectronmicroscopically investigated for calcitonin gene-related peptide (CGRP), a neuropeptide widely distributed in sensory peptidergic fibers. Ultramorphological relation of the osseous CGRP-immunoreactive (ir) nerve terminals and their target cells was comparatively analyzed using healthy, arthritic, and postarthritic bone specimens from control and adjuvant-induced arthritic rats. Terminal-like profiles of the osseous CGRP-ir axons were evidenced in direct contact with the metaphyseal osteoblasts and osteoclasts of the control animals. Terminal-like profiles were also noted in the vicinity of the periosteal lining cells. Nonterminal-like profiles did not make intimate spatial relation to the cells/structures surrounding the nerve. Osseous CGRP-ir terminals and axons, which are either uncovered or thinly ensheathed by the supportive tissues, were extensively degenerated in adjuvant-induced infiltration, whereas larger fibers were relatively resistant. Numerous CGRP-ir axons with distinctive features reinnervated the postarthritic, ossifying periosteum. CGRP-ir axons appeared to reinnervate the eroded surface of metaphyseal bone and cartilage as early as the recruited osteoblasts resume osteogenesis in the postarthritic metaphysis. The observed terminal-target relations in the healthy and diseased bone tissues give an ultramorphological basis for the putative trophic, modulatory actions of CGRP innervation of the bone cells.
The Vitamin D Receptor Start Codon Polymorphism (FokI) and Bone Mineral Density in Premenopausal American Black and White Women
Authors: Harris Susan S. , Eccleshall T. ROSS , Gross Coleman , Dawson-Hughes Bess , Feldman David
Published: April 2006

This study examines the association between bone mineral density (BMD) and a start codon polymorphism (SCP) at the translation initiation site of the vitamin D receptor (VDR) gene. The thymine/cytosine (T/C) polymorphism in the first of two start (ATG) codons can be detected by a restriction fragment length polymorphism (RFLP) using the endonuclease FokI, which recognizes ATG as part of its restriction site. F indicates absence of the first ATG and a VDR that is shorter by three amino acids. The FokI genotype was determined in 154 premenopausal American women (72 black and 82 white) who were 20–40 years old. BMD of the total body, femoral neck, and lumbar spine were measured by dual-energy X-ray absorptiometry. The distribution of the SCP genotypes differed significantly by race (p < 0.001): 4% of blacks versus 18% of whites were ff homozygous and 65% of blacks versus 37% of whites were FF homozygous. There was no statistically significant interaction between race and SCP genotype in analyses of BMD at any skeletal site. In the group as a whole, the ff women had femoral neck BMD that was 7.4% lower than that of the FF women. The ff white women had total body BMD values that were 4.3% lower and femoral neck values that were 12.1% lower than FF white women. Total body and femoral neck BMD did not differ significantly by genotype in black women, and spine BMD did not differ by genotype in either race. Addition of the SCP genotype to analysis of covariance models comparing BMD of the black and white women reduced estimated differences in femoral neck BMD between the two groups by about 35%. In conclusion, the SCP polymorphism, detected with the endonuclease FokI, appears to influence peak bone density, particularly at the femoral neck. Racial differences in its distribution may explain some of the racial difference in femoral neck BMD.
Inhibition of Bone Resorption by Pamidronate Cannot Restore Normal Gain in Cortical Bone Mass and Strength in Tail-Suspended Rapidly Growing Rats
Authors: Kodama Yoshiaki , Nakayama Konosuke , Fuse Hiroaki , Fukumoto Seiji , Kawahara Hajime , Takahashi Hiroo , Kurokawa Takahide , Sekiguchi Chiharu , Nakamura Toshitaka , Matsumoto Toshio
Published: April 2006

To clarify how the changes in bone formation and resorption affect bone volume and strength after mechanical unloading, the effect of inhibition of bone resorption by a potent bisphosphonate, pamidronate, on bone mineral density (BMD), histology, and strength of hind limb bones was examined using tail-suspended growing rats. Tail suspension for 14 days reduced the gain in the BMD of the femur at both the metaphysis rich in trabecular bone and the diaphysis rich in cortical bone. Treatment with pamidronate increased the total BMD as well as that of the metaphysis of the femur but had almost no effect on the BMD of the diaphysis in both control and tail-suspended rats. Histological examinations revealed that 14-day tail suspension caused a loss of secondary cancellous bone with a reduction in the trabecular number and thickness in comparison with control rats. In the femoral diaphysis, the diameter and cortical bone thickness increased to a lesser degree in tail-suspended rats when compared with rats without tail suspension, and a marked reduction in bone formation and the layers of alkaline phosphatase–positive cells was observed at the periosteal side. Pamidronate treatment increased secondary cancellous bone but could not restore normal growth-induced periosteal bone apposition and bone strength. Because the material strength of the femoral diaphysis at the tissue level was not affected by pamidronate treatment, the inability of pamidronate to prevent the reduction in physical strength of the femoral diaphysis does not appear to be due to a change in the quality of newly formed bone. These results demonstrate that tail suspension reduces the growth-induced periosteal modelling drift and that the antiresorptive agent pamidronate is unable to restore normal periosteal bone apposition.
Fractures After Thyroidectomy in Men: A Population-Based Cohort Study
Authors: Nguyen Tu T. , Heath Hunter , Bryant Sandra C. , O'Fallon W. Michael , Melton L. Joseph
Published: April 2006

Bone mass is purportedly reduced by an endogenous or exogenous excess of thyroid hormone or, perhaps, by calcitonin deficiency. Patients who have undergone thyroidectomy could be subject to all of these effects, yet their practical implications in terms of fracture risk are poorly defined. Interpretation is further hampered by the focus on women, where results may be influenced by involutional osteoporosis. Consequently, we assessed the potential for fractures among the 136 Rochester, Minnesota men who underwent thyroidectomy between 1935 and 1979, relative to a group of age-matched control men from the community. With 2194 person-years of follow-up in each group, survival free of any fracture of vertebra, proximal humerus, distal forearm, pelvis, or proximal femur was similar in the two groups (p = 0.23), and the relative risk of any of these fractures for thyroidectomized patients versus their controls was increased only 1.5-fold (95% CI, 0.7–3.2). The difference was entirely accounted for by a statistically significant excess of proximal femur fractures in the men with thyroidectomy. Risk factors for fractures among men with thyroidectomy included greater age at surgery, greater extent of surgery, and the presence of risk factors for secondary osteoporosis. Thus, thyroidectomy, performed mainly for adenoma or goiter, seems to have little overall influence on the risk of age-related fractures in men. However, the association with hip fractures requires further evaluation.
Involvement of α5β1 Integrin in Matrix Interactions and Proliferation of Chondrocytes
Authors: Enomoto-Iwamoto Motomi , Iwamoto Masahiro , Nakashima Kazuhisa , Mukudai Yoshiki , Boettiger David , Pacifici Maurizio , Kurisu Kojiro , Suzuki Fujio
Published: April 2006

Integrins are cell surface receptors involved in cellular processes including adhesion, migration, and matrix assembly. In the present study, we analyzed the possible involvement of α5β1 integrin in the regulation of chondrocyte adhesion, spreading, and proliferation. We found that rabbit growth plate chondrocytes were able to attach to substrates coated with type I collagen, type II collagen, or fibronectin within 24 h of culture. During this time period, attachment to fibronectin appeared to be dependent on α5β1 integrin, whereas adhesion to collagens was not. By day 3 of culture, chondrocytes spread onto all the substrates tested. We found that regardless of the nature of the substrate, cell spreading was reversed by treatment with RGD peptide or antibodies against α5β1 or fibronectin, indicating that cell spreading involved α5β1 and fibronectin endogenously produced and deposited by the chondrocytes themselves. Colony formation by chondrocytes in soft agar was inhibited by treatment with RGD peptides or BIIG2, an antibody that interferes with α5β1 integrin–ligand interactions. Furthermore, DNA content was decreased by treatment with anti-fibronectin antibody in micromass culture of chondrocytes. Immunohistochemical analysis on tissue sections revealed that the α5 subunit was particularly abundant in the proliferative and hypertrophic zones of growth plate. The results of the study indicate that α5β1 integrin plays multiple roles in chondrocyte behavior and function and appears to be involved in the regulation of both chondrocyte–matrix interactions and proliferation.
CD44 Antibodies Inhibit Osteoclast Formation
Authors: Kania Janice R. , Kehat-Stadler Talia , Kupfer Stuart R.
Published: April 2006

Osteoclast differentiation is a complex process requiring multiple factors and sequential regulation. We have determined that CD44, a cell surface glycoprotein that is known to function as an adhesion receptor, is involved in this process. By immunocytochemistry, we show that CD44 is expressed in mouse osteoclasts that develop in primary cultures of bone marrow cells treated with 1α,25-dihydroxyvitamin D3. Monoclonal antibodies to CD44 inhibit osteoclast formation in bone marrow cultures in a dose- and time-dependent manner. In contrast, CD44 Fab monomer antibodies have no effect on osteoclast development, suggesting that the inhibition of differentiation by the whole antibodies is facilitated by cross-linking of CD44 molecules. Cocultures of spleen cells and ST2 bone marrow stromal cells indicate that hematopoietic cells mediate the CD44 antibody inhibitory effect. CD44 antibodies do not inhibit osteoclast resorption of calcified matrix, indicating that CD44 is not absolutely required for resorption activity. These observations demonstrate that CD44 may play a role in osteoclast formation and suggest mechanisms by which CD44 antibody effects are mediated.
Integrin Expression and Function on Human Osteoblast-like Cells
Authors: Gronthos Stan , Stewart Karina , Graves Stephen E. , Hay Shelly , Simmons Paul J.
Published: April 2006

The integrin family of cell adhesion molecules are a series of cell surface glycoproteins that recognize a range of cell surface and extracellular matrix (ECM)-associated ligands. To date, the precise role of individual integrin molecules in bone cell–ECM interactions remains unclear. Cell binding assays were performed to examine the ability of normal human bone cells (NHBCs) to adhere to different ECM proteins in vitro. NHBCs displayed preferential adhesion to fibronectin over collagen types I, IV, and vitronectin and showed low affinity binding to laminin and collagen type V. No binding was observed to collagen type III. The integrin heterodimers α1β1, α2β1, α3β1, α5β1, αvβ3, and αvβ5 were found to be constitutively expressed on the cell surface of NHBCs by flow cytometric analysis. The integrins α4β1 and α6β1 were not expressed by NHBCs. Subsequent binding studies showed that NHBC adhesion to collagen and laminin was mediated by multiple integrins where cell attachment was almost completely inhibited in the presence of a combination of function-blocking monoclonal antibodies (Mabs) to α1β1, α2β1, α3β1, and β1. In contrast, the adhesion of NHBCs to fibronectin was only partially inhibited (50%) in the presence of blocking Mabs to α3β1, α5β1, and β1. The attachment of NHBCs to collagen, laminin, fibronectin, and vitronectin was also found to be unaffected in the presence of a function-blocking Mab to αvβ3. The results of this study indicate that β1 integrins appear to be the predominant adhesion receptor subfamily utilized by human osteoblast-like cells to adhere to collagen and laminin and in part to fibronectin.
Down-Regulation of Protein Kinase C by Parathyroid Hormone and Mezerein Differentially Modulates cAMP Production and Phosphate Transport in Opossum Kidney Cells
Authors: Cole Judith A.
Published: April 2006

We examined the effects of prolonged exposure to parathyroid hormone (PTH) and the protein kinase C (PKC) activator mezerein (MEZ) on cyclic adenosine monophosphate (cAMP) production, PKC activity, and Na+-dependent phosphate (Na/Pi) transport in an opossum kidney cell line (OK/E). A 5 minute exposure to PTH stimulated, while a 6 h incubation reduced, cAMP production. Na/Pi transport was maximally inhibited under desensitizing conditions and was not affected by reintroduction of the hormone. MEZ pretreatment (6 h) enhanced PTH-, cholera toxin (CTX)-, and forskolin (FSK)-stimulated cAMP production, suggesting enhanced Gsα coupling and increased adenylyl cyclase activity. However, PKA- and PKC-dependent regulation of Na/Pi were blocked in MEZ-treated cells. The PTH-induced decrease in cAMP production was associated with a reduction in membrane-associated PKC activity while MEZ-induced increases in cAMP production were accompanied by decreases in membrane and cytosolic PKC activity. Enhanced cAMP production was not accompanied by significant changes in PTH/PTH related peptide (PTHrP) receptor affinity or number, nor was the loss of Na/Pi transport regulation associated with changes in PKA activity. The results indicate that down-regulation of PKC by PTH or MEZ differentially modulates cAMP production and regulation of Na/Pi transport. The distinct effects of PTH and MEZ on PKC activity suggest that agonist-specific activation and/or down-regulation of PKC isozyme(s) may be involved in the observed changes in cAMP production and Na/Pi transport.
Effects of Exercise Involving Predominantly Either Joint-Reaction or Ground-Reaction Forces on Bone Mineral Density in Older Women
Authors: Kohrt Wendy M. , Ehsani Ali A. , Birge Stanley J.
Published: April 2006

This study compared the effects of two exercise training programs, 11 months in duration, on bone mineral density (BMD) in older, sedentary women. Thirty-nine women, aged 60–74 years, were assigned to the following groups: (a) a group that performed exercises that introduced stress to the skeleton through ground-reaction forces (GRF) (i.e., walking, jogging, stairs); (b) a group that performed exercises that introduced stress to the skeleton through joint-reaction forces (JRF) (i.e., weight lifting, rowing); or (c) a no-exercise control group. BMD of the whole body, lumbar spine, proximal femur, and distal forearm was assessed five times at ∼3-month intervals. The GRF and JRF exercise programs resulted in significant and similar increases in BMD of the whole body (2.0 ± 0.8% and 1.6 ± 0.4%, respectively), lumbar spine (1.8 ± 0.7% and 1.5 ± 0.5%, respectively), and Ward's triangle region of the proximal femur (6.1 ± 1.5% and 5.1 ± 2.1%, respectively). There was a significant increase in BMD of the femoral neck only in response to the GRF exercise program (GRF, 3.5 ± 0.8%; JRF, −0.2 ± 0.7%). There were no significant changes in BMD in control subjects. Among all exercisers, there was a significant inverse (r = −0.52, p < 0.01) relationship between increases in whole body BMD and reductions in fat mass, suggesting a dose response effect of exercise on bone mass. Although femoral neck BMD was responsive only to the GRF exercise program, some adaptations (i.e., increase in lean body mass and strength) that were specific to the JRF exercise program may be important in preventing osteoporotic fractures by reducing the risk for falls. It remains to be determined whether all of these benefits can be gained through a training program that combines the different types of exercises employed in this study.
Determinants of Peak Bone Mass in Chinese Women Aged 21–40 Years. III. Physical Activity and Bone Mineral Density
Authors: Ho Suzanne C. , Wong Eric , Chan Sieu GAEN , Lau Joseph , Chan Cynthia , Leung Ping CHUNG
Published: April 2006

Previous studies on the relation between moderate physical activity and bone mass have observed conflicting results. Many of these studies have not dissociated the role of physical activity by age groups and in relation to the period of peak bone mass formation. Our cross-sectional analysis of the baseline data of a longitudinal study of 273 women aged 21–40 attempted to evaluate the role of moderate physical activity on bone mass around the period of peak bone mass attainment. The analyses were carried out separately for the two age groups—21–30 and 31–40—and had also taken into account the effects of age, dietary calcium intake, and lean body mass on bone mineral density (BMD). The total metabolic equivalent values (MET) of leisure time physical activity was based on the MET values for each activity and the reported time spent on each activity in the past year. The results indicated that among the younger group of women, high level of leisure time physical activity was associated with higher bone mass at both the spine and the hip. Additive effects of physical activity and dietary calcium intake on the spine and the hip BMD were observed. Together with age and lean body mass, physical activity and dietary calcium intake accounted for 19% of the variances of bone mineral at the spine and 9–11% at the hip. Among women aged 31–40, presumably after the peak bone mass formation, lean body mass as well as fat mass have independent strong association with BMD. Physical activity was not associated with bone mass in this age group.
Single-Colony Derived Strains of Human Marrow Stromal Fibroblasts Form Bone After Transplantation In Vivo
Authors: Kuznetsov Sergei A. , Krebsbach Paul H. , Satomura Kazuhito , Kerr Janet , Riminucci Mara , Benayahu Dafna , Robey Pamela Gehron
Published: April 2006

Populations of marrow stromal fibroblasts (MSFs) can differentiate into functional osteoblasts and form bone in vivo. It is not known, however, what proportion of MSF precursor cells, colony forming units-fibroblast (CFU-Fs), have osteogenic potential. In the present study, analysis of bone formation in vivo by single-colony derived strains of human marrow stromal fibroblasts (HMSFs) has been performed for the first time. Each strain originated from an individual CFU-F and underwent four passages in vitro prior to subcutaneous implantation into immunodeficient mice within vehicles containing hydroxyapatite-tricalcium phosphate ceramic. Multicolony derived HMSF strains were also transplanted to serve as positive controls. After 8 weeks, abundant bone formation was found in the transplants of all multicolony derived HMSF strains, whereas 20 out of 34 (58.8%) single-colony derived strains from four donors formed bone. Immunostaining with antibody directed against human osteonectin and in situ hybridization for human-specific alu sequences demonstrated that cells forming new bone were of human origin and were vital for at least 45 weeks post-transplantation. Both the incidence of bone-forming colonies and the extent of bone formation by single-colony derived HMSF strains were increased by cultivation with dexamethasone and ascorbic acid phosphate. Other factors, including type of transplantation vehicle, morphology, size, and structure of the original HMSF colonies showed no obvious correlation with the incidence or extent of bone formation. Hematopoietic tissue within the newly formed bone was developed in the transplants exhibiting exuberant bone formation. These results provide evidence that individual human CFU-Fs have osteogenic potential and yet differ from each other with respect to their osteogenic capacity.
Assessment of Articular Cartilage and Subchondral Bone: Subtle and Progressive Changes in Experimental Osteoarthritis Using 50 MHz Echography In Vitro
Authors: Saïed A. , Chérin E. , Gaucher H. , Laugier P. , Gillet P. , Floquet J. , Netter P. , Berger G.
Published: April 2006

The main objectives of this work were to demonstrate the potential of 50 MHz echography for assessing initial and progressive morphological and structural changes of articular cartilage and bone developed in an experimental model of osteoarthritis (OA). Degenerative lesions were induced in rat knees by the unilateral intra-articular injection of a 3 mg dose of mono-iodo-acetic acid. To assess the lesion progression, the animals (n = 30) were sacrificed at different time intervals up to 8 weeks after the injection. Three-dimensional echographic data were acquired in vitro on patellar cartilage and bone at various stages of the remodeling process using a scanning ultrasound microscope. Changes involving the OA cartilage characteristics are discussed relative to those of the contralateral control joint which received a placebo. Images of control cartilage showed a smooth hyperechoic articular surface and an echoic matrix. The cartilage thickness was 266 ± 44 μm (mean ± SD) in the central region of the tissue. The precision of ultrasonic thickness measurements was better than 1.3%. First changes in cartilage internal structure and subchondral bone appeared on ultrasound images 3 days after the injection and were even more evident by day 7. They resulted in a slight thinning of the cartilage, a 30% increase of its internal structure echogenicity, and the appearance of echoic zones in subchondral bone. Histologic findings confirmed chondrocyte depletion and degeneration, decrease of matrix proteoglycans, and fibrovascular connective tissue proliferation at the subchondral plate. Progressive and severe lesions at both bone and cartilage surface and internal structure were assessed and correlated to histologic features. These results show that high resolution echography is sensitive to subtle and progressive osteochondral remodeling. This technique has the potential to be used for intra-articular quantitative imaging and assessment of early changes in bone and cartilage structure associated with natural human disease.
Peptide Aldehyde Inhibitors of Cathepsin K Inhibit Bone Resorption Both In Vitro and In Vivo
Authors: Votta Bartholomew J. , Levy Mark A. , Badger Alison , Bradbeer Jeremy , Dodds Robert A. , James Ian E. , Thompson Scott , Bossard Mary J. , Carr Thomas , Connor Janice R. , Tomaszek Thaddeus A. , Szewczuk Lawrence , Drake Fred H. , Veber Daniel F. , Gowen Maxine
Published: April 2006

We have shown previously that cathepsin K, a recently identified member of the papain superfamily of cysteine proteases, is expressed selectively in osteoclasts and is the predominant cysteine protease in these cells. Based upon its abundant cell type-selective expression, potent endoprotease activity at low pH and cellular localization at the bone interface, cathepsin K has been proposed to play a specialized role in osteoclast-mediated bone resorption. In this study, we evaluated a series of peptide aldehydes and demonstrated that they are potent cathepsin K inhibitors. These compounds inhibited osteoclast-mediated bone resorption in fetal rat long bone (FRLB) organ cultures in vitro in a concentration-dependent manner. Selected compounds were also shown to inhibit bone resorption in a human osteoclast-mediated assay in vitro. Cbz-Leu-Leu-Leu-H (in vitro enzyme inhibition Ki,app = 1.4 nM) inhibited parathyroid hormone (PTH)-stimulated resorption in the FRLB assay with an IC-50 of 20 nM and inhibited resorption by isolated human osteoclasts cultured on bovine cortical bone slices with an IC-50 of 100 nM. In the adjuvant-arthritic (AA) rat model, in situ hybridization studies demonstrated high levels of cathepsin K expression in osteoclasts at sites of extensive bone loss in the distal tibia. Cbz-Leu-Leu-Leu-H (30 mg/kg, intraperitoneally) significantly reduced this bone loss, as well as the associated hind paw edema. In the thyroparathyriodectomized rat model, Cbz-Leu-Leu-Leu-H inhibited the increase in blood ionized calcium induced by a 6 h infusion of PTH. These data indicate that inhibitors of cathepsin K are effective at reducing osteoclast-mediated bone resorption and may have therapeutic potential in diseases of excessive bone resorption such as rheumatoid arthritis or osteoporosis.
Histomorphometry of Distraction Osteogenesis in a Caprine Tibial Lengthening Model
Authors: Dr. Welch Robert D. , Birch John G. , Makarov Marina R. , Samchukov Mikhail L.
Published: April 2006

Standardized histomorphometry of bone formation and remodeling during distraction osteogenesis (DO) has not been well characterized. Increasing the rhythm or number of incremental lengthenings performed per day is reported to enhance bone formation during limb lengthening. In 17 skeletally immature goats, unilateral tibial lengthenings to 20 or 30% of original length were performed at a rate of 0.75 mm/day and rhythms of 1, 4, or 720 times per day using standard Ilizarov external fixation and an autodistractor system. Two additional animals underwent frame application and osteotomy without lengthening and served as osteotomy healing controls. Histomorphometric indices were measured at predetermined regions from undecalcified tibial specimens. Within the distraction region, bone formation and remodeling activity were location dependent. Intramembranous bone formed linearly oriented columns of interconnecting trabecular plates of woven and lamellar type bone. Total new bone volume and bone formation indices were significantly increased within the distraction and osteotomy callus regions (Tb.BV/TV, 226% [p < 0.05]; BFR/BS, 235–650% [p < 0.01]) respectively, compared with control metaphyseal bone. Bone formation indices were greatest adjacent to the mineralization zones at the center of the distraction gap; mineral apposition rate 96% (p < 0.01); mineralized bone surfaces 277% [p < 0.001]); osteoblast surfaces 359% [p < 0.001]); and bone formation rate (650% [p < 0.01]). There was no significant difference (p < 0.14; R = 0.4) in the bone formation rate of the distracted callus compared with the osteotomy control callus. Within the original cortices of the lengthened tibiae, bone remodeling indices were significantly increased compared with osteotomy controls; activation frequency (200% [p < 0.05]); osteoclast surfaces (295% [p < 0.01]); erosion period (75%); porosity (240% [p < 0.001]). Neither the rhythm of distraction nor the percent lengthening appeared to significantly influence any morphometric parameter evaluated. Distraction osteogenesis shares many features of normal fracture gap healing. The enhanced bone formation and remodeling appeared to result more from increased recruitment and activation of bone forming and resorbing cells rather than from an increased level of individual cellular activity.
Induction of Human Osteoclast-like Cells by Treatment of Blood Monocytes with Anti-Fusion Regulatory Protein-1/CD98 Monoclonal Antibodies
Authors: Higuchi Shigeomi , Tabata Nobutada , Tajima Masatoshi , Ito Morihiro , Tsurudome Masato , Sudo Akihiro , Uchida Atsumasa , Ito Yasuhiko
Published: April 2006

We have developed a new and simple system of human osteoclast formation by fusing peripheral blood monocytes with anti-Fusion Regulatory Protein-1 (anti–FRP-1) monoclonal antibody (mAb). When human blood monocytes were cultured in the presence of anti-FRP–1/CD98 mAbs, polykaryocytes began to appear at approximately 15 h and increased in size with time until 3–4 days of incubation with anti–FRP-1 mAb. These fused cells showed positive staining in tartrate-resistant acid phosphatase, possessed numerous calcitonin receptors, and were capable of bone resorption. These results strongly suggest that anti–FRP-1 antibody-induced multinucleated cells are osteoclasts. Furthermore, FRP-1 antigens were detected in osteoclasts isolated from human bone and in the osteoclast-like cells obtained from human giant cell tumors of bone.
The Migration of Purified Osteoclasts Through Collagen Is Inhibited by Matrix Metalloproteinase Inhibitors
Authors: Sato Takuya , Foged Niels Tækker , Delaissé Jean-Marie
Published: April 2006

The most obvious proteolytic event controlled by the osteoclast is bone matrix removal in the resorption compartment. Here, however, we investigated whether matrix metalloproteinase (MMP) activity of the osteoclast might be involved in its migration to its future bone resorption site. We seeded either nonpurified or purified osteoclasts onto either uncoated or collagen-coated dentine slices and cultured them in the presence or absence of specific MMP inhibitors. When nonpurified osteoclasts were cultured on uncoated dentine, MMP inhibitors did not prevent pit formation, as previously reported. However, when collagen-coated dentine was used, pit formation was strongly inhibited by MMP inhibitors. The same results were obtained when performing these experiments with purified osteoclasts, thus demonstrating the ability of osteoclasts by themselves to migrate through collagen via an MMP-dependent pathway. This demonstration was confirmed by using collagen-coated invasion chambers. In addition, the invasions were not, or only slightly, inhibited by inhibitors of serine proteinases, cysteine proteinases, and carbonic anhydrase, though the latter two are well established bone resorption inhibitors that strongly inhibited pit formation. It is concluded that osteoclasts can migrate through collagen in the absence of other cells and that this migration relies on MMP activity, whereas other enzymes typically required for bone removal in the resorption compartment are not essential for migration. Some of the osteoclast MMPs might thus be relevant to the migratory/invasive activity of the osteoclast, rather than to its bone resorptive activity itself.
High Fatty Acid Content in Rabbit Serum Is Responsible for the Differentiation of Osteoblasts Into Adipocyte-like Cells
Authors: Diascro Dominic D. , Vogel Robert L. , Johnson Timothy E. , Witherup Keith M. , Pitzenberger Steven M. , Rutledge Su Jane , Prescott David J. , Rodan Gideon A. , Schmidt Azriel
Published: April 2006

Osteoblasts and adipocytes originate from common mesenchymal precursors. With aging, there is a decrease in osteoprogenitor cells that parallels an increase of adipocytes in bone marrow. We observed that rabbit serum (RS) induces adipocyte-like differentiation in human osteosarcoma SaOS-2/B10 and MG-63 cell lines, in rat ROS17/2.8 cells, and in mouse calvaria-derived osteoblastic MB1.8 cells, as evidenced by the accumulation of Oil Red O positive lipid vesicles and the decrease in alkaline phosphatase expression. Both SaOS-2/B10 and MG-63 cells, but not ROS17/2.8 nor MB1.8 cells, express significant levels of PPARγ mRNA, a member of the peroxisome proliferator activated receptor (PPAR) family that has been implicated in the control of adipocyte differentiation. However, both ROS17/2.8 and MG-63 cells express significant levels of the adipocyte selective marker, aP2 fatty acid binding mRNA, which can be further increased by RS. These cell types express PPARδ/NUC-1 but not PPARα, indicating that cells that do not express either PPARγ or PPARα are capable of differentiating into adipocyte-like cells. Transfection experiments in COS cells showed that compared with fetal bovine serum (FBS), RS is rich in agents that stimulate PPAR-dependent transcription. The stimulatory activity was ethyl acetate extractable and was 35-fold more abundant in RS than in FBS. Purification and analysis revealed that the major components of this extract are free fatty acids. Furthermore, the same fatty acids, a mixture of palmitic, oleic, and linoleic acids, activate the PPARs and induce adipocyte-like differentiation of both ROS17/2.8 and SaOS-2/B10 cells. These findings suggest that fatty acids or their metabolites can initiate the switch from osteoblasts to adipocyte-like cells.
A BMP Responsive Transcriptional Region in the Chicken Type X Collagen Gene
Authors: Volk Susan W. , Luvalle Phyllis , Leask Todd , Leboy Phoebe S.
Published: April 2006

Bone morphogenetic proteins (BMPs) were originally identified by their ability to induce ectopic bone formation and have been shown to promote both chondrogenesis and chondrocyte hypertrophy. BMPs have recently been found to activate a membrane serine/threonine kinase signaling mechanism in a variety of cell types, but the downstream effectors of BMP signaling in chondrocyte differentiation remain unidentified. We have previously reported that BMP-2 markedly stimulates type X collagen expression in prehypertrophic chick sternal chondrocytes, and that type X collagen mRNA levels in chondrocytes cultured under serum-free (SF) conditions are elevated 3- to 5-fold within 24 h. To better define the molecular mechanisms of induction of chondrocyte hypertrophy by BMPs, we examined the effect of BMPs on type X collagen production by 15-day chick embryo sternal chondrocytes cultured under SF conditions in the presence or absence of 30 ng/ml BMP-2, BMP-4, or BMP-7. Two populations of chondrocytes were used: one representing resting cartilage isolated from the caudal third of the sterna and the second representing prehypertrophic cartilage from the cephalic third of the sterna. BMP-2, BMP-4, and BMP-7 all effectively promoted chondrocyte maturation of cephalic sternal chondrocytes as measured by high levels of alkaline phosphatase, diminished levels of type II collagen, and induction of the hypertrophic chondrocyte-specific marker, type X collagen. To test whether BMP control of type X collagen expression occurs at the transcriptional level, we utilized plasmid constructs containing the chicken collagen X promoter and 5′ flanking regions fused to a reporter gene. Constructs were transiently transfected into sternal chondrocytes cultured under SF conditions in the presence or absence of 30 ng/ml BMP-2, BMP-4, or BMP-7. A 533 bp region located 2.4–2.9 kb upstream from the type X collagen transcriptional start site was both necessary and sufficient for strong BMP responsiveness in cells destined for hypertrophy, but not in chondrocytes derived from the lower sterna.
Mouse Osteoblastic Cell Line (MC3T3-E1) Expresses Extracellular Calcium (Ca2+o)–Sensing Receptor and Its Agonists Stimulate Chemotaxis and Proliferation of MC3T3-E1 Cells
Authors: Yamaguchi Toru , Chattopadhyay Naibedya , Kifor Olga , Butters Robert R. , Sugimoto Toshitsugu , Brown Edward M.
Published: April 2006

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the “reversal” phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3-E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3-E1 cells. We also identified CaR transcripts in MC3T3-E1 cells by Northern analysis using a CaR-specific riboprobe and by reverse transcription-polymerase chain reaction with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3-E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3-E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3-E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o.
17β-Hydroxysteroid Dehydrogenases in Human Bone Cells
Authors: Dong Yu , Qiu Qing Qing , Debear Joanna , Lathrop William F. , Bertolini Donald R. , Tamburini Paul P.
Published: April 2006

Interconversion of estrogens by osteoblasts may play a role in regulating bone mass. As a first step toward exploring this possibility, we investigated the expression and activity of 17β-hydroxysteroid dehydrogenases (17β-HSDs) in cultured human osteoblasts (HOB) and osteoblast-like osteosarcoma cells (MG63, TE85, and SaOS-2). Significant 17β-HSD activity was detected in cell-free extracts of all bone cells with oxidation of estradiol to estrone predominating over reduction. Reverse transcription-polymerase chain reaction (RT-PCR) experiments showed that the mRNA for 17β-HSD I was detectable only in MG63 cells, albeit at low levels, while 17β-HSD II was present in MG63, TE85, and HOB, but not SaOS-2, and 17β-HSD III was absent from each bone cell type. 17β-HSD IV was the only isoform present in all bone cells analyzed. Further analysis of the expression of 17β-HSD IV in these bone cells by immunoblotting revealed both the full-length 83 kDa protein and the proteolytic 38 kDa form. The kinetic parameters for estradiol oxidation by purified recombinant 17β-HSD IV (Km = 49.7 μM, Vmax = 79.4 nmol/minute/mg of protein) and its HSD-domain (Km = 79.4 μM, Vmax = 476 nmol/minute/mg of protein) were significantly higher than previously reported, but consistent with the values obtained with crude cell-free extracts of SaOS-2 cells (Km = 98.8 μM, Vmax = 0.07 nmol/minute/mg of protein) which contain only 17β-HSD IV based on RT-PCR. These studies show that bone cells have the capacity to interconvert circulating estrogens and suggest that bone cell 17β-HSDs serve primarily to attenuate the continuing actions of estradiol through conversion to its less potent form, estrone, under certain conditions.
Osteocyte Shape Is Dependent on Actin Filaments and Osteocyte Processes Are Unique Actin-Rich Projections
Authors: Tanaka-Kamioka Kayo , Kamioka Hiroshi , Ris Hans , Lim Soo-Siang
Published: April 2006

Osteocytes are derived from a select group of osteoblasts that have undergone a final differentiation. Due to their inaccessibility when embedded in the bone matrix, very little is known about the osteocyte cytoskeleton. This study provides an extensive analysis of the osteocyte cytoskeleton, based on the successful isolation of osteocytes from 16-day embryonic chick calvariae. We used OB7.3, a chicken osteocyte-specific monoclonal antibody, to confirm the osteocytic phenotype of the isolated cells and established culture conditions to promote growth of cells that most resemble osteocytes in vivo. Immunofluorescence staining with antitubulin, antivimentin, and antiactin showed the relative distribution of the microtubules, intermediate filaments, and actin filaments in both osteocyte cell body and processes. Field emission scanning electron microscopy revealed the three-dimensional relationships of the cytoskeletal elements and a unique organization of actin bundles that spanned the cell body and osteocyte processes. When combined with drug studies, these experiments demonstrate that actin filaments are crucial for the maintenance of osteocyte shape. Furthermore, we identified two actin-bundling proteins, alpha-actinin and fimbrin, in osteocyte processes. The prominence and unique distribution of fimbrin in osteocyte processes provides the possibility of its use as an intracellular marker to distinguish osteocytes from osteoblasts.
A Novel Polymorphism in the Promoter Region for the Human Osteocalcin Gene: The Possibility of a Correlation with Bone Mineral Density in Postmenopausal Japanese Women
Authors: Dohi Yoshiko , Iki Masayuki , Ohgushi Hajime , Gojo Satoshi , Tabata Shiro , Kajita Etsuko , Nishino Harumi , Yonemasu Kunio
Published: April 2006

We present a polymorphism of the human osteocalcin gene (also known as BGP, for bone Gla protein) due to a 1 base pair (bp) substitution from cytosine to thymine at position 298 nucleotides (nt), which is at position 198 nt upstream from the BGP exon 1. This mutation was detected by single-strand conformation polymorphism analysis after polymerase chain reaction for the osteocalcin gene fragment (326 bp) and sequencing analysis. The cytosine/thymine polymorphism can be defined by restriction fragment length polymorphism analysis using a modified primer pair and the restriction endonuclease HindIII. The osteocalcin genotype was determined in 160 postmenopausal Japanese women (age 48–80 years). Osteocalcin alleles were designated according to the absence (H) or presence (h) of the HindIII restriction site. There were 12 HH, 49 Hh, and 99 hh individuals, and the allele frequencies were 22.8% for H and 77.2% for h. To determine if genetic variation influences bone mineral density (BMD) and thus can be a determinant of susceptibility to osteoporosis in older women, we examined the association of BMD with the osteocalcin genotypes found in the present study. The subjects with genotype HH had the smallest BMD and those with hh had the greatest BMD among subjects, but these differences did not reach statistical significance. The HindIII genotype showed a significant effect on the prevalence of osteopenia in the subjects, that is, women with genotype HH had a 5.74 times greater risk for osteopenia (p < 0.05) and those with genotype Hh had a 1.59 times greater risk than women with genotype hh. We identified the osteocalcin gene polymorphism, detected with the HindIII genotype, which was suggested to influence bone density and is a possible genetic marker for bone metabolism.
Activation of Phospholipase D Signaling Pathway by Epidermal Growth Factor in Osteoblastic Cells
Authors: Carpio Lillian Carol , Dziak Rosemary
Published: April 2006

The receptor-mediated activation of phospholipase D (PLD) is a major signaling pathway in several cell systems. This study determined the effects of epidermal growth factor (EGF) on PLD activity in normal rat osteoblastic cells. Primary cultures were obtained from fetal rat calvaria by sequential collagenase digestion and seeded in BGJb media supplemented with 10% fetal calf serum. PLD activity was assayed by the transphosphatidylation reaction in [H3]myristic acid (5 μCi/ml)-labeled cells treated with EGF in the presence of 5% ethanol and measuring the production of phosphatidylethanol (PEtOH). Lipids were extracted and separated by thin-layer chromatography, detected by iodine staining, and the areas of interest were scraped off and transferred to vials for scintillation counting. EGF significantly increased PEtOH production in a dose-dependent manner and at short (10–60 s) and long (up to 30 minutes) incubation periods (p < 0.05). Phosphatidic acid levels were also significantly increased (p < 0.05) compared with unstimulated controls, but the levels were ∼60% less than those of PEtOH. 4b-phorbol 12-myristate, 13-acetate (PMA) also produced a significant increase in PEtOH levels when compared with unstimulated control cultures, but when PMA was added together with EGF, the production of PEtOH was reduced about 30%. Pretreatment of cells with the protein kinase C (PKC) inhibitor H-7 caused a significant increase in PEtOH levels, compared with cells stimulated with EGF alone. Preincubation of cells with pertussis toxin produced a partial decrease in PEtOH levels. This study demonstrates that EGF activates the PLD signaling cascade in normal rat osteoblastic cells and that the pathway appears to involve, at least in part, a PKC- and Gi protein-dependent mechanism.