bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effects of Fibroblast Growth Factor-2 on Human Neonatal Calvaria Osteoblastic Cells Are Differentiation Stage Specific
Authors: Debiais F. , Hott M. , Graulet A. M. , Marie P. J.
Published: April 2006

Fibroblast growth factors (FGFs) appear to play an important role in human cranial osteogenesis. We therefore investigated the effects of recombinant human FGF-2 (rhFGF-2) on human calvaria (HC) osteoblastic cells. Immunocytochemical analysis showed that confluent HC cells express both FGF receptors -1 and -2. In short-term culture, rhFGF-2 (0.1–100 ng/ml, 2–5 days) increased HC cell growth and decreased alkaline phosphatase (ALP) activity and type I collagen (ColI) synthesis, as evaluated by P1CP levels. When HC cells were induced to differentiate in long-term culture in the presence of 50 μg/ml ascorbic acid and 3 mM phosphate, HC cells initially proliferated, then ALP activity and ColI synthesis decreased and calcium content in the extracellular matrix increased. Continuous treatment with rhFGF-2 (50 ng/ml) for 1–28 days, or a transient rhFGF-2 treatment for 1–7 days, slightly increased DNA synthesis at 7 days, whereas a late treatment for 8–28 days had no effect on cell growth. The continuous and transient treatments with rhFGF-2 decreased ALP activity, ColI synthesis, and matrix mineralization. This was associated with a transient fall in osteocalcin (OC) production at 7 days. In contrast, the late rhFGF-2 treatment for 8–28 days only slightly inhibited ALP activity and increased matrix mineralization. In addition, both continuous and late treatments with rhFGF-2 increased OC production in more mature cells at 3–4 weeks of culture. We also found that the early and late treatments with rhFGF-2 had opposite effects on transforming growth factor β2 production in proliferating cells and more mature cells. The results show that rhFGF-2 slightly stimulates cell growth and reduces the expression of osteoblast markers in less mature cells, whereas it induces OC production and matrix mineralization in more mature cells, indicating that the effects of FGF-2 are differentiation stage specific and that FGF-2 may modulate HC osteogenesis by acting at distinct stages of cell maturation.
Concentration of Transforming Growth Factor Beta in Human Bone Tissue: Relationship to Age, Menopause, Bone Turnover, and Bone Volume
Authors: Pfeilschifter Johannes , Diel Ingo , Scheppach Beate , Bretz Anette , Krempien Robert , Erdmann Johannes , Schmid Gerald , Reske Nicole , Bismar Hanadi , Seck Thomas , Krempien Burkhard , Ziegler Reinhard
Published: April 2006

Transforming growth factor β (TGF-β) is thought to play an important role in bone metabolism, but its relationship to human bone turnover and bone mass has not been examined yet. In this study, we measured the concentration of TGF-β in 811 samples of male and female bone from four representative sites of the human skeleton and in the supernatants of 72 short-term human bone marrow cultures from the iliac crest. The concentrations of TGF-β1 and TGF-β2 in the bone matrix were positively correlated with histomorphometric indices of bone resorption and bone formation and with serum levels of osteocalcin and bone-specific alkaline phosphatase. We also observed a positive association between the release of TGF-β in the bone marrow cultures and serum osteocalcin. Changes in the rate of cancellous or cortical bone remodeling with age or menopause were accompanied by corresponding changes in skeletal TGF-β. In contrast, there was no significant relationship between the concentration of TGF-β and bone volume at any skeletal site. In conclusion, our study supports the hypothesis that TGF-β plays an important role in human bone remodeling, but fails to demonstrate an association between the skeletal concentration of TGF-β and human bone mass.
Human Growth Hormone Locally Released in Bone Sites by Calcium-Phosphate Biomaterial Stimulates Ceramic Bone Substitution Without Systemic Effects: A Rabbit Study
Authors: Guicheux Jérôme , Gauthier Olivier , Aguado Eric , Pilet Paul , Couillaud Séverine , Jegou Dominique , Daculsi Guy , Heymann Dominique
Published: April 2006

Calcium-phosphate bone replacement biomaterial has been used as a drug carrier for therapeutic agents. This study investigated the efficacy of local administration of human growth hormone (hGH) by macroporous biphasic calcium phosphate (MBCP) implants in improving the bone substitution qualities of ceramics. hGH release from MBCP implants loaded with 1 μg of hGH was rapid during the first 48 h and then sustained for a total of 9 days. Immunolocalization of hGH in vitro and in vivo by transmission electron microscopy showed its presence inside the material, indicating that it was able to penetrate within the porosity of the ceramic during the adsorption process. MBCP cylinders (6 × 6 mm) were loaded with 0.1, 1, and 10 μg of hGH and implanted into rabbit femurs (n = 40). The effects of locally released hGH on bone ingrowth and ceramic resorption were evaluated by scanning electron microscopy and image analysis. The results indicated that hGH increased bone ingrowth (+65%) and ceramic resorption (+140%) significantly in comparison with control implants and that the increase was dose dependent. Biochemical parameters monitored in rabbit plasma and urine, as well as the absence of any significant difference between contralateral implants and the control, indicated that hGH did not produce detectable systemic effects. Thus, the use of MBCP appears to be effective for local delivery of hGH, resulting in improved bone substitution.
A Unitary Model for Involutional Osteoporosis: Estrogen Deficiency Causes Both Type I and Type II Osteoporosis in Postmenopausal Women and Contributes to Bone Loss in Aging Men
Authors: Riggs B. Lawrence , Khosla Sundeep , Melton L. Joseph
Published: April 2006

We propose here a new unitary model for the pathophysiology of involutional osteoporosis that identifies estrogen (E) deficiency as the cause of both the early, accelerated and the late, slow phases of bone loss in postmenopausal women and as a contributing cause of the continuous phase of bone loss in aging men. The accelerated phase in women is most apparent during the first decade after menopause, involves disproportionate loss of cancellous bone, and is mediated mainly by loss of the direct restraining effects of E on bone cell function. The ensuing slow phase continues throughout life in women, involves proportionate losses of cancellous and cortical bone, and is associated with progressive secondary hyperparathyroidism. This phase is mediated mainly by loss of E action on extraskeletal calcium homeostasis which results in net calcium wasting and increases in the level of dietary calcium intake required to maintain bone balance. Because elderly men have low circulating levels of both bioavailable E and bioavailable testosterone (T) and because recent data suggest that E is at least as important as T in determining bone mass in aging men, E deficiency may also contribute substantially to the continuous bone loss of aging men. In both genders, E deficiency increases bone resorption and may also impair a compensatory increase in bone formation. For the most part, this unitary model is well supported by observational and experimental data and provides plausible explanations to traditional objections to a unitary hypothesis.
Combination of Interleukin-6 and Soluble Interleukin-6 Receptors Induces Differentiation and Activation of JAK-STAT and MAP Kinase Pathways in MG-63 Human Osteoblastic Cells
Authors: Nishimura Riko , Moriyama Keiji , Yasukawa Kiyoshi , Mundy Gregory R. , Yoneda Toshiyuki
Published: April 2006

Studies on the role of interleukin-6 (IL-6) in bone metabolism have been accumulating. However, its effects on osteoblasts are still unclear because the results are conflicting depending on the study models employed. We reasoned that these conflicting data are due to variable expression levels of membrane-bound IL-6 receptors (IL-6Rs). In the present study, we found that IL-6 in combination with soluble IL-6R (sIL-6R) consistently caused a marked elevation of alkaline phosphatase and a decrease in proliferation in the human osteoblastic cell line MG-63, which expressed no detectable membrane-bound IL-6R and failed to respond to IL-6. These effects of IL-6/sIL-6R were blocked by neutralizing antibodies to the IL-6 signal transducer gp130, suggesting an involvement of IL-6 signaling in the elicitation of the effects of IL-6/sIL-6R. Upon stimulation with IL-6/sIL-6R, the gp130, cytoplasmic Janus kinases JAK1 and JAK2 were tyrosine phosphorylated. Moreover, signal transducers and activators of transcription STAT1 and STAT3 were also tyrosine phosphorylated, translocated to the nucleus, and bound to the putative STAT-binding DNA elements. In addition, mitogen-activated protein (MAP) kinase was also activated in response to IL-6/sIL-6R. These data demonstrate that sIL-6R may enhance the responsiveness of MG-63 cells to IL-6. Thus, IL-6 in collaboration with sIL-6R may modulate differentiation and proliferation of osteoblastic cells, presumably by activating two distinct signaling pathways of JAK-STAT and MAP kinase.
Alendronate/Interleukin-1β Cotreatment Increases Interleukin-6 in Bone and UMR-106 Cells: Dose Dependence and Relationship to the Antiresorptive Effect of Alendronate
Authors: Sanders Jennifer L. , Tarjan Gabor , Foster Shirley A. , Stern Paula H.
Published: April 2006

Aminobisphosphonates inhibit bone resorption but have been shown to elicit acute-phase-like elevations in interleukin-6 (IL-6) in bone in vitro. The current studies were carried out to determine the relationship between the antiresorptive effects of the aminobisphosphonate alendronate and its effects on IL-6. Resorption was elicited in cultured 19-day fetal rat limb bones by 72 h treatment with interleukin-1β (IL-1β). Bone mass was quantitated at the end of the culture period to assess resorption. IL-6 was determined by bioassay (7TD1 cell proliferation). IL-1β (18 and 180 pM) stimulated bone resorption and increased IL-6. Alendronate (70 μM) inhibited the IL-1β–stimulated resorption. Alendronate alone did not affect IL-6 production by the bones. The IL-6 production from bones stimulated with 18 pM IL-1β was not significantly affected by alendronate, but the IL-6 production from bones stimulated with 180 pM IL-1β plus alendronate (21 and 70 μM) was higher than with IL-1β alone. Indomethacin (1 mM) inhibited the IL-6 increase elicited by 180 pM IL-1β and the enhanced IL-6 production elicited by cotreatment with IL-1β and alendronate. Since bone cultures contain multiple cell types, further experiments were carried out to determine whether alendronate could increase IL-1β–stimulated IL-6 production in an osteoblast cell line, UMR-106. Alendronate alone did not affect IL6 in UMR-106 cells. Alendronate (70 μM) in combination with IL-1β (180, 1.8, or 8 nM), or 7 μM alendronate, in combination with 8 nM IL-1β, significantly increased IL-6 in 48 h cell cultures. The results from the bone organ cultures show that alendronate can enhance IL-6 production elicited by higher concentrations of the cytokine IL-1β in bone, but that this effect on IL-6 does not prevent the inhibitory actions of alendronate on bone resorption. The results with the UMR106 cells indicate that one cellular site at which this enhancement of IL-6 production can occur is the osteoblast.
Vascular Pericytes Express Osteogenic Potential In Vitro and In Vivo
Authors: Doherty Mary Jo , Ashton Brian A. , Walsh Susan , Beresford Jon N. , Grant Michael E. , Canfield Ann E.
Published: April 2006

At postconfluence, cultured bovine pericytes isolated from retinal capillaries form three-dimensional nodule-like structures that mineralize. Using a combination of Northern and Southern blotting, in situ hybridization, and immunofluorescence we have demonstrated that this process is associated with the stage-specific expression of markers of primitive clonogenic marrow stromal cells (STRO-1) and markers of cells of the osteoblast lineage (bone sialoprotein, osteocalcin, osteonectin, and osteopontin). To demonstrate that the formation of nodules and the expression of these proteins were indicative of true osteogenic potential, vascular pericytes were also inoculated into diffusion chambers and implanted into athymic mice. When recovered from the host, chambers containing pericytes were found reproducibly to contain a tissue comprised of cartilage and bone, as well as soft fibrous connective tissue and cells resembling adipocytes. This is the first study to provide direct evidence of the osteogenic potential of microvascular pericytes in vivo. Our results are also consistent with the possibility that the pericyte population in situ serves as a reservoir of primitive precursor cells capable of giving rise to cells of multiple lineages including osteoblasts, chondrocytes, adipocytes, and fibroblasts.
Colony-Stimulating Factor-1 Stimulates the Fusion Process in Osteoclasts
Authors: Amano Hitoshi , Yamada Shoji , Felix Rolf
Published: April 2006

Colony-stimulating factor-1 (CSF-1), also called macrophage colony-stimulating factor, is required for growth, differentiation, activation, and survival of cells of the mononuclear phagocytic system. This cytokine has been shown to be essential for osteoclast development as well as for inducing both proliferation and differentiation of osteoclast progenitors. It also sustains survival of mature osteoclasts and stimulates spreading and migration of these cells. In the present in vitro study, the formation of large tartrate-resistant acid phosphatase (TRAP)-positive cells with a high number of nuclei was observed when osteoclasts isolated from rat long bones were incubated with CSF-1. These large cells, cultured on plastic, bind calcitonin and form F-actin along the edges of the cells. Fusion to such large TRAP-positive multinucleated cells in the presence of CSF-1 and the formation of pits were also observed on dentine slices. Quantitative data obtained from cultures on plastic demonstrated that the number of osteoclasts slightly increased in the course of 72 h in the presence of 250 pM CSF-1, whereas it decreased rapidly after 24 h in the absence of CSF-1, which confirms that this cytokine is required for the survival of osteoclasts. The number of nuclei per osteoclast was maximal after 16 h of incubation with CSF-1, namely twice the value found in the absence of CSF-1. The maximal effect of the cytokine on the fusion process was observed at a concentration of 250 pM. A calculation of the medians of the average frequency of nuclei distribution per osteoclast resulted in four nuclei per osteoclast in the absence and six in the presence of CSF-1. Genistein and herbimycin A, inhibitors of tyrosine kinases, inhibited the fusion induced by CSF-1. The data suggest that CSF-1 induces osteoclast fusion and that tyrosine kinase(s) are involved in this process. The fusion process may continue throughout the entire life of an osteoclast.
Regulation of Interleukin-6 Production by Prostaglandin E2 in Fetal Rat Osteoblasts: Role of Protein Kinase A Signaling Pathway
Authors: Millet Isabelle , Mccarthy Thomas L. , Vignery AgnÈS
Published: April 2006

Prostaglandin E2 (PGE2) is an abundant eicosanoid in bone that has been implicated in a number of pathological states associated with bone loss. Interleukin-6 (IL-6) is a cytokine that plays a critical role in bone remodeling and appears to act as a downstream effector of most bone-resorbing agents. In light of the evidence that PGE2 induces IL-6 in the bone environment, this study was designed to investigate whether PGE2 regulated IL-6 expression by osteoblasts. Here we demonstrate that PGE2 is a potent inducer of IL-6 production by fetal rat osteoblasts and synergizes with lipopolysaccharide to enhance IL-6. We show that PGE2 stimulates the activity of the IL-6 promoter in osteoblasts, suggesting that PGE2 controls IL-6 gene expression at least at the transcriptional level. Moreover, we show that PGE2-mediated IL-6 induction is prevented by the cAMP antagonist, Rp-cAMP, and the protein kinase A (PKA) inhibitors, KT5720 and H89. Thus, our data indicate that PGE2 involves the cAMP–PKA signaling pathway to regulate IL-6 gene expression in osteoblasts.
Generation of Mouse Osteoclastogenic Cell Lines Immortalized with SV40 Large T Antigen
Authors: Chen Wei , Li Yi-Ping
Published: April 2006

Progress in the field of osteoclast gene regulation has been hampered significantly by the lack of such cell lines. In this study, mouse osteoclast precursor cells were elicited in an osteoclast-inductive coculture system and immortalized using SV40 large T antigen. One of the osteoclast precursor cell lines (MOCP-5) forms 95% tartrate-resistant acid phosphatase positive (TRAP+) multinuclear osteoclast-like cells (OCLs) in the coculture system. The yield of TRAP+ OCLs was 4.5–7 × 104 cells per 10 cm2 dish. Expression of SV40 large T antigen was visualized in the nucleus of MOCP-5 cells and OCLs by immunohistochemistry. MOCP-5 cells were positive for MoMa-2 antigen and nonspecific esterase but negative for F4/80 antigen. OCLs derived from MOCP-5 cells were able to form extensive resorption bone pits on bone slices. The resorbing activity of the OCLs was comparable to that of authentic mouse osteoclasts. Pit formation was inhibited by salmon calcitonin (CT). Acid production by OCLs was demonstrated by vital staining with acridine orange. The OCLs expressed cathepsin K and CT receptors. MOCP-5 cells could be transfected by a construct that carries the β-galactosidase gene. Transfected MOCP-5 cells expressing β-galactosidase retain the ability to differentiate into OCLs, indicating a useful model for osteoclast gene regulation. To date, the MOCP-5 cell line has been maintained in continuous culture for 23 months and has maintained the capacity to differentiate into osteoclasts throughout this time. In summary, these data show that a stable immortalized osteoclast precursor cell line has been established and that the immortalization with SV40 large T oncogene does not prevent osteoclast precursor cell differentiation.
Bone Mineral Density and Its Change in Pre- and Perimenopausal White Women: The Michigan Bone Health Study
Authors: Sowers Maryfran , Crutchfield Mary , Bandekar Rajesh , Randolph John F. , Shapiro Brahm , Schork M. Anthony , Jannausch Mary
Published: April 2006

There is a need to better understand potential bone mineral density (BMD) loss during the menopausal transition since this period may include the initiation of interventions. The study purpose was to determine if there was BMD loss at the femoral neck, lumbar spine, or total body bone sites in a population-based study of women approaching or transitioning the midlife. The 583 enrollees were 25–45 years of age at the first of four annual measurements from 1992 through 1996. Bone mineral content and bone width were measured using dual-energy X-ray absorptiometry. Considering all enrollees collectively, there was a significant 3-year decline (1%) in BMD at the femoral neck over the 3-year period (p = 0.0076). There was no significant annual change in the lumbar spine (p = 0.11), and a significant annual increase in the total body BMD (p = 0.0003). Within subgroups and cross-sectionally, BMD values of the femoral neck were 5% lower in women classified as perimenopausal compared with premenopausal enrollees; BMD was 3% and 1% lower at the lumbar spine and total body sites, respectively. Longitudinally, among perimenopausal women, a double oophorectomy was associated with BMD loss in the spine (p = 0.0003), even though 75–85% of these women had a hormone replacement prescription at some time during the study period. In summary, the site with evidence of loss was the femoral neck, specifically among perimenopausal women. There was little evidence of substantial total body or lumbar spine BMD loss in premenopausal women with ovaries who maintained follicle-stimulating hormone levels < 20 mIU/l in the early follicular period. Double oophorectomy, even with hormone replacement, was associated with bone loss.
Type of Physical Activity, Muscle Strength, and Pubertal Stage as Determinants of Bone Mineral Density and Bone Area in Adolescent Boys
Authors: Nordström Peter , Pettersson Ulrika , Lorentzon Ronny
Published: April 2006

The present study was conducted to evaluate the influence of different types of weight-bearing physical activity, muscle strength, and puberty on bone mineral density (BMD, g/cm2) and bone area in adolescent boys. Three different groups were investigated. The first group consisted of 12 adolescent badminton players (age 17.0 ± 0.8 years) training for 5.2 ± 1.9 h/week. The second group consisted of 28 ice hockey players (age 16.9 ± 0.3 years) training for 8.5 ± 2.2 h/week. The third group consisted of 24 controls (age 16.8 ± 0.3 years) training for 1.4 ± 1.4 h/week. The groups were matched for age, height, and pubertal stage. BMD, bone mineral content (BMC, g), and the bone area of the total body, lumbar spine, hip, femur and tibia diaphyses, distal femur, proximal tibia, and humerus were measured using dual-energy X-absorptiometry. When adjusting for the difference in body weight between the groups, the badminton players were found to have significantly higher BMD (p < 0.05) of the trochanter and distal femur compared with the ice hockey players despite a significantly lower weekly average training. The badminton players had higher BMD compared with the control group at all weight-bearing BMD sites, except at the diaphyses of the femur and tibia and lumbar spine. The independent predictors of bone density were estimated by adjusting BMC for the bone area in a multivariate analysis among all subjects (n = 64). Accordingly, the bone density of all sites except the spine was significantly related to muscle strength and height, and the bone density of the total body, neck, trochanter, distal femur, and proximal tibia was significantly related to type of physical activity (β = 0.09–0.33, p < 0.05). The bone area values at different sites were strongly related to muscle strength and height and less strongly related to the type of physical activity and pubertal stage. In conclusion, it seems that during late puberty in adolescent boys the type of weight-bearing physical activity is an important determinant of bone density, while the bone area is largely determined by parameters related to body size. The higher BMD at weight-bearing sites in badminton players compared with ice hockey players, despite significantly less average weekly training, indicates that physical activity including jumps in unusual directions has a great osteogenic potential.
A Dual-Label Immunofluorometric Assay for Human Osteocalcin
Authors: Ylikoski Alice , Hellman Jukka , Matikainen Terhi , Käkönen Sanna-Maria , Karp Matti , Väänänen H. Kalervo , Lövgren Timo , Pettersson Kim
Published: April 2006

Circulating human osteocalcin (hOC) has been shown to be comprised of two main forms: the intact 1–49 form and the proteolytic N-terminal midfragment (N-mid) spanning amino acid residues 1–43 or 1–44. We used three monoclonal antibodies (MAbs) raised against hOC and bovine osteocalcin in developing a dual-label assay for the simultaneous measurement of the proportions of the intact and N-mid forms in serum samples. The assay is based on time-resolved fluorescence utilizing differently labeled tracer MAbs. Biotinylated MAb 2H9 is used as a capture antibody for both the intact hOC and the N-mid. Tracer MAb 6F9 labeled with a Europium(III)-chelate binds to the N-mid and the intact hOC, whereas tracer MAb 3G8 labeled with a Terbium(III)-chelate binds to the intact hOC only. The simultaneous binding of the antibodies was tested by comparing full-length hOC purified from human bone and hOC shortened from the C terminus by four amino acid residues with carboxypeptidase Y. Serum hOC measurements with the dual-label assay were in agreement with the corresponding single-label assays (r = 0.96 for intact + N-mid assays and r = 0.81 for intact assays, n = 91). The lower correlation between the intact assays was attributable to proteolytic susceptibility of the intact form due to one additional freezing and thawing cycle in carrying out the dual-label assay. As measured with the dual-label assay, the levels (mean ± SD) of serum intact + N-mid OC were 6.2 ± 2.1 ng/ml in the premenopausal group (n = 44), 13.9 ± 4.9 ng/ml in the postmenopausal group without hormone replacement therapy (HRT; n = 13), and 7.5 ± 3.4 ng/ml in the postmenopausal group with HRT (n = 13). The levels of intact hOC in the same groups were 4.8 ± 1.4 ng/ml, 9.8 ± 2.9 ng/ml, and 5.3 ± 2.1 ng/ml, respectively. Whether the main forms of OC or their relative proportions in serum can be used for predicting bone diseases or for monitoring the progression and management of diseases awaits further investigations.
Extracellular Calcium (Ca2+o)-Sensing Receptor in a Mouse Monocyte-Macrophage Cell Line (J774): Potential Mediator of the Actions of Ca2+o on the Function of J774 Cells
Authors: Yamaguchi Toru , Kifor Olga , Chattopadhyay Naibedya , Bai Mei , 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. Macrophage-like mononuclear cells appear at sites of osteoclastic bone resorption during bone remodeling and may play a role 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 bone marrow mononuclear cells in the vicinity, leading us to investigate whether such mononuclear cells express the CaR. In this study, we used the mouse J774 cell line, which exhibits a pure monocyte-macrophage phenotype. Both immunocytochemistry and Western blot analysis, using polyclonal antisera specific for the CaR, detected CaR protein in J774 cells. The use of reverse transcriptase-polymerase chain reaction with CaR-specific primers, including a set of intron-spanning primers, followed by nucleotide sequencing of the amplified products, also identified CaR transcripts in J774 cells. Exposure of J774 cells to high Ca2+o (2.8 mM or more) or the polycationic CaR agonist, neomycin (100 μM), stimulated both chemotaxis and DNA synthesis in J774 cells. Therefore, taken together, our data strongly suggest that the monocyte-macrophage cell line, J774, possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney.
Cysteine Proteinases and Matrix Metalloproteinases Play Distinct Roles in the Subosteoclastic Resorption Zone
Authors: Everts Vincent , Delaissé Jean-Marie , Korper Wolf , Beertsen Wouter
Published: April 2006

Digestion of calvarial bone by osteoclasts depends on the activity of cysteine proteinases and matrix metalloproteinases (MMPs). It is unknown, however, whether these enzymes act simultaneously or in a certain (time) sequence. In the present study, this was investigated by culturing mouse calvarial bone explants for various time intervals in the presence or absence of selective low molecular weight inhibitors of cysteine proteinases (E-64, Z-Phe-Tyr(O-t-Bu)CHN2 or CA074[Me]) and MMPs (CI-1, CT1166, or RP59794). The explants were morphometrically analyzed at the electron microscopic level. All proteinase inhibitors induced large areas of nondigested demineralized bone matrix adjacent to the ruffled border of actively resorbing osteoclasts. The appearance of these areas proved to be time dependent. In the presence of the cysteine proteinase inhibitors, a maximal surface area of demineralized bone was seen between 4 and 8 h of culturing, whereas the metalloproteinase inhibitors had their maximal effect at a later time interval (between 16 and 24 h). Because different inhibitors of each of the two classes of proteolytic enzymes had the same effects, our data strongly suggest that cysteine proteinases attack the bone matrix prior to digestion by MMPs. In line with the view that a sequence may exist were differences in the amount of proteoglycans (shown with the selective dye cuprolinic blue) in the subosteoclastic demineralized areas induced by the inhibitors. In the presence of the cysteine proteinase inhibitor, relatively high levels of cuprolinic blue precipitates were found, whereas this was less following inhibition of metalloproteinases. These data suggested that cysteine proteinases are important for digestion of noncollagenous proteins. We propose the following sequence in the digestion of calvarial bone by osteoclasts: after attachment of the cell to the mineralized surface an area with a low pH is created which results in dissolution of the mineral, then cysteine proteinases, active at such a low pH, digest part of the bone matrix, and finally, when the pH has increased somewhat, MMPs exert their activity.
Bone Loss, Physical Activity, and Weight Change in Elderly Women: The Dubbo Osteoporosis Epidemiology Study
Authors: Nguyen T. V. , Sambrook P. N. , Eisman J. A.
Published: April 2006

The present study examined the effects of physical activity, weight, and weight change on femoral bone loss in relation to age in elderly women. Baseline and follow-up measurements at an average interval of 2.7 years of femoral neck bone mineral density (BMD) were reanalyzed for 827 women who were part of the Dubbo Osteoporosis Epidemiology Study. Physical activity was assessed based on hours per day spent in each of various activities according to its expected oxygen consumption. The rate of loss of BMD progressively increased with age, i.e., −0.6 ± 0.1, −1.1 ± 0.2, and −2.1 ± 0.6% per year (mean ± SEM) for the 60–69, 70–79, and ≥80 age groups, respectively (p < 0.001). After adjustment for age, physically inactive women lost more (−1.4 ± 0.2% per year; p < 0.001), compared with physically active women (−0.5 ± 0.3% per year; p = 0.15). Thinner women experienced more rapid bone loss, and women whose weight decreased (≥5%) over the study period lost more bone (−1.7 ± 0.4% per year) than those whose weight was stable (−0.8 ± 0.1% per year) or increased (+0.1 ± 0.3% per year; p < 0.01, analysis of variance). Furthermore, women whose BMD was high (>0.81 g/cm2) at baseline experienced greater loss (−1.1 ± 0.2%) compared with those in the middle tertile (1.0 ± 0.2%) or lowest tertile (−0.5 ± 0.3%). Independent predictors of rate of bone loss included age, baseline BMD, weight, weight change, and physical activity; collectively these factors accounted for 13% of total variance of bone loss by multiple regression analysis. It is concluded that a physically active lifestyle and stable weight in the later decades of life may retard proximal femur bone loss and thus contribute to reduction of fracture risk.
Falls Among Community-Dwelling Elderly in Japan
Authors: Aoyagi Kiyoshi , Ross Philip D. , Davis James W. , Wasnich Richard D. , Hayashi Takuo , Takemoto Tai-Ichiro
Published: April 2006

Japanese have a lower incidence of hip fracture than Caucasians despite having lower bone mass. Hip fractures usually occur after a fall, and differing incidence rates of falls might explain the observed differences in hip fracture rates. To explore this hypothesis, we studied falls and related conditions among 1534 (624 men, 910 women) community-dwelling people aged 65 years and over in Japan and compared the prevalence of falls to Japanese-Americans living in Hawaii and to published studies of Caucasians. In Japan, 9% of the men and 19% of the women reported one or more falls during the past year. The prevalence of falls increased with age in both genders and was greater among women compared with men. In logistic regression models, having musculoskeletal disease, physical disability or limited activity increased the risk of falls by two to four times in both genders. Most fallers (92%) reported fear of future falls, and about one third of fallers reported that they went out less often as a result of their falls. Compared with native Japanese, the age-standardized prevalence of falls among Japanese-Americans was similar but about twice as high for Caucasians, which may explain the lower hip fracture risk of Japanese.
Randomized Controlled Study of Effects of Sudden Impact Loading on Rat Femur
Authors: Järvinen Teppo L. N. , Kannus Pekka , Sievänen Harri , Jolma Pasi , Heinonen Ari , Järvinen Markku
Published: April 2006

Physical loading creating high peak strains on the skeleton at high strain rates is suggested to be the most effective type of activity in terms of bone mineral acquisition. This study assessed the effects of sudden impact loading on mineral and mechanical bone properties in 13-week-old Sprague-Dawley rats. The rats were randomly assigned as sedentary controls (SED, n = 10), control animals receiving low-intensity exercise (EX, n = 15), and experimental animals receiving low-intensity exercise combined with sudden impact-loading (EX + IMP, n = 15). In the EX group, the rats walked in a walking mill at a speed of 10 cm/s for 20 minutes/day, 5 days/week for 9 weeks. In the EX + IMP group, the program was identical to the EX group except for the additional sudden impacts administered to their skeleton during the walking exercise. At the start, there were 50 impacts per session, after which their number was gradually increased to 200 impacts per session by week 6 and then kept constant until the end of the experiment, week 9. These horizontally and vertically directed body impacts were produced by a custom-made walking mill equipped with computer-controlled high-pressure air cylinders. After sacrifice, both femora of each rat were removed and their dimensions, bone mineral content (BMC) by dual-energy X-ray absorptiometry, and mechanical properties by femoral shaft three-point bending and femoral neck compression were determined. The cortical wall thickness increased significantly in the EX and EX + IMP groups as compared with SEDs (+7.6%, p = 0.049 and +10%, p = 0.020, respectively). The EX + IMP group showed +9.0% (p = 0.046) higher cross-sectional moment of inertia values than the EX group. No significant intergroup differences were seen in the BMC values, while the breaking load of the femoral shaft (EX + IMP vs. SED +8.8%, p = 0.047) and femoral neck (EX + IMP vs. SED +14.1%, p = 0.013) was significantly enhanced by the impact loading. In conclusion, this study indicates that mechanical loading can substantially improve the mechanical characteristics of a rat femur without simultaneous gain in its mineral mass. If this is true in humans too, our finding gives an interesting perspective to the numerous longitudinal exercise studies (of women) in which the exercise-induced gains in bone mass and density have remained mild to moderate only.
Effect of Recombinant Human Bone Morphogenetic Protein-2 on Fracture Healing in a Goat Tibial Fracture Model
Authors: Welch Robert D. , Jones Alan L. , Bucholz Robert W. , Reinert Charles M. , Tjia Jane S. , Pierce William A. , Wozney John M. , Li X. Jian
Published: April 2006

Bone morphogenetic proteins (BMPs) are considered to have important regulatory roles in skeletal embryogenesis and bone healing. Recombinant human BMPs (rhBMPs) have been shown to heal critical size defects and promote spinal fusion. We studied the effects of rhBMP-2 in an absorbable collagen sponge (ACS) on bone healing in a large animal tibial fracture model. Bilateral closed tibial fractures were created in 16 skeletally mature goats and reduced and stabilized using external fixation. In each animal, one tibia received the study device (0.86 mg of rhBMP-2/ACS or buffer/ACS), and the contralateral fracture served as control. The device was implanted as a folded onlay or wrapped circumferentially around the fracture. Six weeks following fracture, the animals were sacrificed and the tibiae harvested for torsional testing and histomorphologic evaluation. Radiographs indicated increased callus at 3 weeks in the rhBMP-2/ACS treated tibiae. At 6 weeks, the rhBMP-2/ACS wrapped fractures had superior radiographic healing scores compared with buffer groups and controls. The rhBMP-2/ACS produced a significant increase in torsional toughness (p = 0.02), and trends of increased torsional strength and stiffness (p = 0.09) compared with fracture controls. The device placed in a wrapped fashion around the fracture produced significantly tougher callus (p = 0.02) compared with the onlay application. Total callus new bone volume was significantly increased (p = 0.02) in the rhBMP-2/ACS fractures compared with buffer groups and controls regardless of the method of device application. The rhBMP-2/ACS did not alter the timing of onset of periosteal/endosteal callus formation compared with controls. Neither the mineral apposition rates nor bone formation rates were affected by rhBMP-2/ACS treatment. The increased callus volume associated with rhBMP-2 treatment produced only moderate increases in strength and stiffness.
Dual Signaling and Ligand Selectivity of the Human PTH/PTHrP Receptor
Authors: Takasu Hisashi , Guo Jun , Bringhurst F. Richard
Published: April 2006

Parathyroid hormone (PTH) activates PTH/PTH-related peptide-related receptors (PTHRs) to stimulate both adenylyl cyclase (AC) and phospholipase C (PLC). How these parallel signals mediate specific cellular and tissue responses to PTH, such as the complex anabolic versus catabolic actions of PTH on bone, remains unsettled. Previous studies of PTHR signaling and function employed mainly rodent or other cell lines that express endogenous PTHRs and, possibly, alternate species of PTH receptors. To preclude confounding effects of such receptors, we stably expressed recombinant human PTHRs (hPTHRs) at different levels of surface density in LLC-PK1 porcine renal epithelial cells that lack endogenous PTH responsiveness. hPTH(1–34) induced concentration-dependent activation of both AC and PLC via transfected hPTHRs. Maximal intensity of each signal increased with receptor density, but more hPTHRs were required for PLC than for AC activation. Coupling to AC was saturated at receptor densities too low to detect sustained PLC activation. hPTH(3–34), found by others to be a PLC/protein kinase C (PKC)-selective peptide in rat cells, did not activate PLC via human (or rat) PTHRs under conditions (1 μM peptide, 106 hPTHRs/cell) where hPTH(1–34) stimulated PLC severalfold. Other cellular responses that require PKC activation in these cells, such as sodium-dependent phosphate transport and cAMP-independent secretion of plasminogen activator, were induced by PTH(1–34) but not by hPTH(3–34) or hPTH(7–34). We conclude that amino-truncated PTH analogs reported to activate PKC cannot directly activate phosphatidylinositol-specific PLC via the human or rat PTHR and therefore that PTH receptors may access alternate, PLC-independent pathways of PKC activation in some target cells. The relative intensity of AC and PLC signaling via the hPTHR may be strongly regulated by changes in its surface expression.
Site-Specific Effects of Strength Training on Bone Structure and Geometry of Ultradistal Radius in Postmenopausal Women
Authors: Adami Silvano , Gatti Davide , Braga Vania , Bianchini Donatella , Rossini Maurizio
Published: April 2006

Knowledge of the effects of exercise on bone mass in postmenopausal women is limited and controversial. Animal studies have shown that the response of bone to bending strain is an alteration of bone geometry. We studied 250 postmenopausal women, aged 52–72 years, willing to participate in a 6-month exercise program. The first 125 started the program immediately and the remaining 125 served as controls. The training program included exercises designed to maximize the stress on the wrist. One hundred and eighteen of the active group and 116 of the control group completed the study and were reassessed 6 months later. Bone mineral density (BMD) of the femoral neck, lumbar spine, ultradistal and proximal radius was measured by dual-energy X-ray absorptiometry (DXA) both before and at the end of the exercise program. The forearm was also evaluated by peripheral quantitative computed tomography, which measures the area, bone mineral content (BMC), and volumetric density for both the cortical and the trabecular component. The results showed that the DXA measurements at the femoral neck, lumbar spine, ultradistal and proximal radius were similar between the two groups. No significant difference was detected after the exercise program at the proximal radius. At the ultradistal radius, the cross-sectional area of cortical bone rose by 2.8 ± 15.0% (SD, p < 0.05), apparently for both periosteal apposition and corticalization of the trabecular tissue. The volumetric density of cortical bone rose by 2.2 ± 15.8% (p < 0.1), and that of trabecular bone decreased by 2.6 ± 10.7% (p < 0.01). The combined changes in both bone volume and density in the exercise group were associated with marked increase in cortical BMC (3.1 ± 10.7%, p < 0.01) and decrease in trabecular BMC (−3.4 ± 14.2%, p < 0.05), which were statistically different from those observed in the control group (p < 0.05). In conclusion, these results confirm that site-specific moderate physical exercises have very little effect on bone mass. However, it appears that some exercises may reshape the bone segment under stress by increasing both the cross-sectional area and the density of the cortical component. These structural changes are theoretically associated with increases in the bending strength.
Skeletal Unloading Causes Resistance of Osteoprogenitor Cells to Parathyroid Hormone and to Insulin-like Growth Factor-I
Authors: Kostenuik Paul J. , Harris Jonathan , Halloran Bernard P. , Turner Russell T. , Morey-Holton Emily R. , Bikle Daniel D.
Published: April 2006

Skeletal unloading decreases bone formation and osteoblast number in vivo and decreases the number and proliferation of bone marrow osteoprogenitor (BMOp) cells in vitro. We tested the ability of parathyroid hormone (PTH) to stimulate BMOp cells in vivo by treating Sprague Dawley rats (n = 32) with intermittent PTH(1–34) (1 h/day at 8 μ g/100 g of body weight), or with vehicle via osmotic minipumps during 7 days of normal weight bearing or hind limb unloading. Marrow cells were flushed from the femur and cultured at the same initial density for up to 21 days. PTH treatment of normally loaded rats caused a 2.5-fold increase in the number of BMOp cells, with similar increases in alkaline phosphatase (ALP) activity and mineralization, compared with cultures from vehicle-treated rats. PTH treatment of hind limb unloaded rats failed to stimulate BMOp cell number, ALP activity, or mineralization. Hind limb unloading had no significant effect on PTH receptor mRNA or protein levels in the tibia. Direct in vitro PTH challenge of BMOp cells isolated from normally loaded bone failed to stimulate their proliferation and inhibited their differentiation, suggesting that the in vivo anabolic effect of intermittent PTH on BMOp cells was mediated indirectly by a PTH-induced factor. We hypothesize that this factor is insulin-like growth factor-I (IGF-I), which stimulated the in vitro proliferation and differentiation of BMOp cells isolated from normally loaded bone, but not from unloaded bone. These results suggest that IGF-I mediates the ability of PTH to stimulate BMOp cell proliferation in normally loaded bone, and that BMOp cells in unloaded bone are resistant to the anabolic effect of intermittent PTH therapy due to their resistance to IGF-I.
Studies of the Murine Homolog of the Multiple Endocrine Neoplasia Type 1 (MEN1) Gene, men1
Authors: Bassett J. H. D. , Rashbass P. , Harding B. , Forbes S. A. , Pannett A. A. J. , Prof. Thakker R. V.
Published: April 2006

The murine homolog of the multiple endocrine neoplasia type 1 (MEN1) gene (men1), which in humans is associated with tumors of the parathyroids, pancreas, and pituitary, has been characterized by isolating 27 clones from a mouse embryonic stem cell cDNA library. The insert sizes ranged from 600–2500 bp, and sequence analysis identified a 1833 bp open reading frame encoding a 611 amino acid protein. In addition, two clones contained an unspliced intron 1, and another two clones contained 20–29 bp of an upstream sequence, which suggested the presence of an alternate exon 1. This was supported by an analysis of the homologous human sequence. The mouse and human coding regions had 89% and 96% identity of the nucleotide and amino acid sequences, respectively. Investigation of clones isolated from a 129ola mouse genomic library, revealed the men1 gene to consist of 10 exons that spanned ∼6 kb. Northern blot analysis demonstrated the ubiquitous expression of 2.9 kb and 3.4 kb transcripts in mouse adult tissues and embryos from 7 days. DNA sequence analysis of the larger 3.4 kb transcript revealed it to result from a retention of intron 1. In situ hybridization confirmed an early ubiquitous expression in whole mount mouse embryos and adult tissues, but in the latter, different levels of cellular expression were observed, e.g., men1 expression was higher in testicular Sertoli cells than in germ cells. Thus, the mouse men1 gene and the basis of alternative transcripts have been defined, and these will help to facilitate studies of a mouse model.
The Integrin αvβ5 Is Expressed on Avian Osteoclast Precursors and Regulated by Retinoic Acid
Authors: Sago Karen , Teitelbaum Steven L. , Venstrom Kristy , Reichardt Louis F. , Ross F. Patrick
Published: April 2006

Osteoclasts arise by proliferation, differentiation, and subsequent fusion of marrow-derived precursors, all processes requiring attachment to matrix. Integrins are important mediators of cell-matrix recognition and bone is rich in proteins containing the Arg-Gly-Asp motif, recognized primarily by αv integrins. Thus, we determined if avian osteoclast precursors express integrins capable of mediating initial attachment to matrix proteins. Early, marrow-derived osteoclast precursors, when first isolated, contain no detectable αvβ3, but express an αv integrin with an 80 kDa associated β subunit. Immunoprecipitation with an antibody raised against the conserved β5 cytoplasmic tail sequence indicates the the αv associated the integrin is αvβ5. Retinoic acid is a resorptive steroid, and its exposure to early osteoclast precursors prompts a time- and dose-dependent decrease in αvβ5 expression, while simultaneously stimulating αvβ3 expression. Northern analysis reveals that retinoic acid decreases β5 steady-state mRNA, nontranscriptionally, without altering that of αv. The finding αvβ5 expression decreases under the influence of retinoic acid, an osteoclastogenic steroid, while those of αvβ3 rise, suggests that these closely related integrins play separate and complementary roles during osteoclast differentiation.