bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Protein Kinase C α in Primary Human Osteoblast Proliferation
Authors: Lampasso J. D. , Marzec N. , Margarone J. , Dziak R.
Published: April 2006

Protein kinase C (PKC) isoforms have been shown to have specific expression profiles and individual isoforms are believed to play distinct roles in the cells in which they are found. The goal here was to determine which specific isoform(s) is involved in proliferation of primary human osteoblasts. In primary human osteoblasts, 10 μM of acute sphingosine-1-phosphate (S1P) treatment induced an increase in proliferation that correlated with an increase in PKCα and PKCι expression. To further delineate which isoforms are involved in osteoblastic cell proliferation, the effect of low versus high serum culture conditions on PKC isoform expression was determined. Likewise, the effect of antisense oligodeoxynucleotides (ODNs) to specific PKC isoforms on proliferation and MAPK activation was studied. The effect of S1P on intracellular translocation of activated PKC isoforms was also evaluated. The results indicated that in primary human osteoblasts, PKCα was not expressed under conditions of low proliferative rate while PKC° and PKCι expression was not affected. The specific inhibition of PKCα by antisense ODNs resulted in inhibition of MAPK activity leading to a significant decrease in proliferation. S1P up-regulated antisense ODN inhibited PKCα expression and MAPK activity and led to an increase in proliferation. Subsequent experiments using platelet-derived growth factor (PDGF) as an additional mitogen generated similar data. PDGF stimulation resulted in a significant increase in proliferation that correlated with an up-regulation of inhibited PKCα expression in antisense ODN-treated cells. Immunofluorescence methods showed that mitogenic stimulation of PKCα resulted in nuclear translocation. Our findings present original data that PKCα is the isoform specifically involved in the proliferation of primary human osteoblasts.
Two-Year Results of Once-Weekly Administration of Alendronate 70 mg for the Treatment of Postmenopausal Osteoporosis
Authors:   The Alendronate Once-Weekly Study Group, Rizzoli R
Published: April 2006

The aim of this study was to provide confirmation that once-weekly dosing with 70 mg of alendronate (seven times the daily oral dose) and twice-weekly dosing with 35 mg is equivalent to the 10-mg once-daily regimen and to gain more extensive safety experience with this new dosing regimen. Twelve hundred fifty-eight postmenopausal women (aged 42–95 years) with osteoporosis (bone mineral density [BMD] of either lumbar spine or femoral neck at least 2.5 SDs below peak young adult mean or prior vertebral or hip fracture) were assigned to receive oral once-weekly alendronate, 70 mg (n = 519); twice-weekly alendronate, 35 mg (n = 369); or daily alendronate 10 mg (n = 370) for a total of 2 years of double-blind experience. Mean BMD increases from baseline (95% CI) at 24 months in the once-weekly, twice-weekly, and daily treatment groups, respectively, were 6.8% (6.4, 7.3), 7.0% (6.6, 7.5), and 7.4% (6.9, 7.8) at the lumbar spine and 4.1% (3.8, 4.5), 4.3% (3.9, 4.7), and 4.3% (3.9, 4.7) at the total hip. These increases in BMD as well as the BMD increases at the femoral neck, trochanter, and total body and the reductions of biochemical markers of bone resorption (urinary cross-linked N-telopeptides of type I collagen [NTx]) and bone formation (serum bone-specific alkaline phosphatase [BSAP]) were similar for the three dosing regimens. All treatment regimens were well tolerated with a similar incidence of upper gastrointestinal (GI) adverse experiences. The incidence rates of clinical fractures, captured as adverse experiences, were similar among the groups. The 2-year results confirm the conclusion reached after 1 year that once-weekly alendronate is therapeutically equivalent to daily dosing, providing patients with a more convenient dosing option that may potentially enhance adherence to therapy.
Association of Polymorphisms of the Estrogen Receptor α Gene With Bone Mineral Density and Fracture Risk in Women: A Meta-Analysis
Authors: Ioannidis John P. A. , Stavrou Ioanna , Trikalinos Thomas A. , Zois Christos , Brandi Maria Luisa , Gennari Luigi , Albagha Omar , Ralston Stuart H. , Tsatsoulis Agathocles ,   For the ER-α Genetics Meta-Analysis
Published: April 2006

The contribution of genetic polymorphisms to bone mineral density (BMD) and fracture risk in women is a controversial topic. We evaluated the effect of the XbaI and PvuII polymorphisms of the estrogen receptor α to BMD and fracture risk in a meta-analysis, including published data and additional information from investigators. Five thousand eight hundred thirty-four women from 30 study groups were analyzed with fixed and random effects models. The PvuII polymorphism was not associated with BMD at any skeletal site examined and 95% CIs exclude effects over 0.015 g/cm2 for both the femoral neck and the lumbar spine. Conversely, XX homozygotes (women carrying two copies of the gene variant without an XbaI restriction site) consistently had higher BMD than other subjects. The magnitude of the effect was similar in the femoral neck and lumbar spine (0.014 g/cm2 [95% CI, 0.003–0.025] and 0.015 g/cm2 [95% CI, 0.000–0.030], respectively; no between-study heterogeneity for either). Total body BMD was also significantly higher in XX homozygotes (by 0.039 g/cm2 and 0.029 g/cm2 compared with Xx and xx, respectively). Available data on fractures suggested a protective effect for XX (odds ratio [OR], 0.66 [95% CI, 0.47–0.93] among 1591 women), but not PP (OR, 0.93 [95% CI, 0.72–1.18] among 2229 women). In summary, we have found that XX homozygotes may have higher BMD and also a decreased risk of fractures when compared with carriers of the x allele, whereas the PvuII polymorphism is not associated with either BMD or fracture risk.
When Should Densitometry Be Repeated in Healthy Peri- and Postmenopausal Women: The Danish Osteoporosis Prevention Study
Authors: Abrahamsen B. , Nissen N. , Hermann A. P. , Hansen B. , Bärenholdt O. , Vestergaard P. , Tofteng C. L. , Nielsen S. Pors
Published: April 2006

Intervention should be considered in postmenopausal women with bone mineral density (BMD) ≥1 SD below the reference (T or Z score < −1). However, it is unclear when densitometry should be repeated. This study aimed at determining the need for repeat DXA within 5 years in untreated peri-/postmenopausal women to detect declines of T or Z score to below −1 with 85% confidence. A cohort of 925 healthy women (aged 51.2 ± 2.9 years) were followed within the Danish Osteoporosis Prevention Study (DOPS) for 5 years without hormone-replacement therapy (HRT). DXA of spine, hip, and forearm was done at 0,1, 2, 3, and 5 years (Hologic QDR-1000/2000). The annual loss in SD units was 0.12 ± 0.10 at the spine (1.3%), 0.10 ± 0.09 at the femoral neck (1.2%), and 0.07 ± 0.09 at the ultradistal (UD) forearm (1.0%). Accordingly, T scores below −1 developed earlier at the spine. The need for a future DXA scan to predict declines of T and Z scores below −1 depended strongly on baseline BMD. In subjects with a positive T score, the risk of developing T < −1 remained at <15% for 5 years at all measured sites. A new scan was needed after 1 year if the T score was below −0.5, and after 3 years if the T score was between 0 and −0.5. Slightly longer intervals apply if Z scores are used. Follow-up densitometry in untreated women should be individually targeted from baseline BMD rather than scheduled at fixed time intervals. An algorithm for planning repeat densitometry in perimenopausal women is provided.
In Vivo RANK Signaling Blockade Using the Receptor Activator of NF-κB:Fc Effectively Prevents and Ameliorates Wear Debris-Induced Osteolysis via Osteoclast Depletion Without Inhibiting Osteogenesis
Authors: Childs Lisa M. , Paschalis Eleftherios P. , Xing Lianping , Dougall William C. , Anderson Dirk , Boskey Adele L. , Puzas J. Edward , Rosier Randy N. , O'Keefe Regis J. , Boyce Brendan F. , Schwarz Edward M.
Published: April 2006

Prosthesis failure due to wear debris-induced osteolysis remains a major clinical problem and the greatest limitation for total joint arthroplasty. Based on our knowledge of osteoclast involvement in this process and the requirements of receptor activator of NF-κB (RANK) signaling in osteoclastogenesis and bone resorption, we investigated the efficacy of RANK blockade in preventing and ameliorating titanium (Ti)-induced osteolysis in a mouse calvaria model. Compared with placebo controls we found that all doses of RANK:Fc above 1 mg/kg intraperitoneally (ip) per 48 h significantly inhibited osteoclastogenesis and bone resorption in response to Ti implanted locally. Complete inhibition occurred at 10 mg/kg ip per 48 h, yielding results that were statistically equivalent to data obtained with Ti-treated RANK−/− mice. We also evaluated the effects of a single injection of RANK:Fc on day 5 on established osteolysis and found that Ti-treated were still depleted for multinucleated tartrate-resistant acid phosphatase-positive (TRAP+) cells 16 days later. More importantly, this osteoclast depletion did not affect bone formation because the bone lost from the osteolysis on day 5 was restored by day 21. An assessment of the quantity and quality of the newly formed bone in these calvariae by calcein labeling and infrared (IR) microscopy, respectively, showed no significant negative effect of RANK:Fc treatment. These studies indicate that osteoclast depletion via RANK blockade is an effective method to prevent and reverse wear debris-induced osteolysis without jeopardizing osteogenesis.
Periodontal Ligament Cells Under Mechanical Stress Induce Osteoclastogenesis by Receptor Activator of Nuclear Factor κB Ligand Up-Regulation via Prostaglandin E2 Synthesis
Authors: Kanzaki Hiroyuki , Chiba Mirei , Shimizu Yoshinobu , Mitani Hideo
Published: April 2006

Previously, we discovered that periodontal ligament (PDL) cells not only support osteoclastogenesis through cell-to-cell contact, but also inhibit the formation of tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated cells by a producing soluble factor(s). Furthermore, PDL cells express both receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) messenger RNA (mRNA). Clinically, “ankylosed teeth,” which lack periodontal ligament, cannot be moved with orthodontic tooth treatment. From this, we hypothesized that PDL cells under mechanical stress should play a pivotal role in osteoclast formation during orthodontic tooth movement. This study examined how mechanical stress affects the osteoclastogenesis-supporting activity of PDL cells. PDL cells were compressed continuously and then cocultured with peripheral blood mononuclear cells (PBMCs) for 4 weeks. PDL cells under mechanical stress up-regulated osteoclastogenesis from PBMCs. Furthermore, the expression of RANKL mRNA and protein in PDL cells increased with compressive force in parallel with the change in the number of osteoclasts. In addition, cyclo-oxygenase 2 (COX-2) mRNA expression was induced by compressive force, and indomethacin inhibited the RANKL up-regulation resulting from compressive force. PDL cells under compressive force exhibited significantly increased prostaglandin E2 (PGE2) production in comparison with control PDL cells. Exogenous PGE2 treatment increased RANKL mRNA expression in PDL cells. Interestingly, OPG expression remained constant throughout compressive force or PGE2 treatment. In conclusion, compressive force up-regulated RANKL expression in PDL cells. Furthermore, RANKL up-regulation in mechanically stressed PDL cells was dependent on PGE2.
Recombinant Human Bone Morphogenetic Protein-2 Enhances Osteotomy Healing in Glucocorticoid-Treated Rabbits
Authors: Luppen C. A. , Blake C. A. , Ammirati K. M. , Stevens M. L. , Seeherman H. J. , Wozney J. M. , Bouxsein M. L.
Published: April 2006

The objectives of this study were to evaluate the effect of chronic prednisolone treatment on osteotomy healing in rabbits and to determine whether recombinant human bone morphogenetic protein-2 (rhBMP-2) would enhance healing in the presence of chronic glucocorticoid therapy. Forty-nine skeletally mature, male rabbits were injected with either prednisolone (n = 26; 0.35 mg/kg per day, three times a week) or saline (n = 23). After a 6-week pretreatment period, bilateral ulnar osteotomies were created surgically. One osteotomy was treated with rhBMP-2 (0.2 mg/ml of rhBMP-2, 40 μg of rhBMP-2 total) delivered on an absorbable collage sponge (ACS), whereas the contralateral osteotomy remained untreated. Prednisolone or saline treatment was continued until the rabbits were killed either 6 weeks or 8 weeks after creation of the osteotomy. Osteotomy healing was evaluated by radiography, peripheral quantitative computed tomography (pQCT), torsional biomechanics, and undecalcified histology. Because we observed similar responses to both prednisolone and rhBMP-2/ACS treatment in the 6-week and 8-week cohorts, the results from these time points were combined. Serum osteocalcin and vertebral trabecular bone density were lower in the prednisolone-treated rabbits. Prednisolone treatment dramatically inhibited osteotomy healing. In the untreated ulnas, callus area and torsional strength were 25% and 55% less, respectively, in the prednisolone-treated rabbits than in the saline group (p < 0.001 for both). rhBMP-2/ACS enhanced healing in both the prednisolone- and the saline-treated groups, although the effect was larger in the prednisolone-treated rabbits. In the prednisolone-treated rabbits, callus area and torsional strength were 40% and 165% greater (p < 0.001 for both), respectively, in osteotomies treated with rhBMP-2/ACS compared with the contralateral, untreated osteotomies. Histological evaluation confirmed that osteotomy healing was inhibited by prednisolone and accelerated by rhBMP-2/ACS. In summary, a single application of rhBMP-2/ACS counteracted the inhibition of osteotomy healing caused by prednisolone exposure. These results suggest that rhBMP-2/ACS may be a useful treatment for enhancing fracture healing in patients who are undergoing chronic glucocorticoid therapy.
Ontogeny of Phex/PHEX Protein Expression in Mouse Embryo and Subcellular Localization in Osteoblasts
Authors: Thompson D. L. , Sabbagh Y. , Tenenhouse H. S. , Roche P. C. , Drezner M. K. , Salisbury J. L. , Grande J. P. , Poeschla E. M. , Kumar R.
Published: April 2006

PHEX, a phosphate-regulating gene with homologies to endopeptidases on the X chromosome, is mutated in X-linked hypophosphatemia (XLH) in humans and mice (Hyp). Although recent observations indicate that Phex protein is expressed primarily in bone and may play an important role in osteoblast function and bone mineralization, the pattern of the Phex protein expression in the developing skeleton and its subcellular localization in osteoblasts remain unknown. We examined the ontogeny of the Phex protein in the developing mouse embryo and its subcellular localization in osteoblasts using a specific antibody to the protein. Immunohistochemical staining of mouse embryos revealed expression of Phex in osteogenic precursors in developing vertebral bodies and developing long bones on day 16 postcoitum (pc) and thereafter. Calvaria from day 18 pc mice showed Phex epitopes in osteoblasts. No Phex immunoreactivity was detected in lung, heart, hepatocytes, kidney, intestine, skeletal muscle, or adipose tissue of mouse embryos. Interestingly, embryonic mouse skin showed moderate amounts of Phex immunostaining. In postnatal mice, Phex expression was observed in osteoblasts and osteocytes. Moderate expression of Phex was seen in odontoblasts and slight immunoreactivity was observed in ameloblasts. Confocal microscopy revealed the presence of immunoreactive PHEX protein in the Golgi apparatus and endoplasmic reticulum of osteoblasts from normal mice and in osteoblasts from Hyp mice transduced with a human PHEX viral expression vector. PHEX protein was not detected in untransduced Hyp osteoblasts. These data indicate that Phex protein is expressed in osteoblasts and osteocytes during the embryonic and postnatal periods and that within bone, Phex may be a unique marker for cells of the osteoblast/osteocyte lineage.
Overexpressed LIM Mineralization Proteins Do Not Require LIM Domains to Induce Bone
Authors: Liu Yunshan , Hair Gregory A. , Boden Scott D. , Viggeswarapu Manjula , Titus Louisa
Published: April 2006

Rat LIM mineralization protein 1 (LMP-1, an LIM domain protein) mediates bone morphogenetic protein 6 (BMP-6) induction of bone nodule formation in fetal rat calvarial osteoblast (ROB) cultures. We have isolated the complementary DNA (cDNA) for the human homologue of LMP-1 from an adult human heart cDNA library and showed that when overexpressed it is osteoinductive in the same culture system. The recently revised cDNA sequence of Enigma, the protein product of which binds to the insulin receptor and the tyrosine kinase receptor ret, now matches the nucleotide sequence of human LMP-1 (hLMP-1). A truncated, 223 amino acid (AA) LMP-1(t) protein has identical effects as the full-length protein, despite the deletion of the LIM domains. Two splice variants of human LMP-1 have been detected. Human LMP-2 has a 119-base pair (bp) deletion between bp 325 and 444 and a 17-bp insertion at bp 444. The resulting derived protein contains 423 AA with the LIM domains intact and does not induce bone formation when overexpressed in ROB cultures. Human LMP-3 has the same 17 nucleotide insertion at bp 444, resulting in a shift in the reading frame that causes a stop codon to occur at bp 505-507. The resulting 153 AA protein does not have the LIM domains, but overexpression of hLMP-3 induces bone formation in osteoblast cultures. These findings suggest that the LIM domains are not required for LMPs to induce bone formation. In addition, a small region (36 AA) of the LMP-1 protein may be required for bone formation.
Differential Temporal Expression of Members of the Transforming Growth Factor β Superfamily During Murine Fracture Healing
Authors: Cho Tae-Joon , Gerstenfeld Louis C. , Einhorn Thomas A.
Published: April 2006

Fracture healing is a unique postnatal repair process in which the events of endochondral and intramembranous bone formation follow a definable temporal sequence. The temporal patterns of messenger RNA (mRNA) expression for members of the transforming growth factor β (TGF-β) superfamily were examined over a 28-day period of fracture healing in mouse tibias. Bone morphogenetic protein 2 (BMP-2) and growth and differentiation factor 8 (GDF8) showed maximal expression on day 1 after fracture, suggesting their roles as early response genes in the cascade of healing events. Restricted expression of GDF8 to day 1, in light of its known actions as a negative regulator of skeletal muscle growth, suggests that it may similarly regulate cell differentiation early in the fracture healing process. GDF5, TGF-β2, and TGF-β3 showed maximal expression on day 7, when type II collagen expression peaked during cartilage formation. In contrast, BMP-3, BMP-4, BMP-7, and BMP-8 showed a restricted period of expression from day 14 through day 21, when the resorption of calcified cartilage and osteoblastic recruitment were most active. TGF-β1, BMP-5 and BMP-6, and GDF10 were constitutively expressed from day 3 to day 21. However, during the same time period, GDF3, GDF6, and GDF9 could not be detected, and GDF1 was expressed at extremely low levels. These findings suggest that several members of the TGF-β superfamily are actively involved in fracture healing and although they are closely related both structurally and functionally, each has a distinct temporal expression pattern and potentially unique role in fracture healing.
Down-Regulation of Osteoclast Differentiation by Daidzein via Caspase 3
Authors: Rassi Claudia Maria , Lieberherr Michele , Chaumaz Gilles , Pointillart Alain , Cournot Giulia
Published: April 2006

Phytoestrogens are plant-derived compounds with estrogen-like activity. Phytoestrogen-rich diets may prevent postmenopausal osteoporosis and these molecules maintain bone mass in ovariectomized animals. We compared the effects of the isoflavone daidzein, which has no action on tyrosine kinases, and 17β-estradiol on the development and activity of osteoclasts in vitro. Nonadherent porcine bone marrow cells were cultured on dentine slices or on culture slides in the presence of 10−8 M of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], with or without 10−8 M of daidzein, 10−8 M of 17β-estradiol for 9-11 days. Multinucleated tartrate-resistant acid phosphatase-positive (TRAP+) cells that resorbed bone (osteoclasts) developed in the presence of 1,25(OH)2D3. The number of osteoclasts formed in response to 1,25(OH)2D3 was reduced by 58 ± 8% by daidzein and 52 ± 5% by estrogen (p < 0.01); these effects were reversed by 10−6 M of ICI 182,780. The area resorbed by mature osteoclasts was reduced by 39 ± 5% by daidzein and 42 ± 6% by estradiol (p < 0.01). Both compounds also inhibited the 1,25(OH)2D3-induced differentiation of osteoclast progenitors (mononucleated TRAP+ cells), 53 ± 8% by daidzein and 50 ± 7% by estradiol (p < 0.05). Moreover, daidzein and estradiol promoted caspase-8 and caspase-3 cleavage and DNA fragmentation of monocytic bone marrow cells. Caspase-3 cleavage was reversed by 10−8 M of ICI 182,780. Both compounds up-regulated the expression of nuclear estrogen receptors ER-α and ER-β. Thus, daidzein, at the same concentration as 17β-estradiol, inhibits osteoclast differentiation and activity. This may be caused by, at least in part, greater apoptosis of osteoclast progenitors mediated by ERs.
Incidence of Vertebral Fracture in Europe: Results From the European Prospective Osteoporosis Study (EPOS)
Authors:   Dr. D AJ M G AA JD WC D LI A Armas JB C J R B W S K I J O JA G Vaz A R G P C T G Hap G HH DM W Nave JJ CJ K AD OB J Terence W. Felsenberg Silman Lunt Armbrecht Ismail Finn Cockerill Banzer Benevolenskaya Bhalla Bruges Cannata Cooper Dequeker Eastell Felsch Gowin Havelka Hoszowski Jajic Janott Johnell Kanis Kragl Lopes Lorenc Lyritis Masaryk Matthis Miazgowski Parisi Pols Poor Raspe Reid Reisinger Scheidt Stepan Todd Weber Woolf Yershova Reeve O'Neill
Published: April 2006

Vertebral fracture is one of the major adverse clinical consequences of osteoporosis; however, there are few data concerning the incidence of vertebral fracture in population samples of men and women. The aim of this study was to determine the incidence of vertebral fracture in European men and women. A total of 14,011 men and women aged 50 years and over were recruited from population-based registers in 29 European centers and had an interviewer-administered questionnaire and lateral spinal radiographs performed. The response rate for participation in the study was approximately 50%. Repeat spinal radiographs were performed a mean of 3.8 years following the baseline film. All films were evaluated morphometrically. The definition of a morphometric fracture was a vertebra in which there was evidence of a 20% (+4 mm) or more reduction in anterior, middle, or posterior vertebral height between films—plus the additional requirement that a vertebra satisfy criteria for a prevalent deformity (using the McCloskey-Kanis method) in the follow-up film. There were 3174 men, mean age 63.1 years, and 3614 women, mean age 62.2 years, with paired duplicate spinal radiographs (48% of those originally recruited to the baseline survey). The age standardized incidence of morphometric fracture was 10.7/1000 person years (pyr) in women and 5.7/1000 pyr in men. The age-standardized incidence of vertebral fracture as assessed qualitatively by the radiologist was broadly similar—12.1/1000 pyr and 6.8/1000 pyr, respectively. The incidence increased markedly with age in both men and women. There was some evidence of geographic variation in fracture occurrence; rates were higher in Sweden than elsewhere in Europe. This is the first large population-based study to ascertain the incidence of vertebral fracture in men and women over 50 years of age across Europe. The data confirm the frequent occurrence of the disorder in men as well as in women and the rise in incidence with age.
Why Is the Age-Standardized Incidence of Low-Trauma Fractures Rising in Many Elderly Populations?
Authors: Kannus Pekka , Niemi Seppo , Parkkari Jari , Palvanen Mika , Heinonen Ari , Sievänen Harri , Järvinen Teppo , Khan Karim , Järvinen Markku
Published: April 2006

Low-trauma fractures of elderly people are a major public health burden worldwide, and as the number and mean age of older adults in the population continue to increase, the number of fractures is also likely to increase. Epidemiologically, however, an additional concern is that, for unknown reasons, the age-standardized incidence (average individual risk) of fracture has also risen in many populations during the recent decades. Possible reasons for this rise include a birth cohort effect, deterioration in the average bone strength by time, and increased average risk of (serious) falls. Literature provides evidence that the rise is not due to a birth cohort effect, whereas no study shows whether bone fragility has increased during this relatively short period of time. This osteoporosis hypothesis could, however, be tested if researchers would now repeat the population measurements of bone mass and density that were made in the late 1980s and the 1990s. If such studies proved that women's and men's age-standardized mean values of bone mass and density have declined over time, the osteoporosis hypothesis would receive scientific support. The third explanation is based on the hypothesis that the number and/or severity of falls has risen in elderly populations during the recent decades. Although no study has directly tested this hypothesis, a great deal of indirect epidemiologic evidence supports this contention. For example, the age-standardized incidence of fall-induced severe head injuries, bruises and contusions, and joint distortions and dislocations has increased among elderly people similarly to the low-trauma fractures. The fall hypothesis could also be tested in the coming years because the 1990s saw many research teams reporting age- and sex-specific incidences of falling for elderly populations, and the same could be done now to provide data comparing the current incidence rates of falls with the earlier ones.
Kinetic Characterization of Hypophosphatasia Mutations With Physiological Substrates
Authors: Di Mauro Sonia , Manes Thomas , Hessle Lovisa , Kozlenkov Alexey , Pizauro João Martins , Hoylaerts Marc F. , Millán José Luis
Published: April 2006

We have analyzed 16 missense mutations of the tissue-nonspecific AP (TNAP) gene found in patients with hypophosphatasia. These mutations span the phenotypic spectrum of the disease, from the lethal perinatal/infantile forms to the less severe adult and odontohypophosphatasia. Site-directed mutagenesis was used to introduce a sequence tag into the TNAP cDNA and eliminate the glycosylphosphatidylinositol (GPI)-anchor recognition sequence to produce a secreted epitope-tagged TNAP (setTNAP). The properties of GPI-anchored TNAP (gpiTNAP) and setTNAP were found comparable. After introducing each single hypophosphatasia mutation, the setTNAP and mutant TNAP cDNAs were expressed in COS-1 cells and the recombinant flagged enzymes were affinity purified. We characterized the kinetic behavior, inhibition, and heat stability properties of each mutant using the artificial substrate p-nitrophenylphosphate (pNPP) at pH 9.8. We also determined the ability of the mutants to metabolize two natural substrates of TNAP, that is, pyridoxal-5′-phosphate (PLP) and inorganic pyrophosphate (PPi), at physiological pH. Six of the mutant enzymes were completely devoid of catalytic activity (R54C, R54P, A94T, R206W, G317D, and V365I), and 10 others (A16V, A115V, A160T, A162T, E174K, E174G, D277A, E281K, D361V, and G439R) showed various levels of residual activity. The A160T substitution was found to decrease the catalytic efficiency of the mutant enzyme toward pNPP to retain normal activity toward PPi and to display increased activity toward PLP. The A162T substitution caused a considerable reduction in the pNPPase, PPiase, and PLPase activities of the mutant enzyme. The D277A mutant was found to maintain high catalytic efficiency toward pNPP as substrate but not against PLP or PPi. Three mutations (E174G, E174K, and E281K) were found to retain normal or slightly subnormal catalytic efficiency toward pNPP and PPi but not against PLP. Because abnormalities in PLP metabolism have been shown to cause epileptic seizures in mice null for the TNAP gene, these kinetic data help explain the variable expressivity of epileptic seizures in hypophosphatasia patients.
Differential Expression of Estrogen Receptor-Related Receptor α and Estrogen Receptors α and β in Osteoblasts In Vivo and In Vitro
Authors: Bonnelye Edith , Aubin Jane E.
Published: April 2006

The orphan nuclear estrogen receptor-related receptor (ERR) α is expressed by osteoblastic cells, is known to transactivate at least one osteoblast-associated gene osteopontin (OPN) and plays a functional role in osteoprogenitor cell proliferation and differentiation. To dissect further the role of ERR-α in bone formation, we compared its expression to that of the estrogen receptor (ER) α and ER-β in rat calvaria (RC) and fetal tibia in vivo and in RC and rat bone marrow (RBM) cells in vitro. We found that ERR-α is highly and widely expressed in most, if not all, cells in RC cell cultures from early proliferation stages through mineralized nodule formation; ER-α was localized similarly but at lower levels and ER-β, although present, was barely detectable. These patterns of expression in vitro correlated with what we observed in vivo in sections of 21-day fetal RC, in which ERR-α appeared to be more highly expressed than either of the ERs. Interestingly, ERR-α also is highly expressed in RBM cells, while ER-α and ER-β mRNA is expressed, but at lower levels. Moreover, we found that ERR-α, ER-α, and ER-β were all expressed in osteoblasts in fetal and adult tibia whereas they were expressed differentially in calvaria in vivo in subsets of osteoblasts, supporting the hypothesis that ERR-α may interact with one or both of the ERs in those osteoblasts in which they are coexpressed and that all three receptors may be required for bone formation but at different times and for different functions.
In Vitro Engineering of Human Autogenous Cartilage
Authors: Anderer Ursula , Dr. Libera Jeanette
Published: April 2006

A challenge in tissue engineering is the in vitro generation of human cartilage. To meet standards for in vitro-engineered cartilage, such as prevention of immune response and structural as well as functional integration to surrounding tissue, we established a three-dimensional cell culture system without adding exogenous growth factors or scaffolds. Human chondrocytes were cultured as spheroids. Tissue morphology and protein expression was analyzed using histological and immunohistochemical investigations on spheroid cryosections. A cartilage-like tissue similar to naturally occurring cartilage was generated when spheroids were cultured in medium supplemented only with human serum. This in vitro tissue was characterized by the synthesis of the hyaline-specific proteins collagen type II and S-100, as well as the synthesis of hyaline-specific mucopolysaccharides that increased with prolonged culture time. After 3 months, cell number in the interior of in vitro tissues was diminished and was only twice as much as in native cartilage. Additionally, spheroids quickly adhered to and migrated on glass slides and on human condyle cartilage. The addition of antibiotics to autologous spheroid cultures inhibited the synthesis of matrix proteins. Remarkably, replacing human serum by fetal calf serum resulted in the destruction of the inner part of the spheroids and only a viable rim of cells remained on the surface. These results show that the spheroid culture allows for the first time the autogenous in vitro engineering of human cartilage-like tissue where medium supplements were restricted to human serum.
Autosomal Dominant Hypocalcemia Caused by a Novel Mutation in the Loop 2 Region of the Human Calcium Receptor Extracellular Domain
Authors: Hu Jianxin , Mora Stefano , Colussi Giacomo , Proverbio Maria Carla , Jones Kendra A. , Bolzoni Laura , De Ferrari Maria E. , Civati Giovanni , Spiegel Allen M.
Published: April 2006

We report a novel missense mutation N124K in the extracellular calcium receptor (CaR) identified in two related subjects with the phenotypic features of autosomal dominant hypocalcemia (ADH). Expression of the N124K mutant receptor created by site-directed mutagenesis and transfected into HEK-293 cells was comparable with that of the wild-type (WT) receptor and two other mutant receptors N118K and L125P identified in subjects with ADH. Functional characterization by the extracellular Ca2+ ion ([Ca2+]0)-stimulated phosphoinositide (PI) hydrolysis in transfected HEK-293 cells showed that the N124K mutant receptor was left-shifted in Ca2+ sensitivity. This biochemical gain-of-function is comparable with that seen in other missense mutations of the CaR identified in subjects with ADH. We tested a series of missense substitutions (R, Q, E, and G) in addition to K for N124 and found that only the N124K mutation and to a much lesser extent N124R caused a left shift in Ca2+ sensitivity. Thus, a specific substitution, not merely a mutation of the N124 residue, is required for receptor activation. The N124K mutation is one of eight naturally occurring mutations in subjects with ADH identified in a short segment A116-C129 of the CaR extracellular domain (ECD). We present a hypothesis to explain receptor activation by mutations in this region based on the recently described three-dimensional structure of the related metabotropic glutamate type 1 receptor (mGluR1).
Colocalization of Intracellular Osteopontin With CD44 Is Associated With Migration, Cell Fusion, and Resorption in Osteoclasts
Authors: Suzuki K. , Zhu B. , Rittling S. R. , Denhardt D. T. , Goldberg H. A. , Mcculloch C. A. G. , Sodek J.
Published: April 2006

Although osteopontin (OPN) is recognized generally as a secreted protein, an intracellular form of osteopontin (iOPN), associated with the CD44 complex, has been identified in migrating fibroblastic cells. Because both OPN and CD44 are expressed at high levels in osteoclasts, we have used double immunofluorescence analysis and confocal microscopy to determine whether colocalization of these proteins has functional significance in the formation and activity of osteoclasts. Analysis of rat bone marrow-derived osteoclasts revealed strong surface staining for CD44 and β1- and β3-integrins, whereas little or no staining for OPN or bone sialoprotein (BSP) was observed in nonpermeabilized cells. In permeabilized perfusion osteoclasts and multinucleated osteoclasts, staining for OPN and CD44 was prominent in cell processes, including filopodia and pseudopodia. Confocal microscopy revealed a high degree of colocalization of OPN with CD44 in motile osteoclasts. In cells treated with cycloheximide (CHX), perinuclear staining for OPN and BSP was lost, but iOPN staining was retained within cell processes. In osteoclasts generated from the OPN-null and CD44-null mice, cell spreading and protrusion of pseudopodia were reduced and cell fusion was impaired. Moreover, osteoclast motility and resorptive activity were significantly compromised. Although the area resorbed by OPN-null osteoclasts could be rescued partially by exogenous OPN, the resorption depth was not affected. These studies have identified an intracellular form of OPN, colocalizing with CD44 in cell processes, that appears to function in the formation and activity of osteoclasts.
Prevention of Bone Loss in Ovariectomized Rats by Combined Treatment With Risedronate and 1α,25-Dihydroxyvitamin D3
Authors: Erben Reinhold G. , Mosekilde Lis , Thomsen Jesper S. , Weber Karin , Stahr Kerstin , Leyshon Alyson , Smith Susan Y. , Phipps Roger
Published: April 2006

Bisphosphonates inhibit bone loss through inhibition of osteoclast-mediated bone resorption. At low doses, vitamin D metabolites can prevent bone loss in models of osteopenia in rats by an antiresorptive effect, while at high doses they also stimulate osteoblast activity and show an anabolic effect. Therefore, combined therapy with bisphosphonates and vitamin D analogs might be expected to be more effective than either treatment alone. It was the aim of this study to compare the efficacy of risedronate and of the naturally occurring vitamin D hormone 1α,25-dihydroxyvitamin D3 (calcitriol), alone and in combination, for the prevention of ovariectomy-induced bone loss in rats. One hundred ten female 4-month-old Sprague-Dawley rats were used for this experiment. Ninety rats were bilaterally ovariectomized (OVX), 10 rats were sham-operated (SHAM), and 10 rats were killed at the time of surgery as a baseline control. Groups of rats (10 rats/group) received vehicle or daily doses of 0.1 mg or 0.5 mg of risedronate or 0.05 μg or 0.1 μg of calcitriol/kg body weight, alone and in combination. Both compounds were administered orally via gavage, commencing on the day after surgery. Although estrogen deficiency-induced bone loss was prevented by individual prophylactic administration of risedronate or calcitriol, OVX rats treated with a combination of risedronate and calcitriol had higher bone mineral density (BMD), cancellous bone area (B.Ar), and bone strength in long bones and vertebrae compared with rats receiving risedronate alone. Furthermore, calcitriol enhanced the suppressive effects of risedronate on osteoclast number and partially counteracted the suppressive effects of risedronate on bone formation and histomorphometric indices of osteoblast team performance. Risedronate did not reduce the anabolic effect of calcitriol, and at the high dose it normalized hypercalcemia in calcitriol-treated OVX rats. Therefore, this study in OVX rats suggests that combined therapy with bisphosphonates and vitamin D analogs may offer advantages over the treatment with bisphosphonates or vitamin D analogs alone.
Is Leptin the Link Between Fat and Bone Mass?
Authors: Thomas Thierry , Burguera Bartolome
Published: April 2006

Recently, leptin has emerged as a potential candidate responsible for protective effects of fat on bone tissue. However, it remains difficult to draw a clear picture of leptin effects on bone metabolism because published data are sometimes conflicting or apparently contradictory. Beyond differences in models or experimental procedures, it is tempting to hypothesize that leptin exerts dual effects depending on bone tissue, skeletal maturity, and/or signaling pathway. Early in life, leptin could stimulate bone growth and bone size through direct angiogenic and osteogenic effects on stromal precursor cells. Later, it may decrease bone remodeling in the mature skeleton, when trabecular bone turnover is high, by stimulating osteoprotegerin (OPG) expression. Leptin negative effects on bone formation effected through central nervous system pathway could counterbalance these peripheral and positive effects, the latter being predominant when the blood‐brain barrier permeability decreases or the serum leptin level rises above a certain threshold. Thus, the sex‐dependent specificity of the relationship between leptin and bone mineral density (BMD) in human studies could be, at least in part, caused by serum leptin levels that are two‐ to threefold higher in women than in men, independent of adiposity. Although these hypotheses remain highly speculative and require further investigations, existing studies consistently support the role of leptin as a link between fat and bone.
Expression of a 1,25‐Dihydroxyvitamin D3 Membrane‐Associated Rapid‐Response Steroid Binding Protein During Human Tooth and Bone Development and Biomineralization
Authors: Mesbah Mohand , Nemere Ilka , Papagerakis Petros , Nefussi Jean‐Raphael , Orestes‐Cardoso Silvana , Nessmann Catherine , Berdal Ariane
Published: April 2006

The calciotropic hormone 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] has been established to control skeletal tissue formation and biomineralization via the regulation of gene expression. This action involves the well‐characterized nuclear 1,25(OH)2D3 receptor. However, it has been recognized that several cellular responses to 1,25(OH)2D3 may not to be related to the exclusive nuclear receptor. Indeed, this secosteroid is able to generate rapid responses that have been proposed to be mediated by interactions of the ligand, which is a putative cell membrane‐associated rapid‐response steroid (MARRS) binding protein for 1,25(OH)2D3 [1,25D3‐MARRS]. The nongenomic pathway of 1,25(OH)2D3 was studied here in detail by immunolocalization of the 1,25D3‐MARRS during the specific context of human prenatal development. Western blotting with proteins extracted from 4 week‐ to 27‐week‐old embryos was performed, evidencing a 65‐kDa molecular species recognized by antibody Ab 099 generated against synthetic peptides corresponding to the N terminus of the 1,25D3‐MARRS from chick intestinal basolateral membranes. Based on this biochemical conservation of protein in the human species, the temporospatial expression patterns were established in the craniofacial skeleton at the same ages. Comparative analysis was performed in teeth and bones from early morphogenesis to terminal cell differentiation and extracellular biomineralization. The data show the potential implication of 1,25D3‐MARRS in the heterogeneous cell population including ameloblasts, odontoblasts, osteoblasts, and osteoclasts. The epithelial‐mesenchymal cascade related to odontogenesis was coincident with a sequence of up‐ and down‐regulation of immunoreactive 1,25D3‐MARRS. Biomineralization was associated with a striking up‐regulation in the adjoining secretory cells in all tissues. Finally, osteoclasts appeared also to express the 1,25D3‐MARRS during these early phases of bone modeling. Previously obtained data of the nuclear vitamin D receptor (VDR) expression and this study on 1,25D3‐MARRS suggest the existence of cross‐talk between the genomic and nongenomic pathways during human development.
Radius Bone Strength in Bending, Compression, and Falling and Its Correlation With Clinical Densitometry at Multiple Sites
Authors: Lochmüller Eva‐Maria , Lill Christoph A. , Kuhn Volker , Schneider Erich , Eckstein Felix
Published: April 2006

This study comprehensively analyzes the ability of site‐specific and nonsite‐specific clinical densitometric techniques for predicting mechanical strength of the distal radius in different loading configurations. DXA of the distal forearm, spine, femur, and total body and peripheral quantitative computed tomography (pQCT) measurements of the distal radius (4, 20, and 33%) were obtained in situ (with soft tissues) in 129 cadavers, aged 80.16 ± 9.8 years. Spinal QCT and calcaneal quantitative ultrasound (QUS) were performed ex situ in degassed specimens. The left radius was tested in three‐point bending and axial compression, and the right forearm was tested in a fall configuration, respectively. Correlation coefficients with radius DXA were r = 0.89, 0.84, and 0.70 for failure in three‐point bending, axial compression, and the fall simulation, respectively. The correlation with pQCT (r = 0.75 for multiple regression models with the fall) was not significantly higher than for DXA. Nonsite‐specific measurements and calcaneal QUS displayed significantly (p < 0.01) lower correlation coefficients, and QUS did only contribute to the prediction of axial failure stress but not of failure load. We conclude that a combination of pQCT parameters involves only marginal improvement in predicting mechanical strength of the distal radius, nonsite‐specific measurements are less accurate for this purpose, and QUS adds only little independent information to site‐specific bone mass. Therefore, the noninvasive diagnosis of loss of strength at the distal radius should rely on site‐specific measurements with DXA or pQCT and may be the earliest chance to detect individuals at risk of osteoporotic fracture.
A New Method for In Vivo Analysis of Parathyroid Hormone–Calcium Set Point in Mice
Authors: Imanishi Yasuo , Hall Charles , Sablosky Marilyn , Brown Edward M. , Arnold Andrew
Published: April 2006

Although methods for measuring the parathyroid hormone (PTH)‐calcium set point in vivo in humans and large animals exist, translating such methods to the increasingly important mouse model poses considerable challenges. We also found that manipulation of dietary calcium does not yield sufficiently high or low serum calcium levels to achieve the minimum and maximum PTH levels needed for set point estimations. Therefore, we developed a new method for in vivo evaluation of the relative set point in mice. Intraperitoneal injection of calcium gluconate caused progressive increases in serum calcium over 120 minutes, with corresponding decreases in serum PTH levels. Intraperitoneal injection of Na2‐EGTA caused a nadir of serum calcium at 30 minutes and a corresponding peak value of serum PTH. The lowest and highest serum calcium concentrations achieved were 6.7 mg/dl and 10.0 mg/dl, respectively. Linear regression analyses indicated high correlation coefficients (serum calcium vs. serum PTH; r = 0.969). To obtain the additional data points needed for set point estimation, blood was collected 30 minutes after intraperitoneal injection by tail nicking from three mice once a day, on 8 consecutive days, using multiple doses of calcium gluconate or Na2‐EGTA. The lowest and highest serum calcium concentrations achieved were 5.0 mg/dl and 11.4 mg/dl. Maximum and minimum PTH levels were indeed observed, and a sigmoidal curve with a set point of 7.8 mg/dl was readily generated by the four‐parameter model that was fit using a nonlinear mixed effects statistical approach. This protocol for in vivo set point analysis should be applicable in the future study of multiple genetically engineered and pharmacologically treated mouse models.
Differential Regulation of Chemokine Secretion in Tuberculous and Staphylococcal Osteomyelitis
Authors: Wright Kathleen M. , Friedland Jon S.
Published: April 2006

Bone infection or osteomyelitis is characterized by uncontrolled inflammation and destructive bone loss although little is known about immunopathogenesis of infection. We investigated control of chemokine secretion from osteoblasts infected with either Mycobacterium tuberculosis, which normally elicits a granulomatous host response, or Staphylococcus aureus, which drives a host response dominated by neutrophil influx. We show that M. tuberculosis infection of cultured and primary osteoblasts induces extensive secretion of the chemokines interleukin (IL)‐8, inducible protein (IP) 10, RANTES, and monocyte chemoattractant protein (MCP) 1 within 72 h (1630 ± 280 pg/ml per 4 × 105 cells, 74,130 ± 8480 pg/ml per 4 × 105 cells, 18,330 ± 3040 pg/ml per 4 × 105 cells, and 13,8670 ± 13,340 pg/ml per 4 × 105 cells, respectively, for MG‐63 osteoblasts). S. aureus infection also results in secretion of these chemokines but secretion is delayed and of lesser magnitude (210 ± 10 pg/ml per 4 × 105 cells, 11,570 ± 1240 pg/ml per 4 × 105 cells, 930 ± 34 pg/ml per 4 × 105 cells, and 13,770 ± 720 pg/ml per 4 × 105 cells for IL‐8, IP‐10, RANTES, and MCP‐1, respectively). The minimal up‐regulation of secretion of the neutrophil attractant IL‐8 in staphylococcal infection is both striking and unexpected. In both infections, chemokine secretion was dependent on the presence of live organisms. Differences in kinetics and magnitude of chemokine secretion are associated with distinct patterns of mRNA expression, as assessed by ribonuclease protection assay (RPA) and reverse‐transcription polymerase chain reaction (RT‐PCR). In addition, nuclear localization of the transcription factor activator protein (AP) 1 in M. tuberculosis‐infected osteoblasts also is distinct as compared with S. aureus‐infected cells. In summary, this study shows that osteoblasts have an important pathogen‐specific role in control of chemokine gene expression and secretion during the human immune response to osteomyelitis.