bluuebunny/crossref_metadata_2025 huggingface dataset:
Bisphosphonate Therapy for Paget's Disease in a Patient with Hypoparathyroidism: Profound Hypocalcemia, Rapid Response, and Prolonged Remission
Authors: Stuckey B. G. A. , Lim E. M. , Kent G. N. , Ward L. C. , Gutteridge D. H.
Published: April 2006

Bisphosphonate treatment for severe Paget's disease leads to hypocalcemia followed by a secondary hyperparathyroid response to restore normocalcemia. A case is presented of a 60-year-old woman with polyostotic Paget's disease and postsurgical hypoparathyroidism. In 1993 her Paget's disease—alkaline phosphatase (ALP), 1260 U/liter (35–135 U/liter), and fasting urinary hydroxyproline excretion, 13.7 μmol/liter GF (0.4–1.9 μmol/liter)—was treated with intravenous pamidronate. Symptomatic hypocalcemia followed the first 60-mg dose, requiring large doses of calcium supplementation and calcitriol. Pamidronate therapy to a total dose of 360 mg was followed by rapid and prolonged remission with indices of bone turnover in the normal range within 2 months and persisting for at least 19 months after treatment. In 1999 relapse of Paget's disease—ALP, 511 U/liter (35–135 U/liter), and fasting urinary deoxypyridinoline/creatinine 53.1 μmol/mol (5–27 μmol/mol)—was treated with alendronate, 10 mg daily. Symptomatic hypocalcemia occurred again, requiring increased calcium and calcitriol therapy. Indices of bone turnover were within the normal range 9 weeks after the start of therapy. These responses were significantly more rapid and sustained than those observed in euparathyroid subjects. This case suggests that the lack of parathyroid response may modify the response to bisphosphonates by: (a) increasing intrinsic uptake of bisphosphonate into the pagetic skeleton, allowing response to a smaller dose; (b) increasing duration and severity of hypocalcemia after bisphosphonate therapy; and (c) removing the hyperparathyroid drive to reactivation of pagetic osteoclasts, leading to a prolonged remission. These observations have implications for optimizing bisphosphonate therapy both in Paget's disease and in osteoporosis.
Historical Vignette: Hypophosphatasia: Molecular Diagnosis of Rathbun's Original Case
Authors: Mumm Steven , Jones Jonathan , Finnegan Patrick , Whyte Michael P.
Published: April 2006

In 1948, Dr. John Campbell Rathbun characterized the disorder “hypophosphatasia” when he reported paradoxically low levels of alkaline phosphatase (ALP) activity in blood and in several tissues from an infant who died with rickets and epilepsy, which seemed to reflect “a new developmental anomaly.” Hypophosphatasia is now recognized to be an inborn error of metabolism featuring deficient activity of the tissue-nonspecific isoenzyme of ALP (TNSALP) caused by deactivating mutations in TNSALP. Here, we show, more than 50 years after Rathbun's case report, that analysis of the parental DNA indicates compound heterozygosity involving two missense mutations (G340A and A881C) in TNSALP caused the death of Rathbun's patient.
Alendronate in the Prevention of Bone Loss After a Fracture of the Lower Leg
Authors: Van Der Poest Clement E. , Van Engeland M. , Adèr H. , Roos J. C. , Patka P. , Lips P.
Published: April 2006

Fracture of a leg and the consequent absence from weight-bearing lead to local bone loss. A 1-year, single-center, prospective, randomized, double-blind study was conducted, to determine whether bone loss would occur in the proximal femur and the calcaneus after a fracture of the lower leg and whether this loss could be prevented by the antiresorptive drug bisphosphonate alendronate. Twenty-three men and 18 women with a recent unstable fracture of the lower leg were randomized to receive either 10 mg of alendronate daily or placebo. Bone mineral density (BMD) of both hips and the lumbar spine was measured at baseline and 6 weeks and 3, 6, and 12 months after start of the treatment. Quantitative ultrasound (QUS) measurements of the calcaneus were performed at baseline on the noninjured side and at 6 weeks and 3, 6, and 12 months after start of treatment on both sides. After 1 year, in the placebo group, there was a significant decrease from baseline in BMD of the hip on the side of the fracture. In the alendronate group, there was no significant change from baseline. The differences in BMD between the two treatment groups on the side of the fracture were significant in all sites of the hip: 4.4% (p = 0.016) in the trochanter, 4.6% (p = 0.016) in the femoral neck, and 3.9% (p = 0.009) in the total hip. In the hip on the contralateral side, there were no significant changes from baseline in either treatment group and there was no difference between the two treatment groups. BMD in the lumbar spine increased in the alendronate group, and after 1 year there was a significant difference between the active treatment and placebo group of 3.4% (p = 0.04). One year after fracture, ultrasound parameters of the calcaneus in the placebo group were significantly lower on the fractured side compared with the contralateral side (p < 0.01). In the alendronate group, no significant difference between the two sides was observed. In conclusion, BMD of the proximal femur was still decreased 1 year after a fracture of the lower leg. Alendronate prevented this bone loss.
Quantitative Magnetic Resonance Imaging in the Calcaneus and Femur of Women With Varying Degrees of Osteopenia and Vertebral Deformity Status
Authors: Wehrli Felix W. , Hilaire Luna , Fernández-Seara Maria , Gomberg Bryon R. , Song Hee Kwon , Zemel Babette , Loh Louise , Snyder Peter J.
Published: April 2006

Quantitative magnetic resonance imaging (QMRI) allows measurement of two parameters that are related to the integrity of the trabecular bone: R2, the rate constant of the free induction signal, and trabecular bone volume fraction (BVF), the counterpart of apparent density. In this work, R2 and BVF were measured in 68 women (mean age, 58.2 ± 9.5 years) of varying spinal bone mineral density (BMD) T scores (mean, −1.37 ± 1.54) and vertebral fracture status on a commercial 1.5 T whole-body imager using customized image acquisition and processing techniques. Twenty-five of the patients had vertebral fractures, characterized by the total cumulative deformity burden exceeding 200%. R2 was measured in the calcaneus and proximal femur and BVF could be measured in the calcaneus only. On a pixel-by-pixel basis, calcaneal R2 and BVF within each subject were highly positively correlated (r2 = 0.61 ± 0.11) but the correlation of region-of-interest (ROI) means for different calcaneal sites among patients was weaker (r2 = 0.34; p < 0.0001). The strongest discriminator of vertebral deformity was R2 of the calcaneus, which was lower in the fracture group, consistent with lower trabecular density. Among the calcaneal sites examined, the subtalar region, a location characterized by dense nearly horizontal trabeculae that transmit the stresses imparted by body weight from the tibia to the heel, best discriminated the two groups (p = 0.0001), with 77% diagnostic accuracy as determined from the area under the receiver operating characteristic (ROC) curve (compared with 66% for vertebral BMD). The cavum calcanei, an anterior site of low trabecular density, and the tuber calcanei (the location ordinarily used for ultrasound measurements) also had significantly reduced R2 in the fracture group (p < 0.005 and p = 0.01, respectively). The R2av, computed as the average of all pixels in the calcaneus, was a strong discriminator as well (p < 0.005). On the other hand, calcaneal BVF was only marginally discriminating (p = 0.05). Among the BMD sites examined, the lumbar spine (average L1-L4) was significant (p = 0.005, 66% diagnostic accuracy), as was the femoral neck (p = 0.01). The data suggest the calcaneus to be suited as a surrogate site to assess vertebral osteoporosis and that R2 is sensitive to alterations in bone quality not captured by density.
Cell Proliferation and Apoptosis During Fracture Healing
Authors: Li Gang , White Grainne , Connolly Chris , Marsh David
Published: April 2006

This study investigated the relation between cell proliferation and apoptosis during fracture healing in a mouse femoral fracture model. Left femoral osteotomies were performed in 30 mature male CFLP mice immobilized with uniplanar external fixators. Six animals were killed on days 2, 4, 8, 16, and 24 postfracture for examination. Localization of cell proliferation was examined using immunohistochemistry with proliferating cell nuclear antigen (PCNA) monoclonal antibody. Apoptotic cells were visualized with the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end-labeling (TUNEL) method. Images of each time-specific specimen were captured. The total callus area, the positively labeled cells by PCNA, and TUNEL per high-power field were quantified. Cell proliferation and apoptosis were found coexisting during the entire period of study. In the early phases of fracture healing (days 2-8), PCNA-positive labeling was predominant and peaked at day 8 and the TUNEL-positive labeling was minimal. In later stages of fracture healing (days 16-24), PCNA expression declined at day 16 as callus ossification and remodeling spread within the fracture site and apoptosis was the dominant cell activity with the TUNEL-positive labeling peaking at day 16 and declining sharply at day 24. These cell activities were reflected by the change of fracture callus, where there was a continuous increase in total callus area to day 16 and subsequent decrease at day 24. This study indicated that cell proliferation and apoptosis are coupled events during fracture repair, cell proliferation is active at the early stages, and apoptosis is active during the phase of callus remodeling.
Anabolic Action of Parathyroid Hormone Is Skeletal Site Specific at the Tissue and Cellular Levels in Mice
Authors: Iida-Klein Akiko , Zhou Hua , Lu Shi Shou , Levine Lance R. , Ducayen-Knowles Mercedes , Dempster David W. , Nieves Jeri , Lindsay Robert
Published: April 2006

The cellular and molecular events triggering the anabolic response of the skeleton to exogenous parathyroid hormone (PTH) are not well understood. Despite the numerous bone mass studies in rats, few data are available for mice. Therefore, we treated 10-week-old female intact C57BL/6J mice with human PTH(1-34) delivered subcutaneously at a dose of 40 μg/kg per day 5 days a week for 3 weeks and 7 weeks. Bone mineral density (BMD) of total bone, femur, tibia, and lumbar vertebrae was measured weekly by PIXImus. Bone turnover was examined by histomorphometry, and gene expression of bone formation and resorption markers and osteoclastogenesis regulators in the excised femur and tibia was assessed by reverse-transcription polymerase chain reaction (RT-PCR) at 3 weeks and 7 weeks. The PTH-stimulated increase in BMD was more prominent in the tibia and femur than in the lumbar vertebrae, with an anabolic effect detected within 1-2 weeks and BMD continuing to increase. The appearance of a detectable PTH-stimulated increase in BMD was slower in the lumbar vertebrae where the increase was only significant after 7 weeks of treatment. Histomorphometric analysis of the proximal tibia at both 3 weeks and 7 weeks indicated significant time-dependent increases in trabecular area, trabecular number, trabecular and cortical widths, and osteoblast and osteoid perimeters. In the lumbar vertebrae, these stimulatory effects of PTH on trabecular area, trabecular number, and cortical width were smaller and not detected until 7 weeks. PTH-stimulated increases in bone turnover were evident by increased gene expression of osteocalcin (OC), tartrate-resistant acid phosphatase (TRAP), and receptor of activator nuclear factor κB (NF-κB) ligand (RANKL) in the tibia and femur. No significant difference in gene expression was observed between the two long bone sites. In conclusion, PTH exerts an anabolic action at the tissue and cellular levels in intact mice and the magnitude and temporal pattern of this anabolic action, as assessed by densitometry and histomorphometry, are skeletal site specific.
Bone Mineral Response to a 7-Month Randomized Controlled, School-Based Jumping Intervention in 121 Prepubertal Boys: Associations With Ethnicity and Body Mass Index
Authors: Mackelvie K. J. , Mckay H. A. , Petit M. A. , Moran O. , Khan K. M.
Published: April 2006

We examined the effects of a 7-month jumping intervention (10 minutes, 3 times per week) on bone mineral gain in prepubertal Asian and white boys (10.3 ± 0.6 years, 36.0 ± 9.2 kg) at 14 schools randomized to control (n = 60) and intervention (n = 61) groups. Intervention and control groups had similar mean baseline and change in height, weight, lean mass and fat mass, baseline areal bone mineral density (aBMD; g/cm2), bone mineral content (BMC; g; dual-energy X-ray absorptiometry [DXA], QDR 4500W), and similar average physical activity and calcium intakes. Over 7 months, the intervention group gained more total body (TB) BMC (1.6%, p < 0.01) and proximal femur (PF) aBMD (1%, p < 0.05) than the control group after adjusting for age, baseline weight, change in height, and loaded physical activity. We also investigated the 41 Asian and 50 white boys (10.2 ± 0.6 years and 31.9 ± 4.4 kg) who were below the 75th percentile (19.4 kg/m2) of the cohort mean for baseline body mass index (BMI). Boys in the intervention group gained significantly more TB and lumbar spine (LS) BMC, PF aBMD, and trochanteric (TR) aBMD (+ ∼2%) than boys in the control group (adjusted for baseline weight, final Tanner stage, change in height, and loaded physical activity). Bone changes were similar between Asians and whites. Finally, we compared the boys in the control group (n = 16) and the boys in the intervention group (n = 14) whose baseline BMI fell in the highest quartile (10.5 ± 0.6 years and 49.1 ± 8.2 kg). Seven-month bone changes (adjusted as aforementioned) were similar in the control and intervention groups. In summary, jumping exercise augmented bone mineral accrual at several regions equally in prepubertal Asian and white boys of average or low BMI, and intervention effects on bone mineral were undetectable in high BMI prepubertal boys.
Neuromuscular and Psychomotor Function in Elderly Subjects Who Fall and the Relationship With Vitamin D Status
Authors: Dr. Dhesi J. K. , Bearne L. M. , Moniz C. , Hurley M. V. , Jackson S. H. D. , Swift C. G. , Allain T. J.
Published: April 2006

Vitamin D and calcium supplementation significantly reduces the incidence of fractures. Evidence suggests vitamin D deficiency impairs neuromuscular function, causing an increase in falls and thereby fractures. The relationship between vitamin D, functional performance, and psychomotor function in elderly people who fall was examined in a prospective cross-sectional study. Patients were recruited from a falls clinic and stratified according to serum 25-hydroxyvitamin-D levels (25OHD): group 1, 25OHD < 12 μg/liter; group 2 25OHD, 12-17 μg/liter; and group 3, 25OHD > 17 μg/liter. Healthy elderly volunteers with 25OHD > 17 μg/liter comprised group 4 (n = 20/group). Measures included aggregate functional performance time (AFPT, seconds), isometric quadriceps strength (Newtons), postural sway (degrees), and choice reaction time (CRT, seconds). Serum bone biochemistry, 25OHD, and parathyroid hormone levels were measured. Patients who fell had significantly impaired functional performance, psychomotor function, and quadriceps strength compared with healthy subjects (AFPT: 51.0 s vs. 32.8 s, p < 0.05; CRT: 1.66 s vs. 0.98 s, p < 0.05; strength: 223N vs. 271N, t = 2.35, p = 0.02). Group 1 had significantly slower AFPT (66.0 s vs. 44.8 s, t = 4.15, p < 0.05) and CRT (2.37 s vs. 0.98 s, t = 3.59, p < 0.05) than groups 2 and 3. Group 1 had the greatest degree of postural sway and the weakest quadriceps strength, although this did not reach significance. Multivariate analysis revealed 25OHD as an independent variable for AFPT, CRT, and postural sway. PTH was an independent variable for muscle strength. Older people who fall have impaired functional performance, psychomotor function, and muscle strength. Within this group, those with 25OHD < 12 μg/liter are the most significantly affected.
Bone Mineral Content per Muscle Cross-Sectional Area as an Index of the Functional Muscle-Bone Unit
Authors: Schoenau Eckhard , Neu Christina Maria , Beck Bodo , Manz Friedrich , Rauch Frank
Published: April 2006

Bone densitometric data often are difficult to interpret in children and adolescents because of large inter- and intraindividual variations in bone size. Here, we propose a functional approach to bone densitometry that addresses two questions: Is bone strength normally adapted to the largest physiological loads, that is, muscle force? Is muscle force adequate for body size? To implement this approach, forearm muscle cross-sectional area (CSA) and bone mineral content (BMC) of the radial diaphysis were measured in 349 healthy subjects from 6 to 19 years of age (183 girls), using peripheral quantitative computed tomography (pQCT). Reference data were established for height-dependent muscle CSA and for the variation with age in the BMC/muscle CSA ratio. These reference data were used to evaluate results from three pediatric patient groups: children who had sustained multiple fractures without adequate trauma (n = 11), children with preterminal chronic renal failure (n = 11), and renal transplant recipients (n = 15). In all three groups mean height, muscle CSA, and BMC were low for age, but muscle CSA was normal for height. In the multiple fracture group and in renal transplant recipients the BMC/muscle CSA ratio was decreased (p < 0.05), suggesting that bone strength was not adapted adequately to muscle force. In contrast, chronic renal failure patients had a normal BMC/muscle CSA ratio, suggesting that their musculoskeletal system was adapted normally to their (decreased) body size. This functional approach to pediatric bone densitometric data should be adaptable to a variety of densitometric techniques.
Localization of the Gene Causing Autosomal Dominant Osteopetrosis Type I to Chromosome 11q12-13
Authors: Van Hul Els , Gram Jeppe , Bollerslev Jens , Van Wesenbeeck Liesbeth , Mathysen Danny , Andersen Poul Erik , Vanhoenacker Filip , Van Hul Wim
Published: April 2006

The osteopetroses are a heterogeneous group of genetic conditions characterized by increased bone density due to impaired bone resorption by osteoclasts. Within the autosomal dominant form of osteopetrosis, the radiological type I (ADOI) is characterized by a generalized osteosclerosis, most pronounced at the cranial vault. The patients are often asymptomatic but some suffer from pain and hearing loss. ADOI is the only type of osteopetrosis not associated with an increased fracture rate. Linkage analysis in two families with ADOI from Danish origin enabled us to assign the disease-causing gene to chromosome 11q12-13. A summated maximum lod score of +6.54 was obtained with marker D11S1889 and key recombinants allowed delineation of a candidate region of 6.6 cM between markers D11S1765 and D11S4113. Previously, genes causing other conditions with abnormal bone density have been identified from this chromosomal region. The TCIRG1gene was shown to underly autosomal recessive osteopetrosis (ARO), and, recently, mutations in the LRP5gene were found both in the osteoporosis-pseudoglioma syndrome and the high bone mass trait. Because both genes map within the candidate region for ADOI, it can not be excluded that ADOI is caused by mutations in either the TCIRG1or the LRP5gene.
Impaired Vascular Invasion of Cbfa1-Deficient Cartilage Engrafted in the Spleen
Authors: Himeno Miki , Enomoto Hirayuki , Liu Wenguang , Ishizeki Kiyoto , Nomura Shintaro , Kitamura Yukihiko , Komori Toshihisa
Published: April 2006

Chondrocyte maturation and vascular invasion of cartilage are essential in the process of endochondral ossification. Cbfa1-deficient (Cbfa1−/−) mice displayed a complete absence of osteoblast and osteoclast maturation as well as severely inhibited chondrocyte maturation in most parts of the skeleton. Although chondrocyte maturation and mineralization were observed in restricted areas of Cbfa1−/− mouse skeleton, vascular invasion of calcified cartilage was never noted. To investigate the possibility of chondrocyte maturation and vascular invasion in Cbfa1−/− cartilage and the role of the hematopoietic system in the process of vascular invasion, we transplanted embryonic day 18.5 (E18.5) Cbfa1−/− femurs, which are composed of immature chondrocytes, into spleens of normal mice. One week later, the transplanted femurs contained terminally differentiated chondrocytes expressing osteopontin, bone sialoprotein (BSP), and matrix metalloproteinase (MMP) 13. In the diaphyses of the transplants, the cartilage matrix was mineralized and the cartilage was invaded by vascular vessels and osteoclasts. However, chondrocyte maturation and vascular invasion were severely retarded in comparison with transplants of E14.5 wild-type femurs, in which the cartilage was rapidly replaced by bone, and neither mature osteoblasts nor bone formation were observed. In primary culture of Cbfa1−/− chondrocytes, transforming growth factor (TGF) β1, platelet-derived growth factor (PDGF), interleukin (IL)-1β, and thyroid hormone (T3) induced osteopontin and MMP-13 expression. These findings indicated that factors in the hematopoietic system are able to support vascular invasion of cartilage independent of Cbfa1 but are less effective without it, suggesting that Cbfa1 functions in cooperation with factors from bone marrow in the process of growth plate vascularization.
Reduction of Osteoclasts in a Critical Embryonic Period Is Essential for Inhibition of Mouse Tooth Eruption
Authors: Yoshino Miya , Yamazaki Hidetoshi , Yoshida Hisahiro , Niida Shumpei , Nishikawa Shin-Ichi , Ryoke Kazuo , Kunisada Takahiro , Hayashi Shin-Ichi
Published: April 2006

Alveolar bone resorption by osteoclasts is essential for tooth eruption. Osteoclast-deficient Csfmop homozygous (op/op) mice, which lack functional macrophage colony-stimulating factor (M-CSF), suffer from osteopetrosis and completely lack tooth eruption. Although osteoclasts appear, and osteopetrosis is cured with age in op/op mice, tooth eruption is never seen. This fact suggests that there is a critical period when osteoclasts are required for tooth eruption. In this study, to detect the critical period, we administered an antagonistic antibody directed against c-Fms, a receptor for M-CSF, to inbred C57BL/6 mice for various periods. Administration of this antibody decreased tartrate-resistant acid phosphatase-positive (TRAP) osteoclasts, and incisor eruption was completely inhibited by continual administration of this antibody from embryonic day 15.5 (E15.5) until postnatal day 12.5 (D12.5). A 1-day delay of this administration abolished the inhibition of incisor eruption. The number of TRAP-positive osteoclasts was significantly reduced between E16.5 and E18.5 in the mice treated with antibody from E15.5 compared with those treated from E16.5. These results indicate that this period, during which the number of osteoclasts decreases significantly, is critical for inhibiting incisor eruption in C57BL/6 mice.
A Novel Immunoassay for The Determination of Tartrate-Resistant Acid Phosphatase 5b From Rat Serum
Authors: Alatalo Sari L , Peng Zhiqi , Janckila Anthony J , Kaija Helena , Vihko Pirkko , Väänänen H Kalervo , Halleen Jussi M
Published: April 2006

Osteoclasts secrete tartrate-resistant acid phosphatase 5b (TRACP 5b) into the circulation. We have developed an immunoassay for the determination of rat TRACP 5b activity. Intra-assay variation of the immunoassay was 4.5%, interassay variation was 3.8%, dilution linearity was 104.6 ± 7.6%, and recovery of recombinant rat TRACP was 99.1 ± 5.8%. We studied serum TRACP 5b as a marker of bone resorption using orchidectomized (ORC) rats as a model for osteoporosis and age-matched sham-operated rats as controls in a 6-month study. After the operation, trabecular bone mineral density decreased significantly more in the ORC group than in the sham group, whereas cortical bone mineral density increased similarly in both groups. Serum TRACP 5b activity was significantly elevated within the first week after ORC, returned to the control level in the third week, and was not increased above the sham level at any of the later time points. At 6 months, trabecular bone volume was 80% lower in ORC rats than in controls. Osteoclast number per trabecular bone perimeter was slightly increased, but the absolute number of osteoclasts in trabecular bone was significantly decreased. These results suggest that absolute bone resorption is increased within the first week after ORC. Later, it is decreased because there is less bone to be resorbed. However, relative bone resorption (compared with the amount of remaining bone) is still increased, leading to further bone loss. We conclude that serum TRACP 5b is a useful marker for monitoring changes in the bone resorption rate in rat ORC model.
Nitric Oxide Promotes Infectious Bone Resorption by Enhancing Cytokine-Stimulated Interstitial Collagenase Synthesis in Osteoblasts
Authors: Lin Sze-Kwan , Kok Sang-Heng , Kuo Mark Yen-Ping , Lee Ming-Shu , Wang Chih-Chiang , Lan Wan-Hong , Hsiao Michael , Goldring Steven R , Hong Chi-Yuan
Published: April 2006

This experiment was undertaken to determine the role of macrophage-derived nitric oxide (NO) in mediating lipopolysaccharide (LPS)-induced bone resorption by using an in vitro co-culture system and an in vivo model of infectious bone resorption. Our results demonstrated that LPS stimulated the expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-α mRNAs and nitrite synthesis in the J774 mouse macrophage cell line but not in the UMR-106 (rat) and MC3T3-E1 (mouse) osteoblast cell lines. Conditioned media (CM) from LPS-stimulated J774 triggered only low to moderate levels of iNOS mRNAs in MC3T3-E1 and a trivial effect in UMR-106. On the other hand, CM induced matrix metalloproteinase-1 (MMP-1) gene expression in both osteoblast cell lines. The NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) did not alter this effect in MC3T3-E1 and UMR-106, whereas TNF-α antibody diminished the CM-induced MMP-1 gene expression in both cell lines. Interestingly, SNAP, a NO donor, although by itself is not a MMP-1 stimulator for UMR-106, augmented the TNF-α-stimulated MMP-1 mRNA production in UMR-106. In a J774/UMR-106 co-culture system, LPS stimulated significant MMP-1 gene expression in UMR-106, and this upregulation was abolished by L-NMMA and TNF-α antibodies. Immunohistochemical analysis in a rat model of infectious bone resorption (periapical lesion) showed co-distributions of iNOS+ macrophages and MMP-1+ osteoblasts around the osteolytic areas. Administration of L-NMMA markedly reduced the extent of bone loss and the percentage of MMP-1-synthesizing osteoblasts. These data suggest that NO derived from macrophages after LPS stimulation may enhance bone loss by augmenting the cytokine-induced MMP-1 production in osteoblasts.
Chloride Channel ClCN7 Mutations Are Responsible for Severe Recessive, Dominant, and Intermediate Osteopetrosis
Authors: Frattini Annalisa , Pangrazio Alessandra , Susani Lucia , Sobacchi Cristina , Mirolo Massimiliano , Abinun Mario , Andolina Marino , Flanagan Adrienne , Horwitz Edwin M , Mihci Ercan , Notarangelo Luigi D , Ramenghi Ugo , Teti Anna , Van Hove Johan , Vujic Dragana , Young Terri , Albertini Alberto , Orchard Paul J , Vezzoni Paolo , Villa Anna
Published: April 2006

Among 94 osteopetrotic patients presenting with a severe clinical picture and diagnosed early in life, 12 bore mutations in the ClCN7 gene, but only 7 of them had the expected two recessive mutations. The remaining five patients seem to be heterozygous for a ClCN7 mutation, and significant variations were observed in the clinical manifestations of their disease, even within the same family.

Introduction: Human osteopetroses are a heterogeneous group of diseases that include both infantile severe, autosomal recessive (ARO) and adult autosomal dominant (ADO) forms. Two genes, Atp6a3 (TCIRG1) and ClCN7, have been shown to be associated with human ARO, the latter of which is also thought to be responsible for ADO-II. However, patients with an intermediate phenotype have been described: the genetic basis of these observances is unknown.

Materials and Methods: In this study, we report the clinical and molecular analysis of 94 patients in which a diagnosis of severe osteopetrosis was made within the first 2 years of age. Both TCIRG1 and CLCN7 genes were sequenced in all patients and the molecular findings were correlated to clinical parameters.

Results and Conclusions: In 56 of 94 patients with a classical picture of ARO, TCIRG1-dependent recessive mutations were found. In contrast, ClCN7 mutations were found in 12 cases (13%) of severe osteopetrosis, but only 7 of them had two recessive mutations identified: in 6 of these 7 cases, central nervous system manifestations were noted, and these patients had a poor prognosis. The remaining five cases were heterozygous for a ClCN7 mutation, including two brothers from a large family with a history of ADO-II in which the presence of a second ClCN7 mutation was formally excluded. Despite an early and severe clinical presentation, these five patients all reached adulthood, suggesting that the degree of dominant interference with chloride channel function can vary widely. Our findings suggest that recessive ClCN7-dependent ARO may be associated with CNS involvement and have a very poor prognosis, whereas heterozygous ClCN7 mutations cause a wide range of phenotypes even in the same family, ranging from early severe to nearly asymptomatic forms. These findings have prognostic implications, might complicate prenatal diagnosis of human osteopetroses, and could be relevant to the management of these patients.
A Single Nucleotide Polymorphism Under the Reverse Primer Binding Site May Lead to BsmI Mis-Genotyping in the Vitamin D Receptor Gene
Authors: Zajíčková K , Křepelová A , Žofková I
Published: April 2006

The BsmI polymorphism in the VDR gene has been extensively investigated by PCR and restriction digestion in bone genetics. A SNP within the corresponding region for the previously published reverse primer was observed and confirmed by DNA sequencing. BsmI mis-genotyping caused by this SNP could confound genetic findings.

Introduction: By analyzing the FokI, BsmI, ApaI, and TaqI polymorphisms in the vitamin D receptor (VDR) gene, we observed a significantly different genotype distribution in the BsmI polymorphic locus with a deviation from Hardy-Weinberg equilibrium. One of the reasons for polymerase chain reaction (PCR) non-amplification may be a mismatched base at the primer binding region. Therefore, the aim of this study was to analyse whether a single nucleotide polymorphism (SNP), which has been recently described as TruI, is responsible for the discrepancy between expected and observed genotype frequencies.

Materials and Methods: The VDR genotypes were identified in a cohort of 165 peri- and postmenopausal women of white origin. PCR amplification was carried out using the originally published primers and followed by restriction cleavage. The BsmI genotypes were further verified with a reverse primer external to the original binding site. The presence of the TruI polymorphism under the previously published reverse primer was confirmed by a restriction digestion and DNA sequencing. In Bb subjects, the colocalization of b allele with the TruI restriction site on the same chromosome was confirmed by a simultaneous digestion of the PCR product with both BsmI and TruI restriction enzymes.

Results: The BsmI reanalysis with an external primer provided a higher number of heterozygous subjects with a proportionally smaller number of BB subjects, and the changed genotype distribution was under Hardy-Weinberg equilibrium (BB, 31; Bb, 80; bb, 54; r = 0.0203; p = 0.90). In our primary analysis, the presence of the TruI polymorphism led to a drop out of b allele during PCR amplification and thus to the false prevalence of BB genotypes (BB, 50; Bb, 61; bb, 54; r = 11.17; p = 0.01).

Conclusion: The SNP in the region corresponding to the reverse primer may lead to BsmI mis-genotyping, which may have confounded some previous genetic studies.
Bone Remodeling at the Endocortical Surface of the Human Femoral Neck: A Mechanism for Regional Cortical Thinning in Cases of Hip Fracture
Authors: Power J , Loveridge N , Lyon A , Rushton N , Parker M , Reeve J
Published: April 2006

Endocortical remodeling and wall thickness (W.Th.) were measured in femoral neck bone from 12 female fracture cases (81.3 ± 1.5 years) and 12 sex-matched controls (81.9 ± 1.9 years). Regionally, osteoid and eroded surface were increased, whereas W.Th. was reduced. These processes likely contribute to cortical bone loss seen in hip fracture.

Introduction: Because periosteal expression of alkaline phosphatase was similar between cases and controls, we hypothesized that the mechanism causing the marked femoral neck cortical thinning associated with hip fracture may be net endocortical bone loss.

Methods: Twelve female cases of femoral neck fracture (mean age = 81.3 ± 1.5 years) and 12 age- and sex-matched postmortem controls (mean age = 81.9 ± 1.9 years) were included in the study. Samples of their femoral neck bone were embedded in methyl methacrylate, sectioned at 10 μm, and stained with Solochrome cyanine R and Goldner's trichrome for the detection of osteoid (%OS/BS) and resorption surfaces (%ES/BS) respectively. In addition, wall thickness (W.Th.) and lamellar thickness (Lm.Th.) data were also collected from identifiable endocortical bone packets as a measure of formative potential.

Results and Conclusions: %OS/BS was significantly elevated in the anterior (control = 3.4 ± 0.7: fracture = 11.0 ± 2.3; p = 0.0001), inferior (3.4 ± 1.0: 9.9 ± 3.0; p = 0.0009), and posterior quadrants (3.2 ± 0.8: 9.1 ± 2.3; p = 0.0021). Only for anterior region was increased %ES/BS demonstrated in the fracture group (2.8 ± 0.6: 5.3 ± 0.7; p = 0.055). W.Th. (mm) was reduced only in the inferior region of the fracture cases (control = 33.7 ± 1.2: fracture = 30.6 ± 0.9; p = 0.013), whereas Lm.Th. was also reduced inferiorly (control = 2.7 ± 0.08: fracture = 2.5 ± 0.08; p = 0.042). These data suggest that an endocortical remodeling imbalance involving reduced bone formation within inferior region coupled with elevated anterior resorption may make an important contribution to the cortical thinning observed in cases of femoral neck fracture.
Extracellular Calcium Is a Potent Inducer of Cyclo-oxygenase-2 in Murine Osteoblasts Through an ERK Signaling Pathway
Authors: Choudhary Shilpa , Wadhwa Sunil , Raisz Lawrence G , Alander Cynthia , Pilbeam Carol C
Published: April 2006

[Ca2+]e may be important in bone turnover. We found [Ca2+]e induces COX-2 transcription and PGE2 production in primary calvarial osteoblasts through an ERK signaling pathway. Inhibition of PGE2 production inhibited the [Ca2+]e stimulation of osteoblastic differentiation but not the increase in cell number. Hence, some effects of [Ca2+]e on bone may be mediated by COX-2.

Introduction: Local changes in extracellular calcium ([Ca2+]e) may play an important role in bone turnover. We examined the possibility that prostaglandins produced by cyclo-oxygenase-2 (COX-2) could mediate some of the effects of [Ca2+]e on osteoblasts.

Methods: We examined the [Ca2+]e induction of COX-2 expression and prostaglandin E2 (PGE2) production in primary osteoblasts (POBs) obtained by sequential enzymatic digestion of mouse calvariae. We measured mRNA and protein levels by Northern and Western analyses and PGE2 production in culture medium by radioimmunoassay (RIA). COX-2 promoter activity was measured as luciferase activity in calvarial osteoblasts derived from mice transgenic for 371 bp of the COX-2 promoter fused to a luciferase reporter gene.

Results and Conclusions: COX-2 mRNA and protein expression were induced by 3–40 mM of [Ca2+]e. [Ca2+]e (5 mM) induced COX-2 mRNA within 30 minutes; levels peaked at 6–9 h and remained elevated at 24 h. Cumulative medium PGE2 was increased at 3 h, with levels rising to 30 nM at 24 h. PGE2 production in POBs from mice with only COX-1 gene expression was 1/40th of that in POBs from mice with both COX-1 and COX-2 gene expression. [Ca2+]e increased alkaline phosphatase activity and osteocalcin mRNA, and this increase was blocked by inhibiting PGE2 production. [Ca2+]e stimulation of COX-2 promoter activity correlated with the induction of COX-2 mRNA expression. [Ca2+]e induced rapid and transient phosphorylation of extracellular signal-regulated kinase (ERK) in POBs, which peaked at 5–10 minutes. Inhibition of ERK phosphorylation with the specific inhibitors, PD-98059 and U-0126, decreased the [Ca2+]e induction of both COX-2 mRNA and luciferase activity by 70–80 %. Although less effective than [Ca2+]e, strontium [Sr2+]e also induced COX-2 mRNA and promoter activity in POBs through an ERK signaling pathway. We conclude that [Ca2+]e is a potent transcriptional inducer of COX-2 expression and PGE2 production in osteoblasts through an ERK signaling pathway.
Does the Combination of Two BMD Measurements Improve Fracture Discrimination?
Authors: Blake Glen M , Patel Rajesh , Knapp Karen M , Fogelman Ignac
Published: April 2006

Combining information from different types of BMD measurement should improve the evaluation of patients' risk of fracture. This study used a bivariate gaussian model to examine the effect of combining two different BMD measurements. The results show that, in practice, there is little benefit unless the measurements are completely unrelated.

Introduction: Intuitively, the combination of information from two or more different types of bone densitometry investigation should improve our ability to identify patients at high risk of fracture. However, the best way to combine measurements and the resulting gain in fracture discrimination are not known.

Materials and Methods: In this study, we used a bivariate gaussian model to investigate the effect of combining two different types of bone densitometry measurements. The measurements had individual relative risk values RR1 and RR2 and a correlation coefficient r between their Z-scores. Different approaches to the combination of the two measurements were compared by calculating the area under the curve (AUC) for the receiver operating characteristic (ROC) curve, which was obtained by plotting the percentage of fracture patients against the percentage of the whole population with a Z-score below some chosen threshold. ROC curves were calculated for three cases: (1) one type of measurement only; (2) two different types of measurements combined using their mean Z-score weighted according to the theoretical optimum weighting factors predicted by the bivariate gaussian model; and (3) two different types of measurements combined using the conventional World Health Organization (WHO) approach, where one or other measurement is below a set threshold. The theoretical model was tested using measurements of speed of sound (SOS) in the radius, phalanx, and metatarsal in patients with vertebral and Colles' fractures.

Results: Results were calculated for RR values of 1.5, 2.0, and 2.5 and r = 0, 0.5, and 0.7. Although a significant improvement in fracture discrimination was obtained when r = 0 and RR1 = RR2, the improvements obtained when r ≥ 0.5 or RR1 ≠ RR2 were relatively modest. Slightly better fracture discrimination was obtained using the weighted mean Z-score approach compared with the WHO approach, although the differences were small. The results of the in vivo study in Colles' and vertebral fracture patients showed close agreement with the predictions of the bivariate gaussian model.

Conclusion: In practice, from a theoretical point of view, there is unlikely to be any benefit from combining information from different types of bone densitometry measurements unless they are completely unrelated.
Variation in the TNF Gene Promoter and Risk of Osteolysis After Total Hip Arthroplasty
Authors: Wilkinson J Mark , Wilson A Gerard , Stockley Ian , Scott Ian R , Macdonald David A , Hamer Andrew J , Duff Gordon W , Eastell Richard
Published: April 2006

Genetic factors may influence implant failure caused by osteolysis after THA. In an association study of 481 subjects after THA, we found that carriage of the TNF-238A allele was associated with an increased incidence of osteolysis versus noncarriage (odds ratio, 1.7) and was independent of other risk factors. Genetic and environmental factors influence implant survival after THA.

Introduction: Tumor necrosis factor (TNF) is thought to play a role in osteolysis, the major cause of implant failure after total hip arthroplasty (THA). Natural sequence variations at −238 and −308 in the TNF gene promoter are associated with differences in susceptibility to several TNF-mediated diseases. We tested whether these polymorphisms are associated with osteolysis after THA.

Materials and Methods: A total of 481 whites (214 with failed versus 267 with intact implants) were recruited 11.7 ± 4 years after cemented THA. Genomic DNA was extracted from peripheral blood and genotyped for the −238 and −308 polymorphisms using the Taqman 5′ nuclease method. Healthy controls (n = 500) from the background population were also genotyped to establish the local prevalence of these alleles.

Results: The carriage of −238A was 8.8% in the background population and 10.9% in the THA controls (p > 0.05). Carriage of −238A in the osteolysis group was 17.3% (odds ratio, 1.7; 95% CI, 1.0–2.9). Carriage was highest (20.5%) in patients with more widespread osteolysis (OR, 2.1; 1.2–3.8). The association of −238A with osteolysis was independent of other risk factors for osteolysis (logistic regression analysis: OR, 1.8; 1.0–3.2). Carriage of −308A was not associated with osteolysis.

Conclusion: Genetic, as well as environmental factors, influence implant failure after THA. Whether the TNF-238 polymorphism causes a biological change that predisposes to loosening or is in linkage disequilibrium with such a locus is not yet known.
The Renal Clearance of Free and Peptide-Bound Deoxypyridinoline: Response to Pamidronate Treatment of Paget's Disease
Authors: Naylor KE , Jackson B , Eastell R
Published: April 2006

Bisphosphonate treatment of Paget's disease results in a large decrease in urinary peptide-bound pyridinolines but a smaller decrease in urinary free pyridinolines. This discrepancy could be explained by changes in renal handling of pyridinoline forms. We studied eight patients with Paget's disease treated with pamidronate. We collected blood and urine at baseline and at 3 and 14 days after treatment. We measured free and total deoxypyridinoline (DPD) in serum (S) and urine (U) by high-performance liquid chromatography (HPLC). The ratio of free to total DPD at baseline was (mean ± SE) 13 ± 1% in serum and 37 ± 3% in urine; at 3 days, this had increased to 25 ± 3% in serum and 62 ± 7% in urine. Peptide-bound (pb) DPD decreased significantly 3 days after treatment: UpbDPD -63 ± 11%, p < 0.001; SpbDPD -51 ± 8%, p < 0.01. Free DPD decreased in the urine after 14 days: UfDPD −48 ± 5%, p < 0.01; there was no significant change in SfDPD. The fractional excretion of pbDPD relative to creatinine was less than one at all time-points; however, the fractional excretion of fDPD was significantly greater than one throughout the study. As a consequence, the proportion of free DPD in the urine increased as bone turnover decreased. This resulted in a smaller decrease in urine free compared with peptide-bound DPD in response to bisphosphonate therapy. Thus, the conversion of peptide-bound to free DPD in the kidney may become more efficient as bone turnover decreases as a consequence of pamidronate treatment.
Aging Does Not Lessen the Effectiveness of TGFβ2-Enhanced Bone Regeneration
Authors: Sumner DR , Turner TM , Cohen M , Losavio P , Urban RM , Nichols EH , McPHERSON JM
Published: April 2006

Controversy exists over the potency of bone healing in the aged skeleton, and there is concern that enhancement of bone regeneration after use of bone-stimulating growth factors may not be effective in the aged. In this study, 30 skeletally mature beagles (1–2 or 10–12 years old) had titanium implants placed bilaterally in the proximal humerus for a period of 4 weeks in a model of intramembranous bone regeneration. A bony defect made at the time of surgery created a 3-mm gap between the implant surface and the host bone. Some of the implants were treated with recombinant human TGFβ2 (rhTGFβ2) at various does (0.32–35 μg per implant), and some served as paired controls. The dose response was similar in young and old animals. The most effective dose, 35 μg, led to a 3-fold increase in the volume fraction of new bone within the gap in both the young (p = 0.001) and old (p = 0.002) animals. At this dose, there was a 5-fold increase in osteoblast surface. While age did not significantly affect the quantity of new bone formed as assessed by backscatter scanning electron microscopy, the older animals had thinner regenerated trabeculae that tended to be spaced more closely than the younger animals. Coupled with the finding that the increase in osteoid was greater in the old animals compared with the young animals, these qualitative differences suggest that there may have been a slight delay in the rate or a defect of mineralization in the old animals.
Functional Type I PTH/PTHrP Receptor in Freshly Isolated Newborn Rat Keratinocytes: Identification by RT-PCR and Immunohistochemistry
Authors: Errazahi Amina , Bouizar Zhor , Lieberherr Michele , Souil Evelyne , Rizk-Rabin Marthe
Published: April 2006

The presence of identical or distinct type I parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) receptors in keratinocytes is still a matter of debate. We studied the expression and functionality of PTHrP receptors in freshly isolated keratinocytes from newborn rat skin. Four overlapping primers, amplifying different regions in the rat PTH receptor, were used for reverse transcriptase-polymerase chain reaction (RT-PCR). The first region corresponded to the N-terminal extracellular region and the first transmembrane domain (S/M1), the second region amplified the connecting intracellular and extracellular loops transmembrane domain (E2/M5), the third spanned the range from the transmembrane to the intracellular domain (M4/T), and the fourth region amplified the C-terminal tail (M6/7/T). The PCR products from the keratinocyte RNA were identical to those from kidney RNA of the same rats. The cloned four transcripts showed 100% of homologies with the cDNA sequence from bone ROS cells. Keratinocytes, freshly isolated or present in situ in the epidermis, recognized an anti-PTH receptor antibody (PTH-II) directed against the receptor extracellular domain. Western blotting showed the same protein patterns in keratinocytes, kidney, and ROS cell extracts. Low doses of PTHrP(1–34) (10−12–10−9 M) increased the cell number studied by [3H]thymidine incorporation and DNA content. Treatment with the PTH/PTHrP receptor antagonist [Asn10, Leu11, D Trp12] PTHrP(7–34) or two different PTH receptor antibodies inhibited the increase in cell proliferation induced by PTHrP(1–34). All these findings indicate that newborn rat epidermis and keratinocytes express functional PTHrP receptors, which are identical to type I PTH/PTHrP receptor and are recognized by PTHrP(1–34).