bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Predisposition for Adult Lactose Intolerance and Relation to Diet, Bone Density, and Bone Fractures
Authors: Obermayer-Pietsch Barbara M , Bonelli Christine M , Walter Daniela E , Kuhn Regina J , Fahrleitner-Pammer Astrid , Berghold Andrea , Goessler Walter , Stepan Vinzenz , Dobnig Harald , Leb Georg , Renner Wilfried
Published: April 2006

Evidence that genetic disposition for adult lactose intolerance significantly affects calcium intake, bone density, and fractures in postmenopausal women is presented. PCR-based genotyping of lactase gene polymorphisms may complement diagnostic procedures to identify persons at risk for both lactose malabsorption and osteoporosis.

Introduction: Lactase deficiency is a common autosomal recessive condition resulting in decreased intestinal lactose degradation. A −13910 T/C dimorphism (LCT) near the lactase phlorizin hydrolase gene, reported to be strongly associated with adult lactase nonpersistence, may have an impact on calcium supply, bone density, and osteoporotic fractures in the elderly.

Materials and Methods: We determined LCT genotypes TT, TC, and CC in 258 postmenopausal women using a polymerase chain reaction-based assay. Genotypes were related to milk intolerance, nutritional calcium intake, intestinal calcium absorption, bone mineral density (BMD), and nonvertebral fractures.

Results: Twenty-four percent of all women were found to have CC genotypes and genetic lactase deficiency. Age-adjusted BMD at the hip in CC genotypes and at the spine in CC and TC genotypes was reduced by −7% to −11% depending on the site measured (p = 0.04). LCT(T/C−13910) polymorphisms alone accounted for 2–4 % of BMD in a multiple regression model. Bone fracture incidence was significantly associated with CC genotypes (p = 0.001). Milk calcium intake was significantly lower (−55%, p = 0.004) and aversion to milk consumption was significantly higher (+166%, p = 0.01) in women with the CC genotype, but there were no differences in overall dietary calcium intake or in intestinal calcium absorption test values.

Conclusion: The LCT(T/C−13910) polymorphism is associated with subjective milk intolerance, reduced milk calcium intake, and reduced BMD at the hip and the lumbar spine and may predispose to bone fractures. Genetic testing for lactase deficiency may complement indirect methods in the detection of individuals at risk for both lactose malabsorption and osteoporosis.
Expression and Activity of the CDK Inhibitor p57Kip2 in Chondrocytes Undergoing Hypertrophic Differentiation
Authors: Stewart Matthew C , Kadlcek Renata M , Robbins Paul D , Macleod James N , Ballock R Tracy
Published: April 2006

Growth plates ofp57-null mice exhibit several abnormalities, including loss of collagen type X (CollX) expression. The phenotypic consequences of p57 expression were assessed in an in vitro model of hypertrophic differentiation. Adenoviral p57 expression was not sufficient for CollX expression but did augment induction of CollX by BMP-2.

Introduction: During hypertrophic differentiation, chondrocytes pass from an actively proliferative state to a postmitotic, hypertrophic phenotype. The induction of growth arrest is a central feature of this phenotypic transition. Mice lacking the cyclin dependent-kinase inhibitor p57Kip2 exhibit several developmental abnormalities including chondrodysplasia. Although growth plate chondrocytes in p57-null mice undergo growth arrest, they do not express collagen type X, a specific marker of the hypertrophic phenotype. This study was carried out to investigate the link between p57 expression and the induction of collagen type X in chondrocytes and to determine whether p57 overexpression is sufficient for the induction of hypertrophic differentiation.

Materials and Methods: Neonatal rat epiphyseal or growth plate chondrocytes were maintained in an aggregate culture model, in defined, serum-free medium. Protein and mRNA levels were monitored by Western and Northern blot analyses, respectively. Proliferative activity was assessed by fluorescent measurement of total DNA and by3H-thymidine incorporation rates. An adenoviral vector was used to assess the phenotypic consequences of p57 expression.

Results and Conclusions: During in vitro hypertrophic differentiation, levels of p57 mRNA and protein were constant despite changes in chondrocyte proliferative activity and the induction of hypertrophic-specific genes in response to bone morphogenetic protein (BMP)-2. Adenoviral p57 overexpression induced growth arrest in prehypertrophic epiphyseal chondrocytes in a dose-dependent manner but was not sufficient for the induction of collagen type X, either alone or when coexpressed with the related CDKI p21Cip1. Similar results were obtained with more mature tibial growth plate chondrocytes. p57 overexpression did augment collagen type X induction by BMP-2. These data indicate that p57-mediated growth arrest is not sufficient for expression of the hypertrophic phenotype, but rather it occurs in parallel with other aspects of the differentiation pathway. Our findings also suggest a contributing role for p57 in the regulation of collagen type X expression in differentiating chondrocytes.
Fracture Risk in Monoclonal Gammopathy of Undetermined Significance
Authors: Melton L Joseph , Rajkumar S Vincent , Khosla Sundeep , Achenbach Sara J , Oberg Ann L , Kyle Robert A
Published: April 2006

To assess fractures in monoclonal gammopathy of undetermined significance (MGUS), the precursor of multiple myeloma, we followed 488 Olmsted County, MN, residents with MGUS in a retrospective cohort study. There was a 2.7-fold increase in the risk of axial fractures but no increase in limb fractures. The pathophysiologic basis for the increased axial fractures should be determined.

Introduction: Multiple myeloma is often preceded by monoclonal gammopathy of undetermined significance (MGUS). Fractures are common in myeloma as a result of lytic bone lesions, generalized bone loss, and elevated bone turnover from excessive cytokine production. Whether fractures are also increased in MGUS is unknown.

Materials and Methods: In a population-based retrospective cohort study, 488 Olmsted County, MN, residents with MGUS first diagnosed in 1960–1994 (52% men; mean age, 71.4 ± 12.8 years) were followed for 3901 person-years; follow-up was censored at progression to myeloma. The relative risk of fractures was assessed by standardized incidence ratios (SIRs), and risk factors were evaluated in proportional hazards models.

Results and Conclusions: Altogether, 200 patients experienced 385 fractures. Compared with expected rates in the community, statistically significant increases were seen for fractures at most axial sites, for example, vertebrae (SIR, 6.3; 95% CI, 5.2–7.5). There was a slight increase in hip (SIR, 1.6; 95% CI, 1.2–2.2) but not distal forearm fractures (SIR, 0.8; 95% CI, 0.4–1.5). The relative risk (SIR) of any axial fracture was 2.7 (95% CI, 2.3–3.1) compared with only 1.1 (95% CI, 0.9–1.4) for all limb fractures combined. In a multivariate analysis, the independent predictors of any subsequent fracture were age (hazard ratio [HR] per 10-year increase, 1.4; 95% CI, 1.2–1.6) and corticosteroid use (HR, 1.8; 95% CI, 1.2–2.6); greater weight at diagnosis (HR per 10 kg, 0.8; 95% CI, 0.8–0.9), and IgG monoclonal protein (HR, 0.7; 95% CI, 0.5–0.97) were protective. Baseline monoclonal protein level, a determinant of myeloma progression, did not predict fracture risk. Thus, the risk of axial, but not peripheral, fractures is increased among MGUS patients even before progression to myeloma. The pathophysiologic basis for this should be determined because elevated bone turnover, for example, might be treatable.
Isolation of a Human Homolog of Osteoclast Inhibitory Lectin That Inhibits the Formation and Function of Osteoclasts
Authors: Hu Yun Shan , Zhou Hong , Myers Damian , Quinn Julian MW , Atkins Gerald J , Ly Chi , Gange Christine , Kartsogiannis Vicky , Elliott Jan , Kostakis Panagiota , Zannettino Andrew CW , Cromer Brett , Mckinstry William J , Findlay David M , Gillespie Matthew T , Ng Kong Wah
Published: April 2006

Osteoclast inhibitory lectin (OCIL) is a newly recognized inhibitor of osteoclast formation. We identified a human homolog of OCIL and its gene, determined its regulation in human osteoblast cell lines, and established that it can inhibit murine and human osteoclast formation and resorption. OCIL shows promise as a new antiresorptive.

Introduction: Murine and rat osteoclast inhibitory lectins (mOCIL and rOCIL, respectively) are type II membrane C-type lectins expressed by osteoblasts and other extraskeletal tissues, with the extracellular domain of each, expressed as a recombinant protein, able to inhibit in vitro osteoclast formation.

Materials and Methods: We isolated the human homolog of OCIL (hOCIL) from a human fetal cDNA library that predicts a 191 amino acid type II membrane protein, with the 112 amino acid C-type lectin region in the extracellular domain having 53% identity with the C-type lectin sequences of rOCIL and mOCIL. The extracellular domain of hOCIL was expressed as a soluble recombinant protein in E. coli, and its biological effects were determined.

Results and Conclusions: The hOCIL gene is 25 kb in length, comprised of five exons, and is a member of a superfamily of natural killer (NK) cell receptors encoded by the NK gene complex located on chromosome 12. Human OCIL mRNA expression is upregulated by interleukin (IL)-1α and prostaglandin E2 (PGE2) in a time-dependent manner in human osteogenic sarcoma MG63 cells, but not by dexamethasone or 1,25 dihydroxyvitamin D3. Soluble recombinant hOCIL had biological effects comparable with recombinant mOCIL on human and murine osteoclastogenesis. In addition to its capacity to limit osteoclast formation, OCIL was also able to inhibit bone resorption by mature, giant-cell tumor-derived osteoclasts. Thus, a human homolog of OCIL exists that is highly conserved with mOCIL in its primary amino acid sequence (C-lectin domain), genomic structure, and activity to inhibit osteoclastogenesis.
A Genome-Screen of a Large Twin Cohort Reveals Linkage for Quantitative Ultrasound of the Calcaneus to 2q33–37 and 4q12–21
Authors: Wilson SG , Reed PW , Andrew T , Barber MJ , Lindersson M , Langdown M , Thompson D , Thompson E , Bailey M , Chiano M , Kleyn PW , Spector TD
Published: April 2006

A genome-wide screen was performed on a large cohort of dizygous twin pairs to identify regions of the genome that contain QTL for QUS of bone. Suggestive linkage of QUS parameters to 2q33–37 and 4q12–21 highlighted these regions as potentially important for studies of genes that regulate bone.

Introduction: The genetics of osteoporotic fracture is only partly explained by bone mineral density (BMD). Quantitative ultrasound (QUS) of the calcaneus can also be used for independent clinical assessment of osteoporotic fracture risk. Two specific indices are derived from this assessment: broadband ultrasound attenuation (BUA) and velocity of sound (VOS). Both parameters provide information on fracture risk; however, BUA has been studied more extensively and may be favored because it is thought to have a stronger predictive value for osteoporotic fracture and incorporates aspects of trabecular structure and bone quality as well as BMD. Studies of QUS in twins have shown that both derived parameters are under substantial genetic control, independent of BMD.

Materials and Methods: To identify regions of the genome that contain quantitative trait loci (QTL) for QUS of bone, we performed a genome-wide screen on a large cohort of dizygous twin pairs. Unselected female dizygous twins from 1067 pedigrees from the St Thomas' UK Adult Twin Registry were genome scanned (737 highly polymorphic microsatellite markers). Multipoint linkage analyses provided maximum evidence of linkage for BUA (LOD 2.1–5.1) to 2q33–37. Linkage for VOS (LOD 2.2–3.4) was maximal at 4q12–21. Potential evidence of linkage in the cohort indicated five other possible locations of QTL (LOD >2.0) relevant to bone density or structure on chromosomes 1, 2, 13, 14, and X.

Results and Conclusions: This study has identified eight genomic locations with linkage of LOD >2.0. This data should be of value in assisting researchers to localize genes that regulate bone mass and microstructure. These results should complement genome screens of BMD and bone structure and serve to enable further targeted positional candidate and positional cloning studies to advance our understanding of genetic control of bone quality and risk of fracture.
RANK Signaling Is Not Required for TNFα-Mediated Increase in CD11bhi Osteoclast Precursors but Is Essential for Mature Osteoclast Formation in TNFα-Mediated Inflammatory Arthritis
Authors: Li Ping , Schwarz Edward M , O'Keefe Regis J , Ma Lin , Boyce Brendan F , Xing Lianping
Published: April 2006

To address the controversy of whether TNFα can compensate for RANKL in osteoclastogenesis in vivo, we used a TNFα-induced animal model of inflammatory arthritis and blocked RANKL/RANK signaling. TNFα increased osteoclast precursors available for RANK-dependent osteoclastogenesis. RANK signaling is not required for the TNFα-stimulated increase in CD11bhi osteoclast precursors but is essential for mature osteoclast formation.

Introduction: Although critical roles of TNFα in inflammatory arthritis and RANKL in bone resorption have been firmly established, a central controversy remains about the extent to which TNFα can compensate for RANKL during osteoclastogenesis and the stage at which RANK signaling is required for osteoclastogenesis. Here, we used the human TNFα transgenic mouse model (TNF-Tg) of erosive arthritis to determine if there are both RANK-dependent and -independent stages of osteoclastogenesis in TNFα-induced erosive arthritis.

Materials and Methods: Osteoclastogenesis and osteoclast precursor (OCP) frequency were analyzed using histology, fluorescence-activated cell sorting (FACS), and cell culture from (1) TNF-Tg mice treated with the RANKL antagonist, RANK:Fc, or (2) TNF-Tg × RANK−/− mice generated by crossing TNF-Tg mice with RANK−/− mice.

Results: Treatment of TNF-Tg mice, which have increased OCPs in their spleens, with RANK:Fc dramatically reduced osteoclast numbers on the surface of their arthritic joints and within their bones, but did not decrease CD11bhi OCP numbers in their spleens. Long-term RANK:Fc administration alleviated joint erosion. Furthermore, TNF-Tg × RANK−/− mice had severe osteopetrosis, no osteoclasts, and no joint erosion, but increased CD11bhi precursor numbers that failed to form mature osteoclasts in vitro.

Conclusion: RANK signaling is essential for mature osteoclast formation in TNFα-mediated inflammatory arthritis but not for the TNFα-induced increase in CD11bhi OCP that subsequently can differentiate into osteoclasts in inflamed joints.
Biochemical Markers of Bone Metabolism and Prediction of Fracture in Elderly Women
Authors: Gerdhem Paul , Ivaska Kaisa K , Alatalo Sari L , Halleen Jussi M , Hellman Jukka , Isaksson Anders , Pettersson Kim , Väänänen H Kalervo , Åkesson Kristina , Obrant Karl J
Published: April 2006

We studied the ability of various markers of bone turnover to predict fracture in 1040 randomly recruited 75-year-old women. A total of 178 of the women sustained at least one fracture during follow-up (mean, 4.6 years). In elderly women, TRACP5b and urinary fragments of osteocalcin are promising new markers for prediction of fracture, in particular, vertebral fracture.

Introduction: Biochemical markers reflecting bone turnover may improve the prediction of fractures.

Materials and Methods: The ability of 10 markers of bone turnover to predict fracture in 1040 elderly women in the Malmö OPRA study was studied. Serum bone-specific alkaline phosphatase and four different forms of serum osteocalcin (S-OC) were analyzed as markers of bone formation and serum C-terminal cross-linking telopeptides of type I collagen (S-CTX), serum TRACP isoform 5b (S-TRACP5b) and urinary free deoxypyridinoline (U-DPD) as markers of bone resorption. Two novel assays for osteocalcin fragments in urine (U-OC) were analyzed. Areal BMD (aBMD) was measured by DXA in the femoral neck and lumbar spine.

Results: In total, 231 fractures were sustained by 178 of the women during a 3- to 6.5-year (mean, 4.6 years) follow-up period. When women with prospective fractures were compared with women without fractures, S-TRACP5b, S-CTX, one S-OC, and one U-OC were higher in women with a fracture of any type (all p < 0.05), and all bone markers were higher in women with clinical vertebral fracture (all p < 0.05). Markers were not significantly elevated in women with hip fracture. When women within the highest quartile of a bone marker were compared with all others, S-TRACP5b and one U-OC predicted the occurrence of a fracture of any type (odds ratio [OR]), 1.55 and 1.53; p < 0.05). S-TRACP5b, the two U-OCs, and S-CTX predicted vertebral fracture (OR, 2.28, 2.75, 2.71, and 1.94, respectively; all p < 0.05), and the predictive value remained significant for S-TRACP5b and the two U-OCs after adjusting for aBMD (OR, 2.02–2.25; p < 0.05). Bone markers were not able to predict hip fracture.

Conclusion: These results show that biochemical markers of bone turnover can predict fracture, and in particular, fractures that engage trabecular bone. S-TRACP5b and U-OC are promising new markers for prediction of fracture.
Transcriptional Regulation of a BMP-6 Promoter by Estrogen Receptor α
Authors: Ong Delia B , Colley Shane M , Norman Michael R , Kitazawa Sohei , Tobias Jonathan H
Published: April 2006

The effects of 17β-estradiol (E2) and ICI 182,780 (ICI) on activity of a BMP-6 promoter were compared in osteoblast-like and breast cancer cells transiently transfected with ERα. E2 but not ICI stimulated BMP-6 reporter activity in breast cancer cells, whereas the opposite was observed in osteoblast-like cells, associated with lack of AF-2 dependence of the response, and absent intranuclear localization of ERα, suggesting the involvement of a distinct ERα-dependent response mechanism in osteoblasts.

Introduction: Previous studies suggest that the tissue-selective effect of antiestrogens on bone reflects the ability of these compounds to target certain osteoblast regulatory genes. To explore this hypothesis, we examined whether antiestrogens preferentially stimulate the bone morphogenetic protein 6 (BMP-6) promoter in bone cells, and if so, whether this activity is associated with a distinct estrogen receptor (ER)α-dependent response mechanism to that in other cell types.

Materials and Methods: We compared the effects of 17β-estradiol (E2) and ICI 182,780 (ICI) on activity of a 4.3-kb BMP-6 reporter construct in osteoblast-like cells (human MG63 and SaOS-2 cells and rat ROS 17/2.8 cells), human MCF-7 and T47-D breast cancer cell lines, and HepG2 hepatoma cells, after transient transfection with ERα, ERβ, and mutant ER constructs.

Results: E2, but not ICI, stimulated BMP-6 reporter activity by approximately 100% in MCF-7, T47-D cells, and HepG2 cells when transfected with ERα. In contrast, in ERα-transfected osteoblast-like cells, an increase in reporter activity of approximately 75% was observed after treatment with ICI but not E2. The response of MG63 cells to ICI and MCF-7 cells to E2 both required ERα as opposed to ERβ and the ERα activation function (AF)-1 activation domain. However, whereas the AF-2 domain was also required for E2 to stimulate reporter activity in MCF-7 cells, the response to ICI in MG63 cells was AF-2 independent. In further studies where we compared the intracellular distribution of ERα associated with these responses, E2-dependent stimulation of the BMP-6 reporter in MCF-7 cells was associated with intranuclear localization of ERα, whereas extranuclear localization was seen in rat osteosarcoma cells (ROS) cells treated with ICI.

Conclusions: Antiestrogens selectively stimulate BMP-6 reporter activity in osteoblast-like cells through a distinct ERα-dependent mechanism characterized by independence of the AF-2 domain and extranuclear localization of ERα.
Calcium Signaling Pathway Involving Calcineurin Regulates Interleukin-8 Gene Expression Through Activation of NF-κB in Human Osteoblast-Like Cells
Authors: Mitsuyama Hirohito , Kambe Fukushi , Murakami Ryuichiro , Cao Xia , Ishiguro Naoki , Seo Hisao
Published: April 2006

Involvement of aberrant IL-8 production by osteoblasts was demonstrated in pathogenesis of inflammatory joint diseases. We thus investigated intracellular signaling pathways leading to IL-8 expression in human osteoblast-like HOS-TE85 cells. It was demonstrated that Ca2+ signaling pathway involving calcineurin regulates IL-8 gene expression through activation of a transcription factor, NF-κB.

Introduction: Involvement of aberrant interleukin (IL)-8 production by osteoblasts was demonstrated in pathogenesis of inflammatory joint diseases. However, intracellular signaling pathways leading to IL-8 expression in osteoblasts have been poorly explored. Because a variety of external stimuli was shown to increase intracellular Ca2+ in osteoblasts, we investigated effects of Ca2+-ionophore and phorbol-myristate-acetate (Ion/PMA) on IL-8 expression in human osteoblast-like HOS-TE85 cells and compared the effects with those elicited by TNF-α.

Materials and Methods: HOS-TE85 cells were treated with Ion/PMA or TNF-α in the presence and absence of calcineurin inhibitors (CnI), cyclosporin A, and FK506. IL-8 mRNA levels and its promoter activities were examined by Northern blot and luciferase reporter analyses, respectively. Electrophoretic mobility shift assay (EMSA) was used to evaluate DNA binding activities of transcription factors such as NF-κB. Degradation of IκB, a cytoplasmic NF-κB-inhibitory protein, was examined by Western blot analysis.

Results: Ion/PMA and TNF-α induced IL-8 mRNA expression. Interestingly, CnI attenuated the induction by Ion/PMA, but not that by TNF-α. Promoter activity was also increased by both stimuli, and only the Ion/PMA-dependent increase was suppressed by CnI. Introduction of mutations in the promoter demonstrated that one NF-κB site was responsible for the suppression by CnI. EMSA revealed that this site binds with NF-κB containing p65 that was activated by Ion/PMA and TNF-α and that CnI inhibited only Ion/PMA-dependent NF-κB activation. Accordingly, CnI blocked only Ion/PMA-dependent degradation of IκB-α. In addition, the basal and Ion/PMA-dependent IL-8 promoter activities were enhanced by co-transfection of constitutively active calcineurin.

Conclusion: These results show that the Ca2+ signaling pathway involving calcineurin regulates IL-8 gene expression through activation of NF-κB in human osteoblast-like cells.
Estrogen Receptor β Polymorphisms Are Associated With Bone Mass in Women and Men: The Framingham Study
Authors: Shearman Amanda M , Karasik David , Gruenthal Kristen M , Demissie Serkalem , Cupples L Adrienne , Housman David E , Kiel Douglas P
Published: April 2006

ESR2 is expressed in bone cells, yet few studies have tested its variation for association with BMD, an important determinant of osteoporotic fractures. This was investigated in 723 men and 795 women from the Framingham study. Results show association of variation in this gene with BMD in both women and men.

Introduction: Osteoporotic fracture risk is highly dependent on bone density, a quantitative multifactorial trait with a substantial genetic component. In contrast to the growing body of evidence that estrogen receptor α (ESR1) plays a role in bone metabolism, few studies have examined the estrogen receptor β (ESR2) gene for association with BMD. An ESR2 CA repeat polymorphism, D14S1026, was associated with BMD in two small studies, each with <200 women.

Materials and Methods: The objective of this investigation was to assess whether D14S1026 or four other intronic polymorphisms were associated with BMD in 723 men and 795 women (mean age, 60 years) from the offspring cohort of the population-based Framingham Study. BMD was measured at the femur (neck, trochanter, and Ward's area) and the lumbar spine (L2-L4).

Results: In both women and men, there was significant association of D14S1026 genotype with measures of femoral but not spinal BMD. In addition, genotypes of two common single nucleotide polymorphisms, rs1256031 and rs1256059, in strong linkage disequilibrium with one another but not with D14S1026, were associated with measures of femoral BMD in men. The rs1256031 genotypes had up to a 4.0% difference in mean femoral BMD. An inferred rs1256031-D14S1026-rs1256059 haplotype C-23CA-T was significantly associated with reduced femoral BMD in women (p = 0.03, 0.003, and 0.01 for neck, trochanter, and Ward's area, respectively). Haplotype-based BMD differences ranged from 3.0% to 4.3%.

Conclusions: We have observed significant association of common ESR2 variants with measures of femoral BMD in both men and women.
A Synthetic Peptide Fragment of Human MEPE Stimulates New Bone Formation In Vitro and In Vivo
Authors: Hayashibara Tetsuyuki , Hiraga Toru , Yi Bing , Nomizu Motoyoshi , Kumagai Yoshinari , Nishimura Riko , Yoneda Toshiyuki
Published: April 2006

Matrix extracellular phosphoglycoprotein (MEPE) was proposed as a candidate for the phosphaturic hormone phosphatonin. We found that a synthetic peptide fragment of MEPE containing the RGD and SGDG sequence stimulated new bone formation in vitro and in vivo.

Introduction: Matrix extracellular phosphoglycoprotein (MEPE) was recently identified as a candidate for the phosphaturic hormone phosphatonin, which has been implicated in disturbed phosphate metabolism, rickets, and osteomalacia associated with X-linked hypophosphatemic rickets (XLH) and oncogenic hypophosphatemic osteomalacia (OHO). MEPE expression was predominantly found in osteoblasts, and mice deficient in a homolog of MEPE showed increased bone density, suggesting that MEPE produced in osteoblasts negatively regulates bone formation. In this study, we examined the effects of a synthetic 23mer peptide fragment of MEPE (AC-100, region 242–264) containing the RGD (integrin-binding) and SGDG (glycosaminoglycan-attachment) motif on bone formation in vitro and in vivo.

Materials and Methods: The osteogenic activity of AC-100 was examined in organ cultures of neonatal mouse calvariae and in vivo by injecting AC-100 onto the calvariae of mice.

Results: Histomorphometric examination showed that AC-100 stimulated new bone formation with increased numbers of osteoblasts in neonatal mouse calvariae in organ culture. In contrast, synthetic MEPE fragment peptides without either the RGD or SGDG motif failed to increase new bone formation. Repeated daily subcutaneous injections of AC-100 onto the calvariae in mice increased bone thickness and stimulated new bone formation as determined by the calcein double-labeling technique. However, peptides in which the RGD or SGDG sequence was scrambled did not stimulate new bone formation in vivo. AC-100 increased cell proliferation and alkaline phosphatase activity and activated focal adhesion kinase (FAK) and extracellular signal-regulated protein kinase (ERK) in human primary osteoblasts.

Conclusion: Our results show that a synthetic peptide corresponding with the sequence of human MEPE fragment stimulates new bone formation with increased number of osteoblasts. The results also suggest that the RGD and SGDG motifs are critical to the osteogenic activity of AC-100, presumably through activating integrin signaling pathways in osteoblasts. The anabolic effects of AC-100 may be beneficial for bone diseases associated with decreased bone formation.
Genetically Based Influences on the Site-Specific Regulation of Trabecular and Cortical Bone Morphology
Authors: Judex Stefan , Garman Russell , Squire Maria , Donahue Leah-Rae , Rubin Clinton
Published: April 2006

The degree of site-specificity by which genes influence bone quantity and architecture was investigated in the femur of three strains of mice. Morphological indices were highly dependent on both genetic makeup as well as anatomical location showing that the assessment of bone structure from a single site cannot be extrapolated to other sites even within a single bone.

Introduction: The identification of genes responsible for establishing peak BMD will yield critical information on the regulation of bone quantity and quality. Whereas such knowledge may eventually uncover novel molecular drug targets or enable the identification of individuals at risk of osteoporosis, the site-specificity by which putative genotypes cause low or high bone mass (and effective bone morphology) is essentially unknown.

Materials and Methods: μCT was used to determine morphological and microarchitectural features of the femora harvested from three genetically distinct strains of 4-month-old female mice, each with distinct skeletal mass (low: C57BL/6J [B6], medium: BALB/cByJ [BALB], high: C3H/HeJ [C3H]). Two trabecular regions (distal epiphysis and metaphysis) were considered in addition to four cortical regions within the metaphysis and diaphysis.

Results and Conclusions: Comparing morphological properties of the different trabecular and cortical femoral regions between the three strains of mice, it was apparent that high or low values of specific parameters of bone morphology could not be consistently attributed to the same genetic strain. Trabecular metaphyseal bone volume, for instance, was 385% larger in C3H mice than in B6 mice, yet the two strains displayed similar bone volume fractions in the epiphysis. Similarly, BALB mice had 48% more trabecular bone than C3H mice in the epiphysis, but there were no strain-specific differences in cortical bone area at the diaphysis. These data suggest that the genetic control of bone mass and morphology, even within a given bone, is highly site-specific and that a comprehensive search for genes that are indicative of bone quantity and quality may also have to occur on a very site-specific basis.
Comparison of Cross-Sectional Geometry of the Proximal Femur in White and Black Women from Detroit and Johannesburg
Authors: Nelson Dorothy A , Pettifor John M , Barondess David A , Cody Dianna D , Uusi-Rasi Kirsti , Beck Thomas J
Published: April 2006

There are known black-white differences in bone density measured by DXA but less is known about bone architecture. We compared cross-sectional geometric properties of the proximal femur in U.S. black (n = 86) and white (n = 151) and South African black (n = 60) and white (n = 48) postmenopausal women. Results are consistent with greater bone strength in the black groups in both countries.

Introduction: There are well-known ethnic differences in bone density, but little is known about ethnic differences in bone architecture between U.S. and South African blacks and whites.

Materials and Methods: We compared bone density and cross-sectional geometric properties of the proximal femur in 237 U.S. black (n = 86) and white (n = 151) and 108 South African black (n = 60) and white (n = 48) postmenopausal women. The proximal femur (neck, intertrochanteric region, and proximal shaft regions of interest) was measured with DXA and further analyzed with a hip structural analysis program. For each region, BMD, cross-sectional area, outer diameter, section modulus, endosteal diameter, average cortical thickness, and the buckling ratio were estimated.

Results and Conclusions: In the femoral neck, in both countries, the blacks had narrower endosteal diameters (mean difference, 2.6% and 5.1% in U.S. and South African women, respectively), thicker cortices (9.3% and 11.0%), and a lower buckling ratio (11.6% and 15.2%) despite a similar outer diameter. In the intertrochanteric region, the whites had a greater outer diameter (2.2% and 3.0% in U.S. and South African women, respectively), lower cross-sectional area (4.8% and 7.2%), and a higher buckling ratio (7.6% and 3.6%). There are fewer differences in the shaft. Compared with South African whites, U.S. whites had wider (mean difference 2.9%) femoral necks and a greater section modulus (6.4%) in the shaft. U.S. whites also had greater cross-sectional area in both the neck and shaft (5.2% and 4.6%, respectively). The U.S. blacks had significantly greater outer diameters, cross-sectional areas, endosteal diameters, and section moduli in the neck region compared with South African blacks. Our observations are consistent with greater bone strength in the black groups in both countries, and they also suggest that there are fewer differences between the same ethnic groups in the two countries than there are between different ethnic groups within a country.
A Novel Cation-Sensing Mechanism in Osteoblasts Is a Molecular Target for Strontium
Authors: Pi Min , Quarles L Darryl
Published: April 2006

Defining the molecular target for strontium in osteoblasts is important for understanding the anabolic effects of this cation on bone. The current studies demonstrate that a G-protein-mediated response to strontium persists in osteoblasts that lack CASR, suggesting a predominant role for a novel cation-sensing receptor in mediating the osseous response to strontium.

Introduction: Strontium has anabolic effects on bone and is currently being developed for the treatment of osteoporosis. The molecular target for strontium in osteoblasts has not been determined, but the existence of CASR, a G-protein-coupled receptor calcium-sensing receptor, raises the possibility that strontium actions on bone are mediated through this or a related receptor.

Materials and Methods: We used activation of a transfected serum response element (SRE)-luciferase reporter in HEK-293 cells to determine if CASR is activated by strontium. In addition, we examined strontium-mediated responses in MC3T3-E1 osteoblasts and osteoblasts derived from wild-type and CASR null mice to determine if other cation-sensing mechanisms are present in osteoblasts.

Results and Conclusions: We found that strontium stimulated SRE-luc activity in HEK-293 cells transfected with full-length CASR but not in cells expressing the alternatively spliced CASR construct lacking exon 5. In contrast, we found that MC3T3-E1 osteoblasts that lack CASR as well as osteoblasts derived from CASR null mice respond to millimolar concentrations of strontium. The response to strontium in osteoblasts was nonadditive to a panel of extracellular cations, including aluminum, gadolinium, and calcium, suggesting a common mechanism of action. In contrast, neither the CASR agonist magnesium nor the calcimimetic NPS-R568 activated SRE activity in osteoblasts, but the response to these agonists was imparted by transfection of CASR into these osteoblasts, consistent with the presence of distinct cation-sensing mechanisms. Co-expression of the dominant negative Gαq(305–359) minigene also inhibited cation-stimulated SRE activity in osteoblasts lacking known CASR. These findings are consistent with strontium activation of a novel Gαq-coupled extracellular cation-sensing receptor in osteoblasts with distinct cation specificity.
Oxysterols Regulate Differentiation of Mesenchymal Stem Cells: Pro-Bone and Anti-Fat
Authors: Kha Hoa Ton , Basseri Benjamin , Shouhed Daniel , Richardson Jennifer , Tetradis Sotirios , Hahn Theodore J , Parhami Farhad
Published: April 2006

Pluripotent mesenchymal stem cells can undergo lineage-specific differentiation in adult organisms. However, understanding of the factors and mechanisms that drive this differentiation is limited. We show the novel ability of specific oxysterols to regulate lineage-specific differentiation of mesenchymal stem cells into osteogenic cells while inhibiting their adipogenic differentiation. Such effects may have important implications for intervention with osteoporosis.

Introduction: Oxysterols are products of cholesterol oxidation and are formed in vivo by a variety of cells including osteoblasts. Novel pro-osteogenic and anti-adipogenic effects of specific oxysterols on pluripotent mesenchymal cells are demonstrated in this report. Aging and osteoporosis are associated with a decrease in the number and activity of osteoblastic cells and a parallel increase in the number of adipocytic cells.

Materials and Methods: The M2–10B4 pluripotent marrow stromal cell line, as well as several other mesenchymal cell lines and primary marrow stromal cells, was used to assess the effects of oxysterols. All results were analyzed for statistical significance using ANOVA.

Results and Conclusion: Pro-osteogenic and anti-adipogenic effects of specific oxysterols were assessed by the increase in early and late markers of osteogenic differentiation, including alkaline phosphatase activity, osteocalcin mRNA expression and mineralization, and the decrease in markers of adipogenic differentiation including lipoprotein lipase and adipocyte P2 mRNA expression and adipocyte formation. Complete osteogenic differentiation of M2 cells into cells expressing early and late markers of differentiation was achieved only when using combinations of specific oxysterols, whereas inhibition of adipogenesis could be achieved with individual oxysterols. Oxysterol effects were in part mediated by extracellular signal-regulated kinase and enzymes in the arachidonic acid metabolic pathway, i.e., cyclo-oxygenase and phospholipase A2. Furthermore, we show that these specific oxysterols act in synergy with bone morphogenetic protein 2 in inducing osteogenic differentiation. These findings suggest that oxysterols may play an important role in the differentiation of mesenchymal stem cells and may have significant, previously unrecognized, importance in stem cell biology and potential therapeutic interventions.
Clodronate Reduces Vertebral Fracture Risk in Women With Postmenopausal or Secondary Osteoporosis: Results of a Double-Blind, Placebo-Controlled 3-Year Study
Authors: McCloskey Eugene , Selby Peter , Davies Mike , Robinson John , Francis Roger M , Adams Judith , Kayan Karthik , Beneton Monique , Jalava Tarja , Pylkkänen Liisa , Kenraali Juha , Aropuu Sakari , Kanis John A
Published: April 2006

The efficacy of oral clodronate 800 mg daily to reduce vertebral fractures was studied in 593 women with postmenopausal or secondary osteoporosis. The incidence of vertebral fractures was significantly reduced by 46%. The effect was not modified by the underlying cause of osteoporosis or other baseline factors including bone mineral density, QUS, weight, and smoking.

Introduction: This study aimed to determine if the bisphosphonate, clodronate (Bonefos), reduced the incidence of vertebral fractures in osteoporotic women.

Materials and Methods: Women fulfilling the WHO criteria for osteoporosis at the lumbar spine (T-score ≤ −2.5) and/or with at least one prevalent vertebral fracture were recruited to a 3-year double-blind, placebo-controlled study. A total of 593 patients were randomized to two strata comprised of women with postmenopausal osteoporosis (I, n = 483) and secondary osteoporosis (II, n = 110). They received either clodronate 800 mg daily orally (n = 292) or an identical placebo (n = 301). All patients received a calcium supplement of 500 mg daily. BMD was measured at 6, 12, 24, and 36 months, and lateral spine radiographs were obtained at baseline and annually thereafter for vertebral morphometry.

Results: Treatment with clodronate was associated with a significant increase in mean spine BMD over 3 years (percent change from baseline, 4.35 ± 6.34% versus 0.64 ± 6.02% in the placebo group, p < 0.0001). At the hip, clodronate maintained total BMD, whereas a significant decrease was observed in the placebo group (percent change from baseline 0.70 ± 5.67% versus −3.03 ± 6.32% in the placebo group, p < 0.0001). The changes at the spine and hip were similar in both strata. Incident vertebral fractures at 3 years were observed in 63 women in the placebo group and 33 patients receiving clodronate (relative risk, 0.54; 95% CI, 0.37–0.80; p = 0.001). Clodronate significantly reduced vertebral fracture risk in both strata and in women with or without prior vertebral fracture at baseline. Nonvertebral osteoporosis-associated fractures occurred in 21 women in the placebo group and in 14 women treated with clodronate. Treatment was well tolerated, with no significant difference in adverse event rates, including esophagitis, during clodronate treatment.

Conclusion: We conclude that clodronate 800 mg daily is a safe and effective treatment to reduce fracture risk in women with osteoporosis, regardless of causation.
Differential Effects of Teriparatide on BMD After Treatment With Raloxifene or Alendronate
Authors: Ettinger Bruce , Martin San Javier , Crans Gerald , Pavo Imre
Published: April 2006

We investigated the effects of 18 months of treatment with teriparatide in patients previously treated with long-term antiresorptive therapy using bone turnover markers and bone densitometry. Previous raloxifene treatment allowed for teriparatide-induced early bone marker and BMD increases comparable with previously published results for treatment-naïve patients. Conversely, previous alendronate treatment reduced the bone marker and BMD response.

Introduction: Teriparatide [rhPTH(1–34)] has been shown to increase BMD and reduce the risk of fracture in postmenopausal women with osteoporosis. Our objective was to investigate the skeletal effects of 18 months of treatment with teriparatide in women whose osteoporosis was previously treated with either alendronate or raloxifene.

Materials and Methods: Daily subcutaneous injections of 20 μg teriparatide were administered for 18 months to 59 postmenopausal women, 60–87 years of age, with BMD T-scores ≤ −2.0 who had previously received either alendronate (ALN) or raloxifene (RLX) therapy for 18–36 months. All patients received daily calcium (1000 mg) and vitamin D (400 IU) supplementation. The primary study outcome was change in lumbar spine BMD measured by DXA. Secondary outcomes included changes in bone turnover markers, total hip BMD, and safety.

Results: Median baseline bone turnover marker levels in prior ALN patients were about one-half those of prior RLX patients. During teriparatide treatment, bone markers in prior ALN patients increased later and peaked at about one-third lower levels compared with prior RLX patients. During the first 6 months, there were statistically significant (p < 0.05) group differences in BMD change at the hip (prior ALN −1.8% versus prior RLX +0.5%) and at the spine (prior ALN +0.5% versus prior RLX +5.2%). The positive slopes in hip and lumbar spine BMD were similar in both groups between 6 and 18 months. After 18 months, mean lumbar spine BMD increased 10.2% in prior RLX compared with 4.1% in prior ALN (p < 0.05) patients. Furthermore, at 18 months, mean total hip BMD had significantly increased (1.8%, p < 0.05) in prior RLX patients but was not different from baseline in prior ALN patients.

Conclusions: Teriparatide treatment stimulates bone turnover in patients pretreated with both RLX and ALN. Prior treatment with RLX allows for the expected teriparatide-induced BMD increases comparable with those previously reported for treatment-naïve patients. In contrast, prior treatment with ALN prevents increases in BMD, particularly in the first 6 months.
Cortical Bone in the Human Femoral Neck: Three-Dimensional Appearance and Porosity Using Synchrotron Radiation
Authors: Bousson Valérie , Peyrin Françoise , Bergot Catherine , Hausard Marc , Sautet Alain , Laredo Jean-Denis
Published: April 2006

A high-resolution CT system using synchrotron radiation allowed visualization of the 3D cortical bone microarchitecture and measurement of intracortical porosity of femoral neck cortical bone specimens from 19 female cadavers imaged at 10.13-μm resolution. 3D reconstruction of specimens showed osteonal system arrangement. Mean porosity was 15.88%. This technique will provide insights into the mechanisms involved in osteoporotic hip fractures.

Introduction: The purpose of this study was to show that a high-resolution CT system using synchrotron radiation (SR) allows visualization of the 3D cortical bone microarchitecture of the human femoral neck and quantification of intracortical porosity.

Materials and Methods: Bone specimens from the inferior femoral neck were obtained from 19 female cadavers with no hip fracture (mean, 86.9 ± 8.3 years). The specimens, consisting of embedded ∼7 × 7 × 12-mm cortical bone parallelepipeds, were imaged using SR at 10.13-μm resolution. Commercial software was used to visualize both the 660 × 660 × 660-voxel volumes and the 2D axial slices through each volume. Qualitative examination of 2D axial slices focused on the appearance of the vessel canal system, presence of small bright zones (fully mineralized tissue) in the osseous matrix, and presence of cracks. A method was developed to automatically measure 3D intracortical porosity after separating pure bone from pores and cortical bone from trabecular bone.

Results and Conclusions: 3D reconstruction of the specimens showed the entire structure and arrangement of the osteonal systems, parallel to the axis of the femoral neck. Bright zones were seen in the outer cortex. No cracks were observed. Porosity values varied widely from 4.96% to 38.87% (mean, 15.88 ± 9.87%). This study establishes that SR microtomography can be used to display the 3D bone microstructure of the human femoral neck cortex and to quantify intracortical porosity. This technique will provide insights into the mechanisms involved in cortical bone loss and osteoporotic hip fractures.
Treatment of Idiopathic Hyperphosphatasia With Intensive Bisphosphonate Therapy
Authors: Cundy Tim , Wheadon Lisa , King Alan
Published: April 2006

In a family with IH, a rare high turnover bone disease, two older siblings were wheelchair-bound with severe skeletal deformity by age 15. Their youngest affected sibling was treated intensively with intravenous bisphosphonates for 3 years. The treatment was well tolerated and prevented the development of deformity and disability.

Introduction: Idiopathic hyperphosphatasia (IH, also known as juvenile Paget's disease) is a rare genetic bone disease characterized by very high bone turnover and progressive bony deformity. Inhibitors of bone resorption have been used to suppress bone turnover in the short term, but there is no published data on long-term efficacy.

Materials and Methods: An 11-year-old girl with IH, who had two severely affected older siblings, presented with progressive deformity and deafness and long bone fractures. Conventional pediatric doses of pamidronate had failed to prevent clinical deterioration or suppress bone turnover completely. Intensive bisphosphonate therapy (frequent 5-mg ibandronate infusions) was given to try and arrest progression of the skeletal disease. Growth and development, pure tone audiometry, biochemistry, radiology, densitometry (DXA), and bone histology were monitored.

Results: A total of 45 mg ibandronate was given over 3 years until skeletal maturity was reached (20, 15, and 10 mg for years 1–3, respectively). Ibandronate treatment was well tolerated, and biochemical markers of bone turnover suppressed to within the age-appropriate normal range There was some progression of her thoracic kyphosis, but she had no further fractures and remained mobile and active at an age when her siblings had become wheelchair-bound. A significant recovery of hearing (p < 0.01) was documented, particularly at low frequencies. Radiographs showed improvement in spinal osteoporosis and cortical bone dimensions and arrest of progressive acetabular protrusion. Areal bone density increased substantially (lumbar spine z-score from −2.2 to + 1.8). Tetracycline-labeled bone biopsy specimens were taken before and after 18 months of intensive treatment. The second biopsy showed suppression of bone turnover and a doubling of trabecular thickness, with no mineralization defect, and no osteopetrosis.

Conclusions: Intensive bisphosphonate treatment prevented the development of deformity and disability and improved hearing in this child with IH. The dose of bisphosphonate, which is substantially greater than is usually used in pediatric bone disease, had no adverse effects, in particular on bone mineralization.