bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular Machinery for Matrix Degradation in Bone-Resorbing Osteoclasts
Authors: Vääräniemi Jukka , Halleen Jussi M , Kaarlonen Katja , Ylipahkala Hannele , Alatalo Sari L , Andersson Göran , Kaija Helena , Vihko Pirkko , Väänänen H Kalervo
Published: April 2006

In osteoclasts, TRACP co-localized with cathepsin K in transcytotic vesicles and was activated by cathepsin K in vitro, suggesting that TRACP may degrade organic matrix components in transcytotic vesicles in an event regulated by cathepsin K.

Introduction: TRACP is an enzyme with unknown biological function. In addition to its phosphatase activity, TRACP is capable of generating reactive oxygen species (ROS). Bone-resorbing osteoclasts contain large amounts of TRACP, and transgenic animal models suggest that TRACP has a role in bone resorption. Osteoclasts resorb bone by secreting acid and lysosomal enzymes such as cathepsin K into an extracellular resorption lacuna between the cell membrane and bone surface. Matrix degradation products are then endocytosed, transcytosed, and secreted through a functional secretory domain in the basolateral membrane facing bone marrow.

Materials and Methods: We have studied intracellular localization of TRACP in osteoclasts with antibodies against various known endosomal and lysosomal proteins using confocal microscopy. We also studied co-localization of TRACP with cathepsin K and endocytosed bone matrix components and the effect of cathepsin K digestion on the ROS generating activity of TRACP in vitro.

Results: Double-staining experiments of TRACP with endosomal and lysosomal markers showed that, although some endosomal staining was detected, TRACP was not present in lysosomes. However, TRACP was present in transcytotic vesicles, where it co-localized with cathepsin K. Cathepsin K digestion of TRACP in vitro increased the phosphatase activity by 5.6-fold and the ROS generating activity by 2.0-fold.

Conclusions: These results suggest that cathepsin K may activate the ROS-generating activity of TRACP in transcytotic vesicles of resorbing osteoclasts, the ROS being targeted to finalize degradation of organic bone matrix components during their transcytosis.
ApoE Gene Polymorphisms, BMD, and Fracture Risk in Elderly Men and Women: The Rotterdam Study
Authors: Schoofs Mariette WCJ , van der Klift Marjolein , Hofman Albert , van Duijn Cornelia M , Stricker Bruno HCh , Pols Huibert AP , Uitterlinden André G
Published: April 2006

To study the association between the ApoE gene polymorphism and osteoporosis, we performed an association study in 5857 subjects from the Rotterdam Study. We did not observe an association between the ApoE polymorphism and osteoporosis in this study, which is thus far the largest study on ApoE and osteoporosis.

Introduction: The E4 allele of the E2, E3, E4 protein isoform polymorphism in the gene encoding apolipoprotein E (ApoE) has previously been associated with an increased fracture risk. We investigated the association between the ApoE polymorphism and BMD, bone loss, and incident fractures as part of the Rotterdam Study a prospective population-based cohort study of diseases in the elderly.

Materials and Methods: The study population consisted of 5857 subjects (2560 men; 3297 women) for whom data on ApoE genotypes, confounding variables, and follow-up of nonvertebral fractures were available. Data on femoral neck and lumbar spine BMD were available for 4814 participants. Genotype analyses for bone loss (defined as annualized percent change in BMD at the hip and lumbar spine) and BMD were performed using ANOVA. Fractures were analyzed using a Cox proportional-hazards model and logistic regression. All relative risks were adjusted for age and body mass index.

Results and Conclusions: The genotype distribution of the study population was in Hardy-Weinberg equilibrium (p = 0.98) and did not differ by gender. At baseline, mean BMD of the lumbar spine and femoral neck did not differ between the ApoE genotypes of men and women. Bone loss (mean follow-up, 2.0 years) did not differ by ApoE genotype for women and men. During a mean follow-up of 6.6 years, 708 nonvertebral fractures (198 hip fractures and 179 wrist fractures) and 149 incident vertebral fractures occurred. No consistent differences in the distribution of alleles could be observed between subjects with or without these fractures. Our data do not support the hypothesis that the ApoE4 risk allele is associated with BMD, increased bone loss, or an increased risk of osteoporotic fractures.
Influence of LRP5 Polymorphisms on Normal Variation in BMD
Authors: Koay M Audrey , Woon Peng Y , Zhang Yun , Miles Lisa J , Duncan Emma L , Ralston Stuart H , Compston Juliet E , Cooper Cyrus , Keen Richard , Langdahl Bente L , MacLelland Alasdair , O'Riordan Jeffrey , Pols Huibert A , Reid David M , Uitterlinden Andre G , Wass John AH , Brown Matthew A
Published: April 2006

Genetic studies based on cohorts with rare and extreme bone phenotypes have shown that the LRP5 gene is an important genetic modulator of BMD. Using family-based and case-control approaches, this study examines the role of the LRP5 gene in determining normal population variation of BMD and describes significant association and suggestive linkage between LRP5 gene polymorphisms and BMD in >900 individuals with a broad range of BMD.

Introduction: Osteoporosis is a common, highly heritable condition determined by complex interactions of genetic and environmental etiologies. Genetic factors alone can account for 50–80% of the interindividual variation in BMD. Mutations in the LRP5 gene on chromosome 11q12–13 have been associated with rare syndromes characterized by extremely low or high BMD, but little is known about the contribution of this gene to the development of osteoporosis and determination of BMD in a normal population.

Materials and Methods: To examine the entire spectrum of low to high BMD, 152 osteoporotic probands, their families (597 individuals), and 160 women with elevated BMD (T score > 2.5) were recruited. BMD at the lumbar spine, femoral neck, and hip were measured in each subject using DXA.

Results: PAGE sequencing of the LRP5 gene revealed 10 single nucleotide polymorphisms (SNPs), 8 of which had allele frequencies of >5%, in exons 8, 9, 10, 15, and 18 and in introns 6, 7, and 21. Within families, a strong association was observed between an SNP at nucleotide C171346A in intron 21 and total hip BMD (p < 1 × 10−5 in men only, p = 0.0019 in both men and women). This association was also observed in comparisons of osteoporotic probands and unrelated elevated BMD in women (p = 0.03), along with associations with markers in exons 8 (C135242T, p = 0.007) and 9 (C141759T, p = 0.02). Haplotypes composed of two to three of the SNPs G121513A, C135242T, G138351A, and C141759T were strongly associated with BMD when comparing osteoporotic probands and high BMD cases (p < 0.003). An SNP at nucleotide C165215T in exon 18 was linked to BMD at the lumbar spine, femoral neck, and total hip (parametric LOD scores = 2.8, 2.5, and 2.2 and nonparametric LOD scores = 0.3, 1.1, and 2.2, respectively) but was not genetically associated with BMD variation.

Conclusion: These results show that common LRP5 polymorphisms contribute to the determination of BMD in the general population.
Association of the G-174C Variant in the Interleukin-6 Promoter Region With Bone Loss and Fracture Risk in Older Women
Authors: Moffett Susan P , Zmuda Joseph M , Cauley Jane A , Stone Katie L , Nevitt Michael C , Ensrud Kristine E , Hillier Teresa A , Hochberg Marc C , Joslyn Geoff , Morin Phillip , Cummings Steven R
Published: April 2006

We analyzed the association between the IL-6 G-174C polymorphism and osteoporosis phenotypes in 3376 older women. Women with the C/C genotype had a significantly slower rate of decline in hip BMD and a 33% lower risk of wrist fracture than women with the G/G genotype. Variation at the IL-6 locus may contribute to the genetic susceptibility to bone fragility.

Introduction: Interleukin 6 (IL-6) promotes osteoclast formation and bone resorption. The C allele of the G-174C polymorphism in the IL-6 promoter region has been related to lower gene transcription and plasma IL-6 levels.

Materials and Methods: In this study, we evaluated the relationship between the IL-6 G-174C polymorphism and BMD, the rate of decline in BMD, and the risk of fracture in 3376 women 65 years of age and older participating in the Study of Osteoporotic Fractures. BMD was measured at the distal and proximal radius using single photon absorptiometry and at the hip using DXA. Hip BMD was measured again an average of 3.5 years later. Incident fractures over an average of 10.8 years of follow-up were confirmed by physician adjudication of radiology reports.

Results: Distal and proximal radius BMD was lowest among women with the G/G genotype, intermediate in the heterozygotes, and highest in women with the C/C genotype (p = 0.016 and p = 0.049, respectively), although the differences between the genotypes were small. While there were no differences by genotype with initial hip BMD, women with the C/C genotype experienced a slower rate of decline in total hip and femoral neck BMD compared with women with the G/G genotype (p = 0.004 and p = 0.029, respectively). Women with the C/C genotype also had 33% lower risk of wrist fracture compared with women with the G/G genotype, independent of age, body mass index, estrogen use, and study center (RR, 0.67; 95% CI, 0.45, 1.00; p = 0.048), whereas heterozygous women had a more intermediate risk (RR, 0.85; 95% CI, 0.65, 1.12; p = 0.256). No association was found between genotype and risk of hip or all non-spine fractures.

Conclusions: These results suggest that the IL-6 G-174C promoter polymorphism may be a genetic marker for bone loss and wrist fracture among older women.
Maternal and Fetal Outcome After Long-Term Pamidronate Treatment Before Conception: A Report of Two Cases
Authors: Munns Craig FJ , Rauch Frank , Ward Leanne , Glorieux Francis H
Published: April 2006

The pregnancies of two women with osteogenesis imperfecta who received intravenous pamidronate before conception are reported. The mothers suffered no ill effects. One baby had transient asymptomatic hypocalcemia and one had bilateral talipes equinovarus.

This report documents the pregnancy outcomes of two women with osteogenesis imperfecta (OI), types I and IV, who received intravenous pamidronate as part of an observational trial before conception. Pamidronate was not administered after conception. Other than hyperemesis in one woman, the pregnancies and deliveries were uneventful. Both babies inherited OI from their mothers. The baby with OI type IV also had bilateral talipes equinovarus. Biochemical evaluation of the mothers and babies at 24 h and/or 2 weeks postpartum was normal, apart from one baby with asymptomatic hypocalcemia at 24 h of age that had resolved when next measured on day 11 of life. No biochemistry was available on the second child until 13 days of age. Neither baby had skeletal modeling abnormalities consistent with in utero pamidronate exposure. The lumbar spine (L1-L4) areal BMD and anterior to posterior height ratios of lumbar vertebral bodies of both women remained constant during pregnancy. Both the mothers and babies remain well and free of fracture 14 and 16 months postpartum.
Strategies for Directing the Differentiation of Stem Cells Into the Osteogenic Lineage In Vitro
Authors: Heng Boon Chin , Cao Tong , Stanton Lawrence Walter , Robson Paul , Olsen Bjorn
Published: April 2006

A major area in regenerative medicine is the application of stem cells in bone reconstruction and bone tissue engineering. This will require well-defined and efficient protocols for directing the differentiation of stem cells into the osteogenic lineage, followed by their selective purification and proliferation in vitro. The development of such protocols would reduce the likelihood of spontaneous differentiation of stem cells into divergent lineages on transplantation, as well as reduce the risk of teratoma formation in the case of embryonic stem cells. Additionally, such protocols could provide useful in vitro models for studying osteogenesis and bone development, and facilitate the genetic manipulation of stem cells for therapeutic applications. The development of pharmokinetic and cytotoxicity/genotoxicity screening tests for bone-related biomaterials and drugs could also use protocols developed for the osteogenic differentiation of stem cells. This review critically examines the various strategies that could be used to direct the differentiation of stem cells into the osteogenic lineage in vitro.
Isosorbide Mononitrate Increases Bone Formation and Decreases Bone Resorption in Postmenopausal Women: A Randomized Trial
Authors: Jamal Sophie A , Cummings Steven R , Hawker Gillian A
Published: April 2006

NO regulates bone remodeling in cellular and animal models. We examined the effect of administering ISMO, a NO donor, on bone turnover in 144 postmenopausal women. After 3 months, women randomized to ISMO had a greater decrease in bone resorption and a greater increase in bone formation compared with placebo. NO donors may prevent postmenopausal bone loss.

Introduction: NO both stimulates bone formation and inhibits bone resorption in vitro. NO donors (nitrates) are inexpensive and widely available, but their value for postmenopausal osteoporosis has never been evaluated in a randomized trial.

Materials and Methods: We randomly assigned 144 healthy postmenopausal women with a hip BMD T score between 0 and −2.5 to 5 or 20 mg/day of isosorbide mononitrate (ISMO) or placebo for 12 weeks. We measured urine N-telopeptide (NTx), a marker of bone resorption, and serum bone-specific alkaline phosphatase (BSALP), a marker of bone formation. Markers were measured immediately before randomization and after 12 weeks of treatment. We calculated the percent change in NTx and BSALP for each of the treatment groups (placebo, 5 mg ISMO, and 20 mg ISMO). Our primary outcome was the percent change in NTx and BSALP in the 5- and 20-mg ISMO groups compared with placebo.

Results and Conclusions: Compared with women randomized to placebo, women randomized to 20 mg of ISMO had a 45.4% decrease in NTx (95% CI, 25.8-64.9) and a 23.3% increase (95% CI, 8.9-37.8) in BSALP. Women randomized to 5 mg of ISMO had a 36.3% decrease in NTx (95% CI, 14.8-57.8) and a 15.9% increase in BSALP (95% CI, 1.1-30.7). ISMO decreases bone resorption and increases bone formation. These findings suggest that nitrates may be useful for the prevention of postmenopausal osteoporosis.
Physical Activity and Calcium Consumption Are Important Determinants of Lower Limb Bone Mass in Older Women
Authors: Devine Amanda , Dhaliwal Satvinder S , Dick Ian M , Bollerslev Jens , Prince Richard L
Published: April 2006

A population-based study of 1363 older women showed that the 24% who achieved high physical activity and dietary calcium intakes had a 5.1% higher hip BMD than those who did not, supporting the concept that lifestyle factors play an important role in the maintenance of lower extremity bone mass in older women.

Introduction: Although there is general agreement that increased dietary calcium consumption and exercise can slow bone loss in older women, the amount required to have this effect in an older population remains uncertain. This study was devised to examine the effects of calcium consumption (CC) and physical activity (PA) (lifestyle management) on bone mass in an older female population.

Materials and Methods: Using a cross-sectional study design, a population-based sample of older women (mean age, 75 ± 3 years) had hip and heel bone mass measured using DXA (Hologic 4500A; n = 1076) and quantitative ultrasound (QUS, Lunar Achilles; n = 1363), respectively. CC and PA were measured by a validated habitual food frequency and activity questionnaire, respectively. Dose-response effects of PA and CC on bone mass were examined using ANOVA.

Results and Conclusions: Division of the PA and CC into tertiles best described the dose-response effects. After adjustment for CC, age, weight, alcohol consumption, and cigarette smoking, high PA compared with medium or low PA was associated with higher hip BMD and heel QUS (total hip BMD, 3.1%; p < 0.001; QUS stiffness, 2.7%; p = 0.002). After adjustment for PA and covariates, high or medium CC compared with low CC was associated with higher total hip BMD (1.8%; p = 0.027), with no effect at the QUS heel site. PA and CC were dichotomized at the cut-points for effects on BMD. The combination of high PA and CC, achieved by 24% of the population, was associated with a total hip BMD that was 5.1% higher (34% of SD) than those individuals in the low PA and CC group. Stiffness was 3.6% (23% of SD) higher in the high PA and CC group than in the low PA and CC group. If the whole population undertook and achieved a high PA and high CC lifestyle, the population risk of hip fractures may be expected to be reduced by about 17% in this age group as a result of beneficial effects on the musculoskeletal system.
Combined Bone Morphogenetic Protein-2 and −7 Gene Transfer Enhances Osteoblastic Differentiation and Spine Fusion in a Rodent Model
Authors: Zhu Wei , Rawlins Bernard A , Boachie-Adjei Oheneba , Myers Elizabeth R , Arimizu Jun , Choi Eugene , Lieberman Jay R , Crystal Ronald G , Hidaka Chisa
Published: April 2006

To enhance the osteogenic activity of BMP, combination BMP2 and BMP7 gene transfer was performed. This approach led to a significant increase in osteoblastic differentiation of mesenchymal precursors compared with single BMP gene transfer in vitro. When tested in 78 rats, combination gene transfer enhanced mechanically stable spine fusion and bone formation rate versus single BMP gene transfer.

Introduction: Although clinical bone morphogenetic protein (BMP) therapy is effective, required doses are very high. Previous studies have suggested that the co-expression of two different BMP genes can result in the production of heterodimeric BMPs that may be more potent than homodimers. In this study, combined BMP2 and BMP7 gene transfer was performed to test whether this approach improves osteoblastic differentiation and bone formation compared with single BMP gene transfer.

Materials and Methods: A producer cell (A549) was co-transfected with adenovirus vectors encoding BMP2 (AdBMP2) and BMP7 (AdBMP7) or, as controls, each vector alone, AdNull (with no transgene) or no virus. Supernatants were compared for their ability to stimulate osteoblastic differentiation of C2C12 myoblasts and MC3T3-E1 pre-osteoblasts. In a rat posterolateral spine fusion model, co-administration of AdBMP2 and AdBMP7 was compared with treatment with each vector alone, AdNull or no virus in 78 rats. The spines were assessed 8 weeks after surgery for radiographic and mechanical fusion, bone formation, and mineralization.

Results: BMP2 and BMP7 were co-precipitated from supernatants of cells co-transfected with AdBMP2 and AdBMP7, indicating the presence of BMP2/7 heterodimer. Supernatants of co-transfected cells containing relatively low doses (7–140 ng/ml) of BMPs induced osteocalcin expression and alkaline phosphatase activity in both C2C12 and MC3T3-E1 cells, that were up to 6- and 40-fold higher, respectively, than levels induced by maximal doses (200–1000 ng/ml) of either BMP2 or BMP7 alone. In the spine fusion model, co-administration of AdBMP2 and AdBMP7 resulted in a significantly greater number of mechanically stable fusions and also 2-fold higher mineralization rate and bone volume in the fusion mass versus single BMP gene transfer (p < 0.02, all comparisons).

Conclusion: Combined BMP2 and BMP7 gene transfer is significantly more effective in inducing osteoblastic differentiation and spine fusion than individual BMP gene transfer.
Osteoporosis Prevalence and Levels of Treatment in Primary Care: The Australian BoneCare Study
Authors: Eisman John , Clapham Sharon , Kehoe Linda
Published: April 2006

The level of recognition and treatment of osteoporosis is not well characterized in primary care. In data from a large sample of postmenopausal women attending 927 primary care physicians, 29% of women reported one or more fractures after menopause. The great majority (72%) were not on any osteoporosis-specific therapy.

Introduction: Osteoporosis is often first recognized at the time of a low-trauma fracture. However, by this stage, the risk of subsequent fractures has already risen substantially. Moreover, in many countries, only a small proportion of women, who have already sustained fractures, receive a treatment shown to reduce this increased risk of further fractures.

Materials and Methods: This project was initiated to examine the prevalence of osteoporotic fractures, risk factors for osteoporosis, and use of antifracture therapy among postmenopausal Australian women. More than 88,000 women from 927 primary care physicians returned over 69,358 surveys. Of these, 57,088 reported the presence of a postmenopausal fracture or risk factors.

Results: Among these randomly selected postmenopausal women, 29% reported having had one or more low-trauma fractures after menopause (44% substantiated in current records). One-third of these women reported multiple fractures. The prevalence of all types of fractures, except rib and ankle, increased with age and low body weight. Those who reported fractures were also more likely to report early menopause, corticosteroid use, and a family history of osteoporosis. Moreover, those with vertebral fractures were more likely to record height loss, kyphosis, and back pain. Physical inactivity, low calcium intake, and smoking had no consistent relationship with any fracture outcome. Of the women who reported a fracture after menopause, only 28% were on any specific therapy for osteoporosis, and 7% were on calcium alone. Of those who had been told they had osteoporosis by a doctor, 40% were receiving specific osteoporosis therapy.

Conclusions: In this large study of postmenopausal Australian women attending primary care physicians, 29% reported at least one low-trauma fracture after menopause. Less than one-third of these women were on specific treatment for osteoporosis, and only 40% were ever told they had osteoporosis. Therefore, osteoporotic fractures are common in postmenopausal Australian women, and few, despite their substantially increased risk of further fractures, are on any specific anti-osteoporotic therapy. These data support the need for more effective education for the community and medical practitioners of the clinical significance of osteoporotic fractures and alternatives for treatment.
Strontium Ranelate Improves Bone Resistance by Increasing Bone Mass and Improving Architecture in Intact Female Rats
Authors: Ammann Patrick , Shen Victor , Robin Bruno , Mauras Yves , Bonjour Jean-Philippe , Rizzoli Rene
Published: April 2006

Strontium ranelate given to intact rats at doses up to 900 mg/kg/day increases bone resistance, cortical and trabecular bone volume, micro-architecture, bone mass, and total ALP activity, thus indicating a bone-forming activity and an improvement of overall bone tissue quality.

Introduction: Various anti-osteoporotic agents are available for clinical use; however, there is still a need for drugs able to positively influence the coupling between bone formation and bone resorption to increase bone mass and bone strength. Strontium ranelate (PROTELOS), a new chemical entity containing stable strontium (Sr), was tested for its capacity to influence bone quality and quantity.

Materials and Methods: The long-term effects of strontium ranelate on bone were investigated in intact female rats treated with various doses of strontium ranelate (0, 225, 450, and 900 mg/kg/day) for 2 years. In a second series of experiments, the effects of 625 mg/kg/day were evaluated in intact male and female rats for the same period of time. Bone mineral mass and mechanical properties were evaluated at various skeletal sites (vertebra and femur), and bone tissue micro-architecture was evaluated by static histomorphometry at the tibio-fibular junction (cortical bone) and at the tibia metaphysis (trabecular bone). Plasma total alkaline phosphatase (ALP) activity and serum levels of insulin-like growth factor-I (IGF-I) were also assessed.

Results: In female rats treated with strontium ranelate over 2 years, dose-dependent increases of bone strength and bone mass of the vertebral body (containing a large proportion of trabecular bone) and of the midshaft femur (containing mainly cortical bone) were detected without change in bone stiffness. Similar effects were observed in males at the level of the vertebra. This increase in mechanical properties was associated with improvements of the micro-architecture as assessed by increases of trabecular and cortical bone volumes and trabecular number and thickness. Finally, plasma total ALP activity and IGF-I were also increased in treated animals, compatible with a bone-forming activity of strontium ranelate.

Conclusion: A long-term treatment with strontium ranelate in intact rats is very safe for bone and improves bone resistance by increasing bone mass and improving architecture while maintaining bone stiffness.
The COMT val158met Polymorphism Is Associated With Peak BMD in Men
Authors: Lorentzon Mattias , Eriksson Anna-Lena , Mellström Dan , Ohlsson Claes
Published: April 2006

The associations between the functional val158met polymorphism of the estrogen-degrading COMT enzyme and skeletal properties in young men were investigated. BMD was associated with COMT genotype.

Introduction: Peak BMD is an important predictor of future risk of osteoporosis, and it is to a large extent determined by genetic factors. Estrogens are involved in the accretion of bone mass during puberty. Catechol-O-methyltransferase (COMT) is involved in the degradation of estrogens. There is a functional polymorphism in the COMT gene (val158met), resulting in a 60–75% difference in enzyme activity between the val (high activity [H]) and met (low activity [L]) variants. The aim of this cross-sectional study was to investigate the associations between this polymorphism and peak BMD in young men.

Materials and Methods: A total of 458 healthy men (mean age, 19 ± 0.6 years) were genotyped and classified as COMTLL, COMTHL, or COMTHH. Areal BMD (aBMD) was measured by DXA. Cortical and trabecular volumetric BMD (vBMD) were measured by pQCT. The associations between COMT genotype and skeletal phenotypes were determined.

Results and Conclusions: Regression models using physical activity, height, weight, age, and COMT genotype as covariates showed that COMT genotype was an independent predictor of aBMD in the total body and in all femur locations investigated, but not in the spine. The values for COMTHL and COMTHH were very similar, and therefore, they were pooled into one group. aBMD at Ward's triangle, trochanter, and total femur were 4.9%, 4.5%, and 3.7% lower, respectively, in the COMTLL than in the COMTHL/HH group (p < 0.01). pQCT analyses showed that COMT genotype was an independent predictor of trabecular vBMD of the tibia, radius, and fibula. Trabecular vBMD of the radius and fibula in COMTLL was 5.3% and 7.4% lower, respectively, than that of the combined COMTHL/HH group. COMT genotype was associated with cortical vBMD but not with cortical cross-sectional area in the tibia. These findings show that the COMT polymorphism is associated with BMD in young adult men.
CCR1 Chemokines Promote the Chemotactic Recruitment, RANKL Development, and Motility of Osteoclasts and Are Induced by Inflammatory Cytokines in Osteoblasts
Authors: Yu Xuefeng , Huang Yuefang , Collin-Osdoby Patricia , Osdoby Philip
Published: April 2006

Chemoattractants that recruit OC precursors to locally inflamed sites of resorption are not well known. A chemokine receptor, CCR1, was expressed in OC precursors and elevated in mature OCs, and its ligands promoted OC precursor recruitment, RANKL development, and OC motility. Cytokines induced OB release of such chemokines, which may therefore significantly contribute to inflammatory bone loss.

Introduction: Chemokines, primarily of two major (CXC, CC) families, are essential signals for the trafficking and localization of circulating hematopoietic cells into tissues. However, little is known about their potential roles in osteoclast (OC) recruitment, development, or function. Previously, we analyzed CXC receptors in murine OC precursors and found high expression of CXCR4 that mediated their stromal-derived factor-1(SDF-1)-induced chemotaxis and collagen invasion. Here, we investigated if CC receptors and ligands, which are elevated in inflammatory and other osteolytic diseases, also play important roles in the recruitment, formation, or activity of murine bone-resorptive OCs.

Materials and Methods: CC chemokine receptor (CCR) mRNA expression was analyzed during OC formation induced by RANKL in murine RAW 264.7 cells and primary marrow cells. Corresponding CC chemokines were tested for their ability to elicit precursor chemotaxis or OC development, or to influence motility, bone resorption, adhesion, or survival in RANKL-differentiated OCs. Constitutive and inflammatory cytokine-induced release of the chemokines macrophage inflammatory protein-1α (MIP-1α) and regulated on activation, normal T-cell expressed and secreted (RANTES) was measured by ELISA for OCs, osteoblasts (OBs), and their precursor cells.

Results: CCR1 was expressed in murine marrow cells, the most prominent CCR in RAW cells, and upregulated by RANKL in marrow or RAW cells. Chemokines that bind CCR1 (MIP-1α, RANTES, and monocyte chemoattractant protein-3 [MCP-3]) were produced to varying degrees by murine OCs, OBs, and their precursors, and markedly increased by interleukin (IL)-1α and TNFα in differentiating OBs. RANTES, and especially MIP-1α, increased mature OC motility, but did not alter OC resorption activity, adhesion, or survival. All three chemokines stimulated chemotaxis of marrow or RAW cell precursors, leading to the greater formation of OCs (in number and size) after RANKL development of such chemoattracted marrow cells. All three chemokines also directly and dramatically enhanced OC formation in marrow cultures, through a pathway dependent on the presence of RANKL but without altering RANK expression.

Conclusions: Pathological increases in secretion of these chemokines from activated OBs or other cells may potently stimulate the chemotactic recruitment and RANKL formation of bone-resorptive OCs, thereby exacerbating local osteolysis in multiple skeletal diseases.
Amino-Terminal Parathyroid Hormone Fragment Analogs Containing α,α-di-alkyl Amino Acids at Positions 1 and 3
Authors: Shimizu Naoto , Dean Thomas , Khatri Ashok , Gardella Thomas J
Published: April 2006

To define and minimize the N-terminal PTH pharmacophore, the effects of introducing different conformationally constraining di-alkyl amino acids at positions 1 and 3 of PTH(1–14) analogs were studied. Improvements in PTH receptor-binding affinity and signaling potency were found, although some substitutions resulted in partial agonism.

Introduction: The N-terminal portion of parathyroid hormone (PTH) plays a critical role in PTH-1 receptor (P1R) activation. To investigate the mechanisms underlying this action and to minimize the N-terminal PTH pharmacophore, we employed the PTH(1–14) fragment as a scaffold for structure-activity relationship studies, and thus previously found that substitutions of the conformationally constraining, di-alkyl amino acid, α-amino-isobutyric acid (Aib), at positions 1 and 3 increase the P1R-binding affinity and signaling potency of the analog ∼100-fold. Here we extend these findings by investigating the effects of other constrained di-alkyl amino acids at positions 1 and/or 3 on PTH analog activity.

Materials and Methods: The di-alkyl amino acids, 1-aminocycloalkane-carboxylic acid (Acxc, x = 3, 5, or 6) or diethylglycine (Deg), representing alkyl configurations of varying volumes and shape (cyclic and linear), were introduced into the parent peptide, [M]PTH(1–14) (M = Ala1,3,12,Gln10,Har11,Trp14), and the analogs were tested for activity in P1R-expressing cells.

Results: Relative to the binding affinity and cAMP-stimulating potency of the parent peptide (IC50 = 27 mM; EC50 = 220 nM), PTH(1–14) analogs substituted at position 1 exhibited 2- (Ac3c) to 60-fold (Ac5c) increases in affinity and potency, as measured in LLC-PK1 cells stably expressing the cloned P1R. Combining the substitutions of Ac5c1 and Aib3 yielded the highest affinity and most potent PTH(1–14) and shorter-length analogs to date: [Ac5c1, Aib3,M]PTH(1-X) (X = 14, 11, and 10; IC50s = 80 nM, 260 nM, and 850 μM; EC50s = 1.7 nM, 3.1 nM, and 1.9 μM, respectively). The effects of Ac6c1 were similar to those of Ac5c1. A dissociation of binding affinity and signaling activity occurred with Deg, as [Deg1,3,M]PTH(1–14) was a partial agonist.

Conclusion: Constraining the N-terminal PTH backbone conformation with di-alkyl amino acids at positions 1 and 3 may be a general strategy for optimizing and minimizing the PTH pharmacophore; however, inhibitory side-chain effects may be encountered. The new analogs presented should be useful as minimum-length functional probes of the PTH-PTH receptor interaction mechanism.
Mineral Acquisition Rates in Developing Enamel on Maxillary and Mandibular Incisors of Rats and Mice: Implications to Extracellular Acid Loading as Apatite Crystals Mature
Authors: Smith Charles E , Chong Dennis Lee , Bartlett John D , Margolis Henry C
Published: April 2006

The formation rates of mineral in developing enamel were determined by microweighing of incisors of mice and rats. Computations indicated that a large excess of hydrogen ions would result from creating apatite at the calculated rates. Enamel organ cells (ameloblasts), therefore, likely excrete bicarbonate ions to prevent pH in fluid bathing enamel from becoming too acidic.

Introduction: Protons (H+) are generated whenever calcium and phosphate ions combine directly from aqueous solutions to form hydroxyapatite. Enamel is susceptible to potential acid loading during development because the amount of fluid bathing this tissue is small and its buffering capacity is low. The epithelial cells covering this tissue are also believed to form permeability barriers at times during the maturation stage when crystals grow at their fastest rates. The goal of this study was to measure the bulk weight of mineral present in rodent enamel at specific times in development and estimate the amount of acid potentially formed as the apatite crystals mature.

Materials and Methods: Upper and lower jaws of mice and rats were freeze-dried, and the enamel layers on the incisors were partitioned into a series of 0.5 mm (mouse) or 1.0 mm (rat) strips along the length of each tooth. The strips were weighed on a microbalance, ashed at 575°C for 18–24 h to remove organic material, and reweighed to determine the actual mineral weight for each strip.

Results and Conclusions: The data indicated that, despite differences in gross sizes and shapes of maxillary and mandibular incisors in rats and mice, the overall pattern and rates of mineral acquisition were remarkably similar. This included sharply increasing rates of mineral acquisition between the secretory and maturation stages, with peak levels approaching 40 μg/mm tooth length. Computer modeling indicated that quantities of H+ ions potentially generated as apatite crystals grew during the maturation stage greatly exceeded local buffering capacity of enamel fluid and matrix proteins. In other systems, bicarbonate ions are excreted to neutralize highly acidic materials generated extracellularly. Data from this study indicate that ameloblasts, and perhaps cells in other apatite-based hard tissues, use similar bicarbonate release mechanisms to control excess acid arising from mineral formation.
Males Have Larger Skeletal Size and Bone Mass Than Females, Despite Comparable Body Size
Authors: Nieves Jeri W , Formica Carmelo , Ruffing Jamie , Zion Marsha , Garrett Patricia , Lindsay Robert , Cosman Felicia
Published: April 2006

Gender differences in fractures may be related to body size, bone size, geometry, or density. We studied this in 18-year-old males (n = 36) and females (n = 36) matched for height and weight. Despite comparable body size, males have greater BMC and BMD at the hip and distal tibia and greater tibial cortical thickness. This may confer greater skeletal integrity in males.

Introduction: Gender differences in fractures may be related to body size, bone size, geometry, or density. We studied this in males (n = 36) and females (n = 36; mean age = 18 years) pair-matched for height and weight.

Materials and Methods: BMC, bone area (BA), and BMD were measured in the spine and hip using DXA. Distal tibia was measured by pQCT.

Results and Conclusions: Males had a higher lean mass (92%) compared with females (79%). No gender differences were observed for vertebral BMC or vertebral height, although males had greater width and thus BA at the spine. Males had greater BMC and BA at the femoral neck and total femur (p < 0.02). Geometric variables of the hip including neck diameter and neck-axis length were also greater in males (p < 0.02). There was greater cross-sectional moment of inertia, safety factor, and fall index in males (all p < 0.02). Males had greater tibial BMC, volumetric BMD, and cortical area and thickness compared with females (p < 0.01), with both greater periosteal circumference (p = 0.011) and smaller endosteal circumference (p = 0.058). Statistically controlling for lean mass reduced gender differences, but males still had 8% higher hip BMD (p = 0.24) and 5.3% higher total tibial BMD (p = 0.05). A subset of males and females were matched (n = 14 pairs) for total hip BA. Males in this subset still had greater BMC and BMD at the total hip (p < 0.05) than females, despite similar BA. In summary, despite comparable body size, males have greater BMC and BMD than females at the hip and distal tibia but not at the spine. Differences in BMC and BMD were related to greater cortical thickness in the tibia. We conclude that differences in bone mass and geometry confer greater skeletal integrity in males, which may contribute to the lower incidence of stress and osteoporotic fractures in males.
Expression of LRP1 by Human Osteoblasts: A Mechanism for the Delivery of Lipoproteins and Vitamin K1 to Bone
Authors: Niemeier Andreas , Kassem Moustapha , Toedter Klaus , Wendt Dorte , Ruether Wolfgang , Beisiegel Ulrike , Heeren Joerg
Published: April 2006

Accumulating clinical and experimental data show the importance of dietary lipids and lipophilic vitamins, such as vitamin K1, for bone formation. The molecular mechanism of how they enter the osteoblast is unknown. Here we describe the expression of the multifunctional LRP1 by human osteoblasts in vitro and in vivo. We provide evidence that LRP1 plays an important role in the uptake of postprandial lipoproteins and vitamin K1 by human osteoblasts.

Introduction: Chylomicrons (CM) and their remnants (CR) represent the postprandial plasma carriers of dietary lipids. Dietary vitamin K1 is known to be transported in the circulation as part of CM/CR and is required by osteoblasts as an essential co-factor for the γ-carboxylation of bone matrix proteins. The molecular mechanisms underlying the delivery of lipophilic substances to bone are not understood. In this study, the expression and function of CM/CR receptors was examined in human osteoblasts.

Materials and Methods: Four human osteoblast-like cell lines were analyzed: two osteosarcoma lines (MG63, SaOS-2) and two telomerase-immortalized human bone marrow stromal cell lines (hMSC-TERT ‘4’ and ‘20’) after 1,25(OH)2vitamin D3 induction of osteoblastic differentiation (hMSC-TERT-OB). Receptor expression was examined by Western blotting and immunohistochemistry of normal human bone sections. Endocytotic receptor function was analyzed by cellular uptake assays using fluorescent and radiolabeled human CR. Vitamin K1-enriched CR (CR-K1) were generated in vivo after oral vitamin administration and vitamin K1 uptake by osteoblasts was measured by HPLC. The effect of CR-K1 uptake on osteocalcin carboxylation was measured by ELISA.

Results: Osteoblasts exhibit high levels of protein expression of the CR receptors LRP1 and LDLR. VLDLR is expressed to a lower degree. Immunohistochemistry of normal human bone sections showed strong LRP1 expression by osteoblasts and marrow stromal cells. Uptake of fluorescent CR by osteoblasts resulted in the typical pattern of receptor-mediated endocytosis. CR uptake was stimulated by the exogenous addition of the lipoprotein receptor ligands apolipoprotein E and lipoprotein lipase. Uptake was reduced by the known LRP1 inhibitors RAP, lactoferrin, and suramin, but not by LDL, which exclusively binds to the LDLR. Vitamin K1 uptake by hMSC-TERT-OB after incubation with CR-K1 was also shown to be sensitive to LPL stimulation and the LRP1 specific inhibitor lactoferrin. CR-K1 uptake into osteoblasts stimulated the γ-carboxylation of osteocalcin.

Conclusion: Human osteoblasts express receptors of the LDLR family with a capacity for vitamin K1 uptake through CR endocytosis, a novel mechanism for the delivery of dietary lipids and lipophilic vitamins to human bone. The current data suggest that, among the expressed receptors, LRP1 plays a predominant role.
Osteocytic Expression of Constitutive NO Synthase Isoforms in the Femoral Neck Cortex: A Case-Control Study of Intracapsular Hip Fracture
Authors: Caballero-Alías Ana Maria , Loveridge Nigel , Pitsillides Andrew , Parker Martyn , Kaptoge Stephen , Lyon Alan , Reeve Jonathan
Published: April 2006

NO is an osteocytic signaling molecule that can inhibit osteoclasts. The NO synthases eNOS and nNOS were expressed by >50% of osteonal osteocytes in controls. Hip fracture cases showed +NOS osteocytes only in deep osteonal bone, and 25–35% reduced expression overall. These data are consistent with increased osteonal vulnerability to deep osteoclastic attack.

Introduction: Osteocytes may regulate the response to mechanical stimuli in bone through the production of local signaling molecules such as NO derived from the NO synthase eNOS. Because NO is inhibitory to osteoclastic resorption, it has been suggested that osteocytes expressing eNOS act as sentinels, confining resorption within single osteons. Recently, nNOS has been shown to be present in osteocytes of adult human bone.

Materials and Methods: Cross-sections of the femoral neck (eight female cases of intracapsular hip fracture and seven postmortem controls; age, 68–91 years) were analyzed by immunohistochemistry. The percentages of osteocytes expressing each of these two isoforms were calculated, and their distances to the nearest canal surface were measured.

Results: The percentage of +nNOS osteocytes was lower in the fracture cases than in the controls (cases: 43.12 ± 1.49, controls: 56.68 ± 1.45; p < 0.0001). Compared with nNOS, eNOS expression was further reduced (p = 0.009) in the cases but was not different in the controls (cases: 36.41 ± 1.53, controls: 56.47 ± 2.41; p < 0.0001). The minimum distance of +eNOS or +nNOS osteocytes to a canal surface was higher in the cases compared with controls (eNOS: controls; 44.4 ± 2.2 μm, cases: 61.7 ± 2.0 μm; p < 0.0001; nNOS: controls: 52.4 ± 1.7 μm, cases: 60.2 ± 2.1 μm; p = 0.0039). +eNOS osteocytes were closer to the canal surfaces than +nNOS osteocytes in the controls by 8.00 ± 4.0 μm (p = 0.0012).

Conclusion: The proportions of osteocytes expressing nNOS and eNOS were both reduced in the fracture cases, suggesting that the capacity to generate NO might be reduced. Furthermore, the reduction in NOS expression occurs in those osteocytes closest to the canal surface, suggesting that the ability of NO to minimize resorption depth might be impaired. Further studies are needed on the regulation of the expression and activity of these distinct NOS isoforms.
Bone Markers, Calcium Metabolism, and Calcium Kinetics During Extended-Duration Space Flight on the Mir Space Station
Authors: Smith Scott M , Wastney Meryl E , O'Brien Kimberly O , Morukov Boris V , Larina Irina M , Abrams Steven A , Davis-Street Janis E , Oganov Victor , Shackelford Linda C
Published: April 2006

Bone loss is a current limitation for long-term space exploration. Bone markers, calcitropic hormones, and calcium kinetics of crew members on space missions of 4–6 months were evaluated. Spaceflight-induced bone loss was associated with increased bone resorption and decreased calcium absorption.

Introduction: Bone loss is a significant concern for the health of astronauts on long-duration missions. Defining the time course and mechanism of these changes will aid in developing means to counteract these losses during space flight and will have relevance for other clinical situations that impair weight-bearing activity.

Materials and Methods: We report here results from two studies conducted during the Shuttle-Mir Science Program. Study 1 was an evaluation of bone and calcium biochemical markers of 13 subjects before and after long-duration (4–6 months) space missions. In study 2, stable calcium isotopes were used to evaluate calcium metabolism in six subjects before, during, and after flight. Relationships between measures of bone turnover, biochemical markers, and calcium kinetics were examined.

Results: Pre- and postflight study results confirmed that, after landing, bone resorption was increased, as indicated by increases in urinary calcium (p < 0.05) and collagen cross-links (N-telopeptide, pyridinoline, and deoxypyridinoline were all increased >55% above preflight levels, p < 0.001). Parathyroid hormone and vitamin D metabolites were unchanged at landing. Biochemical markers of bone formation were unchanged at landing, but 2–3 weeks later, both bone-specific alkaline phosphatase and osteocalcin were significantly (p < 0.01) increased above preflight levels. In studies conducted during flight, bone resorption markers were also significantly higher than before flight. The calcium kinetic data also validated that bone resorption was increased during flight compared with preflight values (668 ± 130 versus 427 ± 153 mg/day; p < 0.001) and clearly documented that true intestinal calcium absorption was significantly lower during flight compared with preflight values (233 ± 87 versus 460 ± 47 mg/day; p < 0.01). Weightlessness had a detrimental effect on the balance in bone turnover such that the daily difference in calcium retention during flight compared with preflight values approached 300 mg/day (−234 ± 102 versus 63 ± 75 mg/day; p < 0.01).

Conclusions: These bone marker and calcium kinetic studies indicated that the bone loss that occurs during space flight is a consequence of increased bone resorption and decreased intestinal calcium absorption.