bluuebunny/crossref_metadata_2025 huggingface dataset:
Importance of Individual Rods and Plates in the Assessment of Bone Quality and Their Contribution to Bone Stiffness
Authors: Stauber Martin , Rapillard Laurent , van Lenthe G Harry , Zysset Philippe , Müller Ralph
Published: April 2006

Local morphometry based on the assessment of individual rods and plates was applied to 42 human vertebral trabecular bone samples. Results showed that multiple linear regression models based on local morphometry as a measure for bone microstructure helped improving our understanding of the role of local structural changes in the determination of bone stiffness as assessed from direct and computational biomechanics.

Introduction: In a recent study, we proposed a method for local morphometry of trabecular bone, i.e., morphometry as applied to individual rods and plates. In this study, we used this method to study the relative importance of local morphometry in the assessment of bone architecture and its relative contribution to the stiffness of human vertebral bone.

Materials and Methods: We extracted 42 human trabecular bone autopsies from nine intact spinal columns. The cylindrical samples were imaged with μCT to assess bone microstructure. From these images, global and local morphometric indices were derived and related to Young's modulus as assessed by experimental uniaxial compression testing (Emeas) and computational finite element analysis (EFE).

Results: We found the best single predictor for Young's modulus to be apparent bone volume density (BV/TV), which explained 89% of the variance in EFE when fitted with a power law. A multiple linear regression model combining mean trabecular spacing (Tb.Sp), mean slenderness of the rods (), and the relative amount of rod volume to total bone volume (Ro.BV/BV) was able to explain 90% of the variance in EFE. This model could not be improved by adding BV/TV as an independent variable. Furthermore, we found that mean trabecular thickness of the rods was significantly related to EFE (r2 = 0.42), whereas mean trabecular thickness of plates had no correlation to Young's modulus. Because the globally determined trabecular thickness does not discriminate between rods and plates, this index had only a poor predictive power for EFE (r2 = 0.09), showing the importance of local analysis of individual rods and plates.

Conclusions: From these results, we conclude that models based on local morphometry help improving our understanding of the relative importance of local structural changes in the determination of the stiffness of bone. Separate analysis of individual rods and plates may help to better predict age and disease-related fractures as well as to shed new light on the effect of pharmaceutical intervention in the prevention of such fractures beyond BMD.
High Bone Turnover Is an Independent Predictor of Mortality in the Frail Elderly
Authors: Sambrook Philip N , Chen Charles JS , March Lyn , Cameron Ian D , Cumming Robert G , Lord Stephen R , Simpson Judy M , Seibel Markus J
Published: April 2006

Osteoporotic fractures are associated with accelerated bone turnover and excess mortality. In a prospective study of 1112 frail subjects (79% female; mean age, 86 years), high bone turnover was an independent predictor of all-cause mortality. This association seemed to be mainly manifested in deaths from cardiovascular causes.

Introduction: Osteoporotic fractures are associated with accelerated bone turnover and excess mortality. In a prospective cohort study of elderly men and women, we assessed whether the rate of bone turnover measured by markers of bone remodeling is a direct predictor of mortality.

Materials and Methods: We measured serum concentrations of the aminoterminal propeptide of type I collagen (PINP), a marker of bone formation, and of the carboxyterminal telopeptide of type I collagen (CTX-I), a marker of bone resorption, along with serum PTH and 25-hydroxyvitamin D [25(OH)D] levels in 1112 subjects (79% female; mean age, 86 years) living in residential care. Co-morbidity was measured using the Implicit Illness Severity Scale. Fracture data were validated by a radiology report. Mortality and causes of death were ascertained from death certificates.

Results: Over a median follow-up of 817 days, 559 (50.3%) subjects died. In univariate analyses, time to death from all causes was significantly (p < 0.01) associated with age (HR = 1.62 per 10 years), male sex (HR = 1.33), immobility (HR = 1.94), co-morbidity (HR = 0.31, mild versus severe), lower weight (HR = 0.83 per 10-kg increase), impaired cognitive function (HR = 2.14, severe versus normal), number of medications (HR = 1.05 each), hip fracture (HR = 2.26), log serum creatinine (HR = 1.67), log PTH (HR = 1.29), CTX-I (HR = 1.70, highest 25% versus lowest 75%), and PINP (HR = 1.46, highest 25% versus lowest 75%). In multivariate analysis adjusting for age, sex, immobility, co-morbidity, weight, cognitive function, number of medications, PTH, and hip fracture status, the highest quartile was significantly more likely to die than the rest for both serum CTX-I (HR = 1.39; 95% CI: 1.14–1.70; p = 0.002) and PINP (HR = 1.25; 95% CI: 1.02–1.52; p = 0.03). For individual causes of death, CTX-I was significantly associated with deaths from cardiac causes (HR = 1.78: 95% CI: 1.27–2.50; p < 0.001).

Conclusions: We conclude that in the frail elderly, high bone turnover is associated with all cause mortality independently of age, sex, health status, serum PTH levels, and hip fracture status. The mechanism of the effect of bone turnover on mortality seems to be mainly manifested in deaths from cardiovascular causes.
Cortical PQCT Measures Are Associated With Fractures in Dialysis Patients
Authors: Jamal Sophie A , Gilbert Jeremy , Gordon Christopher , Bauer Douglas C
Published: April 2006

To determine if pQCT could identify HD patients with fractures, we conducted a cross-sectional study in 52 men and women on HD. We found that cortical, but not trabecular, pQCT measures were associated with fractures.

Introduction: Fractures are common in hemodialysis (HD) patients, yet DXA is inconsistently associated with fractures. One explanation for this lack of association may be that HD patients have a selective decrease in cortical density not identified by standard DXA.

Materials and Methods: We used pQCT to examine cross-sectional associations between cortical and trabecular measures and fractures in 36 men and 16 women, ≥50 years of age, on HD for at least 1 year. We confirmed low-trauma nonspine fractures since starting HD. Prevalent vertebral fractures were identified by morphometry of lateral spine X-rays. pQCT measurements of the nondominant radius included trabecular density, cortical density, total area, cortical area, and cortical thickness. We also obtained DXA measurements of the hip and lumbar spine. We used logistic regression models, adjusted for age, weight, and sex, to examine the association between fracture (vertebral and/or self-reported nonspine) and each pQCT measure. Results are reported as ORs per SD decrease in the independent variable.

Results: The mean age was 65.8 ± 9.0 (SD) years, the mean weight was 72.3 ± 15.6 kg, most (32 of 52) subjects were white, and there were 32 fractures in 27 subjects (prevalent vertebral fracture or low-trauma fracture) since starting dialysis. A decrease in cortical density was associated with fractures (OR = 16.67; 95% CI: 2.94–83.33), as was a decrease in cortical area (OR = 3.04; 95% CI: 1.28–7.25) and a decrease in cortical thickness (OR = 3.26; 95% CI: 1.36–7.87). Fractures were not associated with pQCT trabecular density (OR = 1.18; 95% CI: 0.6–2.33), total area (OR = 1.1; 95% CI: 0.59–1.7), or DXA measurements of the hip and spine.

Conclusions: Cortical parameters of the radius were associated with fractures in HD patients. If confirmed in prospective studies, these findings may explain the lack of association between fracture and standard DXA measurements and raise the possibility that pQCT could be used to identify HD patients at high risk of fracture.
Bone Marrow Cell Differentiation Induced by Mechanically Damaged Osteocytes in 3D Gel-Embedded Culture
Authors: Kurata Kosaku , Heino Terhi J , Higaki Hidehiko , Väänänen H Kalervo
Published: April 2006

Osteocytes are suggested to have a crucial role in the initial resorptive phase of bone turnover after microdamage. To study the role of osteocytes in targeted remodeling, we developed an in vitro model, in which osteocytes can be locally damaged and their interactions with bone marrow cells studied. Our results show that the damaged osteocytes activate the osteoclast precursors by soluble factors and thus can control the initial phase of targeted remodeling.

Introduction: Microdamage in bone contributes to fractures and acts as a stimulus for bone remodeling. Besides the targeted remodeling, some remodeling may also be random to serve metabolic purposes. Osteocytes have been considered to provide a crucial role in the activation of osteoclastic bone resorption adjacent to the damaged site. This study was aimed to develop a relevant in vitro model of the targeted remodeling and to show that damaged osteocytes can induce the initial bone resorptive stage.

Materials and Methods: We developed a new device, in which osteocyte-like cell line MLO-Y4 cells were 3D cultured, subjected to local scratching, and assayed for cell viability. NIH3T3-3 cells were used as a control. Bone marrow cells were cultured on the top of the mechanically damaged MLO-Y4 cells, and the formation of TRACP+ cells was assayed. Additionally, the conditioned medium from scratched cultures was added to bone marrow cultures, and the TRACP activity in cell lysates was quantified. The macrophage-colony stimulating factor (M-CSF) and RANKL secretion in the conditioned medium was assayed by ELISA.

Results: Scratching induced the death of MLO-Y4 cells. When bone marrow cells were cultured over the gel-embedded MLO-Y4 cells, the application of mechanical scratching induced TRACP+ cell differentiation on gel surface. The cells with TRACP+ could be observed in the very restricted region along the scratching path. Additionally, mechanically damaged osteocytes secreted M-CSF and RANKL, and the conditioned medium showed the potential to induce TRACP+ cells in bone marrow culture.

Conclusions: These findings indicate that soluble factors secreted from damaged osteocytes can locally induce and activate the initial phase of osteoclastic cell formation. This study directly shows the association between the damaged osteocytes and the initiation of resorptive stage in bone remodeling.
Osteocyte Apoptosis Is Induced by Weightlessness in Mice and Precedes Osteoclast Recruitment and Bone Loss
Authors: Aguirre J Ignacio , Plotkin Lilian I , Stewart Scott A , Weinstein Robert S , Parfitt A Michael , Manolagas Stavros C , Bellido Teresita
Published: April 2006

Mechanical stimulation of cultured osteocytic cells attenuates their apoptosis. We report here that, conversely, reduced mechanical forces in the murine model of unloading by tail suspension increases the prevalence of osteocyte apoptosis, followed by bone resorption and loss of mineral and strength.

Introduction: Mechanical loading is critical for the maintenance of bone mass; weightlessness, as with reduced physical activity in old age, bed rest, or space flight, invariably leads to bone loss. However, the cellular and molecular mechanisms responsible for these phenomena are poorly understood. Based on our earlier findings that physiologic levels of mechanical strain prevent apoptosis of osteocytic cells in vitro, we examined here whether, conversely, reduced mechanical forces increase the prevalence of osteocyte apoptosis in vivo and whether this event is linked to bone loss.

Materials and Methods: Swiss Webster mice or OG2-11β-hydroxysteroid dehydrogenase type 2 (OG2-11β-HSD2) transgenic mice and wildtype littermates were tail-suspended or kept under ambulatory conditions. Static and dynamic histomorphometry and osteocyte and osteoblast apoptosis by in situ end-labeling (ISEL) were assessed in lumbar vertebra; spinal BMD was measured by DXA; and bone strength was measured by vertebral compression.

Results: We show that within 3 days of tail suspension, mice exhibited an increased incidence of osteocyte apoptosis in both trabecular and cortical bone. This change was followed 2 weeks later by increased osteoclast number and cortical porosity, reduced trabecular and cortical width, and decreased spinal BMD and vertebral strength. Importantly, whereas in ambulatory animals, apoptotic osteocytes were randomly distributed, in unloaded mice, apoptotic osteocytes were preferentially sequestered in endosteal cortical bone—the site that was subsequently resorbed. The effect of unloading on osteocyte apoptosis and bone resorption was reproduced in transgenic mice in which osteocytes are refractory to glucocorticoid action, indicating that stress-induced hypercortisolemia cannot account for these effects.

Conclusions: We conclude that diminished mechanical forces eliminate signals that maintain osteocyte viability, thereby leading to apoptosis. Dying osteocytes in turn become the beacons for osteoclast recruitment to the vicinity and the resulting increase in bone resorption and bone loss.
Cellular Activity on the Seven Surfaces of Iliac Bone: A Histomorphometric Study in Children and Adolescents
Authors: Rauch Frank , Travers Rose , Glorieux Francis H
Published: April 2006

Transiliac cortical bone histomorphometry was performed in 56 metabolic bone disease-free individuals 1.5–22.9 years of age. During the growing years, the two cortices of an iliac bone specimen differ with regard to bone cell activity on their surfaces, probably reflecting a modeling drift.

Introduction: Standard bone histomorphometry in the clinical setting is typically limited to the analysis of cancellous bone. However, during the growth period, important changes occur also in the cortical compartment.

Materials and Methods: Transiliac bone samples from 56 individuals between 1.5 and 22.9 years of age (25 male; tetracycline labeling present in 42 subjects) and without evidence of metabolic bone disease were analyzed. Each of the three bone surface types (periosteal, intracortical, endocortical) of each cortex was evaluated separately. Results were expressed relative to those obtained in trabecular bone.

Results: A significant increase in cortical width with age was detected only for the internal cortex. Porosity of the external cortex was highest in the 7- to 10.9-year age group and decreased thereafter, whereas there was no clear trend with age for the porosity of the internal cortex. Intracortical remodeling activity decreased after 14 years of age. Periosteal bone formation was very active until 13 years of age, but was close to zero in subjects above that age. As to endocortical surfaces, all bone surface-based parameters of bone formation were higher on the internal cortex than on the external cortex, whereas bone resorption parameters were higher on the external cortex.

Conclusions: In growing subjects, the two cortices of an iliac bone specimen differ with regard to bone cell activity on their surfaces. These data raise fundamental questions about the regulation of bone cell activity in children and adolescents.
Free Testosterone is an Independent Predictor of BMD and Prevalent Fractures in Elderly Men: MrOS Sweden
Authors: Mellström Dan , Johnell Olof , Ljunggren Östen , Eriksson Anna-Lena , Lorentzon Mattias , Mallmin Hans , Holmberg Anna , Redlund-Johnell Inga , Orwoll Eric , Ohlsson Claes
Published: April 2006

The role of androgens for bone health in elderly men is unclear. We show that free testosterone within the normal range is a predictor of BMD at predominantly cortical bone sites and of previous osteoporosis-related fractures in elderly Swedish men.

Introduction: Osteoporosis-related fractures constitute a major health concern not only in women but also in men. Previous studies have clearly shown that serum levels of estradiol are associated with BMD, whereas more conflicting data have been presented regarding the predictive value of testosterone (T) for bone health in elderly men. The aim of this study was to investigate if serum levels of T are associated with BMD and/or prevalent fractures in a large cohort of elderly men.

Materials and Methods: In the Swedish part of the MrOS study (n = 2908; average age, 75.4 years), bone parameters were measured using DXA, and prevalent fractures were recorded using standardized questionnaires and by vertebral X-ray analyses. Serum levels of total T, total estradiol (E2), and sex hormone–binding globulin (SHBG) were measured by radioimmunoassay, and free T (FT) and free E2 (FE2) were derived from the mass action equations. Height, weight, age, physical activity, smoking habits, and calcium intake were included together with FT and FE2 in regression models for BMD.

Results: FT was an independent positive predictor of BMD in total body, total hip, femur trochanter, and arm but not in the lumbar spine. The highest independent predictive value of FT was found in the arm and the hip (with a relatively high content of cortical bone). FE2 was an independent predictor of BMD at all bone sites studied, and the highest predictive value was seen for lumbar spine (with relatively high content of trabecular bone) BMD. FT but not FE2 was a positive predictor of total body bone area and BMC. FT levels below the median were independent predictors of prevalent osteoporosis-related fractures (OR, 1.56; 95% CI, 1.14–2.14; p < 0.01) and X-ray–verified vertebral fractures (OR, 2.00; 95% CI, 1.34–2.86; p < 0.001). The predictive value of FT for prevalent fractures was not affected by adjustment for BMD.

Conclusions: These findings show that variation of FT within the normal range is an independent but modest predictor of BMD at predominantly cortical bone sites and of previous osteoporosis-related fractures in elderly men. Our data indicate that not only estrogens but also androgens are of importance for bone health in elderly men. Longitudinal studies investigating the predictive value of T for fracture risk in elderly men are required.
Increased Prevalence of Radiological Spinal Deformities in Adult Patients With GH Deficiency: Influence of GH Replacement Therapy
Authors: Mazziotti Gherardo , Bianchi Antonio , Bonadonna Stefania , Nuzzo Monica , Cimino Vincenzo , Fusco Alessandra , De Marinis Laura , Giustina Andrea
Published: April 2006

This cross-sectional study shows that a high number of untreated adult patients with GHD develop radiological vertebral deformities. Patients undergoing GH replacement treatment showed a significantly lower prevalence of vertebral deformities versus treated patients in the presence of similar BMD, as assessed by DXA.

Introduction: In this cross-sectional study, we investigated whether the prevalence and degree of spinal deformities in adults with growth hormone deficiency (GHD) were related to the age of patients, degree of bone turnover, BMD, and recombinant human GH (rhGH) replacement therapy.

Materials and Methods: One hundred seven adult hypopituitary patients (67 males and 40 females; mean age, 47 years; range: 16–81 years) with severe GHD and 130 control subjects (39 males, 91 females; mean age: 58.9 years; range: 26–82 years) were evaluated for BMD (DXA) and vertebral deformities (quantitative morphometric analysis). At study entry, 65 patients were on replacement therapy with rhGH, whereas 42 patients had never undergone rhGH.

Results: Vertebral fractures were significantly more frequent in GHD patients versus control subjects (63.6% versus 37.7%; χ2 15.7; p < 0.001). The fracture prevalence, as well as the fracture number, was significantly higher in untreated versus treated patients (78.6% versus 53.8%; χ2: 6.7; p = 0.009), although the two groups of patients did not show any significant difference in median T score. In untreated GHD patients, the prevalence of vertebral deformities was correlated with T score (p = 0.002) and duration of disease (p = 0.003). In treated GHD patients, the prevalence of spinal deformities was correlated only with the timing of the beginning of rhGH replacement.

Conclusions: This cross-sectional study reports high prevalence of vertebral radiological deformities in adult patients with untreated GHD. The replacement treatment of GHD leads to a significant decrease in fracture rate.
Nuclear Matrix Proteins and Osteoblast Gene Expression
Authors: Bidwell Joseph P. , Alvarez Marta , Feister Hilary , Onyia Jude , Hock Janet
Published: April 2006

The molecular mechanisms that couple osteoblast structure and gene expression are emerging from recent studies on the bone extracellular matrix, integrins, the cytoskeleton, and the nucleoskeleton (nuclear matrix). These proteins form a dynamic structural network, the tissue matrix, that physically links the genes with the substructure of the cell and its substrate. The molecular analog of cell structure is the geometry of the promoter. The degree of supercoiling and bending of promoter DNA can regulate transcriptional activity. Nuclear matrix proteins may render a change in cytoskeletal organization into a bend or twist in the promoter of target genes. We review the role of nuclear matrix proteins in the regulation of gene expression with special emphasis on osseous tissue. Nuclear matrix proteins bind to the osteocalcin and type I collagen promoters in osteoblasts. One such protein is Cbfa1, a recently described transcriptional activator of osteoblast differentiation. Although their mechanisms of action are unknown, some nuclear matrix proteins may act as “architectural” transcription factors, regulating gene expression by bending the promoter and altering the interactions between other trans-acting proteins. The osteoblast nuclear matrix is comprised of cell- and phenotype-specific proteins including proteins common to all cells. Nuclear matrix proteins specific to the osteoblast developmental stage and proteins that distinguish osteosarcoma from the osteoblast have been identified. Recent studies indicating that nuclear matrix proteins mediate bone cell response to parathyroid hormone and vitamin D are discussed.
Gene Expression of Bone Matrix Proteins and Endothelin Receptors in Endothelin-1–Deficient Mice Revealed by In Situ Hybridization
Authors: Kitano Yukie , Kurihara Hiroki , Kurihara Yukiko , Maemura Koji , Ryo Yoshimi , Yazaki Yoshio , Harii Kiyonori
Published: April 2006

Endothelin-1 (ET-1) was first found as a vasoconstrictor protein excreted by vascular endothelial cells, but recently ET-1 has been considered to have widespread functions that include regulation of osteochondrogenic metabolism. We analyzed sections of head regions in ET-1 knockout mice that are known to have abnormalities in pharyngeal arch–derived tissues and found that there was severe hypoplasia in facial bones. The hypoplasia suggests that the matrix mineralization system of facial bones is disrupted in ET-1−/− homozygous mice. To elucidate whether osteogenic cells in facial bones are the targets for ET-1 and whether expression of bone matrix genes are modulated by ET-1, we examined gene expression of ET-1 receptors, ETA and ETB, and that of the bone matrix proteins, osteonectin (ON) and osteopontin (OP), both in the head regions of ET-1+/− heterozygous and ET-1−/− homozygous mice by means of in situ hybridization. Different patterns of expression between ETA and ETB mRNAs were observed in both groups. In 18.5 days post coitus fetuses, ETA mRNA was most strongly expressed in osteogenic cells along craniofacial bones, but ETB mRNA was most strongly expressed in trunks of trigeminal nerve. This finding suggests that ET-1 may modulate osteogenic cells through ETA receptor but not through ETB receptor. The expression patterns of ETA, OP, and ON mRNAs were distinct between the two groups. In the lower jaw of ET-1+/− heterozygous mice, the ETA, ON, and OP mRNA positive cells were scattered in the inner and outer regions of the thick bone matrix, but in ET-1−/− homozygous mice, cells containing those mRNAs were located close to each other at the surface of thin bone matrix. However, cellular expression of ON and OP mRNAs in osteogenic cells of ET-1−/− homozygous mice was not suppressed as compared with ET-1+/− heterozygous mice. We conclude that ET-1 may regulate proliferation and migration of osteogenic cells in the maxillofacial region, rather than modulating the expression level of ON and OP mRNAs.
Intestinal Calcium Absorption and Bone Metabolism in Young Adult Men with Childhood-Onset Growth Hormone Deficiency
Authors: De Boer Hans , Blok Geert Jan , Popp-Snijders Corrie , Sips Adriënne , Lips Paul , Van Der Veen Eduard
Published: April 2006

Suboptimal growth hormone (GH) replacement therapy during childhood is a major cause of osteopenia in young adults with childhood-onset GH deficiency (CO-GHD). This is primarily attributed to reduced bone formation in childhood. It is currently not known whether GHD also has adverse effects on bone metabolism in adult life. To examine the impact of GHD on calcium and bone metabolism in adults, we evaluated 50 men with CO pituitary failure at a mean age of 28.2 ± 4.5 years, i.e., 8.8 ± 4.1 years after the discontinuation of previous GH treatment for short stature. Thirty-three patients had multiple pituitary hormone deficiencies (MPHD) for which they received conventional replacement therapy, seventeen patients had isolated GHD (IGHD), and forty-nine age-matched men served as controls. Intestinal calcium absorption, serum calcium concentration, serum phosphate levels, and renal calcium and phosphate excretion were normal in IGHD and MPHD patients. IGHD patients had marginally elevated serum levels of the carboxy-terminal cross-linked telopeptide of type I collagen (ICTP: 5.0 ± 1.2 vs. 4.2 ± 1.2 μg/l, p < 0.05), but other indices of bone turnover were normal. In contrast, MPHD patients had reduced levels of the carboxy-terminal propeptide of type I procollagen (PICP: 137 ± 76 vs. 179 ± 72 μg/l, p < 0.01), elevated serum ICTP levels (6.0 ± 3.8 vs. 4.2 ± 1.2 μg/l, p < 0.001), and reduced serum 1,25-dihydroxyvitamin D levels (55.1 ± 16.7 vs. 73.0 ± 23.0 pmol/l, p < 0.001). Multivariate regression analysis showed that the serum levels of bone resorption and bone formation markers in MPHD patients were correlated with the hydrocortisone, thyroxine, and testosterone replacement doses. There was no relationship with serum insulin-like growth factor I concentration. Panhypopituitary adults receiving conventional hormone replacement therapy are at risk to develop osteopenia either caused by reduced bone formation or by increased bone resorption activity. Predominantly, these abnormalities result from nonoptimal thyroid, gonadal, or adrenal hormone replacement therapy. GHD is not an important factor. In adults, GHD does not adversely affect intestinal calcium absorption or bone formation activity. Bone resorption activity may be slightly higher than normal, but the abnormality is too small to expect substantial bone loss as a consequence of GHD.
Apatite Induction by Insoluble Dentin Collagen
Authors: Saito Takashi , Yamauchi Mitsuo , Crenshaw Miles A.
Published: April 2006

Phosphoproteins are thought to play a role in mineral formation in dentin. A portion of this phosphoprotein is bound to collagen. We have investigated the requirement for bound phosphate in mineral induction by isolated dentin collagen. Insoluble bovine dentin collagen obtained by ethylene-diamino-tetra-acetic acid (EDTA) demineralization had 19.5 mol of P/mol of collagen that could not be extracted with 0.5 M EDTA in 4 M guanidine HCl. When this collagen was incubated in supersaturated solutions that did not spontaneously precipitate, apatite was induced. With progressive enzymatic dephosphorylation, induction times for mineral formation became progressively longer. The dentin did not induce mineral formation when 90% of the ester phosphate was removed. Insoluble bone collagen, which had even less phosphate, also did not induce mineral formation. Mineral induction times by dentin collagen increased with decreasing solution saturations. Using these data, the interfacial tension for mineral induction was determined to be 90 ergs/cm2. This value approximated that of phosphatidic acid liposomes and of phosvitin cross-linked to agarose beads, and it might reflect the energetics of heterogeneous nucleation on a highly phosphorylated surface. Sequestering of calcium-phosphate clusters on the phosphoprotein probably accounts for the observed calcium binding by dentin collagen in excess of that required to neutralize the phosphate esters of the collagen. Because the phosphoprotein is immobilized at a low density on the collagen, it cannot self-associate in calcium-phosphate solutions as it does when it is free in solution. This immobilized phosphoprotein allows the mineral clusters formed on its surface to grow into a crystalline order.
Impaired Expression of Noncollagenous Bone Matrix Protein mRNAs During Fracture Healing in Ascorbic Acid–Deficient Rats
Authors: Sugimoto Mizuo , Hirota Seiichi , Sato Motohiko , Kawahata Hirohisa , Tsukamoto Ikuyo , Yasui Natsuo , Kitamura Yukihiko , Ochi Takahiro , Nomura Shintaro
Published: April 2006

In scorbutic patients, fractures are slow to heal because of impaired collagen synthesis. To investigate the influence of impaired collagen synthesis on the differentiation and proliferation of osteogenic and chondrogenic cells, we examined the expression of genes encoding bone matrix proteins, including osteonectin (ON), osteopontin (OPN), osteocalcin (OC), and matrix Gla protein (MGP), as differentiation markers for osteogenic and chondrogenic cells during fracture healing in Osteogenic Disorder Shionogi (ODS) rats, which have a hereditary defect in the ability to synthesize ascorbic acid (Asc). In ODS rats without Asc supplementation, intramembranous ossification was completely inhibited. Although a few fibroblast-like cells expressing ON mRNA were observed, no OPN mRNA-expressing cells were detected. During endochondral ossification, a small amount of metachromatic staining cartilage appeared at the fracture site, but there was no provisional calcification zone in the cartilage. Chondrocytes expressed ON and MGP mRNAs, but not OPN mRNA. When Asc was given to these rats, callus formation was soon detected around the fracture site, while OPN mRNA was expressed by differentiated osteoblasts and hypertrophic chondrocytes. Our data indicate that impaired collagen synthesis due to Asc deficiency inhibited the increase of ON and MGP mRNA-expressing cells as well as the appearance of OPN mRNA-expressing cells. Since OPN is considered to play an important role in normal and pathological mineralization, lack of OPN mRNA expression accompanying impaired collagen synthesis may have a role in defective mineralization and delayed fracture healing in scurvy.
Clodronate Prevents Osteopenia and Loss of Trabecular Connectivity in Estrogen-Deficient Rats
Authors: Kippo Katriina , Hannuniemi Ritva , Isaksson Päivi , Laurén Leena , Österman Thua , Peng Zhiqi , Tuukkanen Juha , Kuurtamo Pirjo , Väänänen H. Kalervo , Sellman Raija
Published: April 2006

Daily oral clodronate treatment was evaluated in Sprague-Dawley rats for its ability to inhibit estrogen-deficiency–induced changes in femoral neck, femoral diaphysis, and lumbar vertebrae (L4–L5). Six-month-old ovariectomized (OVX) rats were administered by gavage a vehicle (Veh) or clodronate (100 or 500 mg/kg/day). Sham-operated (SHAM) control rats received the vehicle (n = 15/group). Treatment was started on the day of operation and continued for 3 months. Trabecular bone volume (BV/TV) and structural variables (trabecular number, Tb.N; thickness, Tb.Th; separation, Tb.Sp; and trabecular bone pattern factor, Tb.Pf) were assessed on secondary spongiosa of the right femoral neck. Furthermore, cantilever bending test of the left femoral neck and compression test of L4, ash weight of L5, and morphometric studies of femoral diaphysis were carried out, and serum and urinary markers of bone turnover were determined. The OVX/Veh group had higher levels of serum osteocalcin and alkaline phosphatase and higher urinary excretion of deoxypyridinoline/creatinine than the SHAM/Veh group at 3 months postsurgery, and clodronate reduced these changes. BV/TV of femoral neck, bone mass of L5, and the maximum loads of the femoral neck and L4 were lower after OVX than SHAM operation. Although clodronate prevented trabecular bone loss in the femoral neck and preserved Tb.Pf at the SHAM control level, it failed to preserve the mechanical strength at the femoral neck. However, in lumbar vertebrae, clodronate prevented the loss of bone mass and mechanical properties. Furthermore, there was a good positive correlation between maximum load of L4 and the ash weight of L5 (n = 58, r = 0.69, p < 0.001). In the femoral neck (n = 55), Tb.Pf correlated negatively with BV/TV and Tb.N (r = −0.59 and r = −0.55; p < 0.001, respectively) and positively with Tb.Sp (r = 0.61, p < 0.001). In femoral mid-diaphysis, there were no significant changes in cortical bone geometry in any of the groups. We conclude that orally administered clodronate suppresses the enhanced bone turnover in adult OVX rats and preserves trabecular bone volume and connectivity in the femoral neck. In the axial skeleton, clodronate has a beneficial effect on lumbar vertebral bone mass and strength.
Seasonal Deficiency of Vitamin D in Children: A Potential Target for Osteoporosis-Preventing Strategies?
Authors: Docio Suceso , Riancho José A. , Pérez Amabilia , Olmos José M. , Amado José A. , González-Macías Jesús
Published: April 2006

Peak bone mass attained after skeletal growth is a major determinant of the risk of developing osteoporosis later in life, hence the importance of nutritional factors that contribute to bone mass gain during infancy and adolescence. An adequate supply of vitamin D is essential for normal bone homeostasis. This study was undertaken to determine what the levels are of 25-hydroxyvitamin D (25(OH)D) that may be considered desirable in children and to assess if normal children maintain these levels throughout the year. Vitamin D metabolites and parathyroid hormone (PTH) serum levels were measured in 21 children in March and October, prior to and after the administration of a daily supplement of 25(OH)D (40 μg for 7 consecutive days). There were inverse correlations between basal 25(OH)D levels and supplementation-induced changes in serum 1,25(OH)2D (r = 0.57, p < 0.05) and PTH (r = 0.41, p < 0.05). When basal levels of 25(OH)D were below 20 ng/ml, the supplement induced an increase in serum 1,25(OH)2D; with basal 25(OH)D under 10–12 ng/ml, the supplement also decreased serum PTH. The lowest serum level of 25(OH)D in 43 normal children studied in summer was 13 ng/ml. Those results suggested that the lowest limit for desirable levels of 25(OH)D in children was somewhere between 12 and 20 ng/ml. However, 31% of 51 normal children studied in winter had levels below 12 ng/ml, and 80% had levels lower than 20 ng/ml. Those children are likely to have suboptimal bioavailability of vitamin D, which might hamper their achievement of an adequate peak bone mass. Since cutaneous synthesis of vitamin D is rather limited in winter, oral vitamin D supplementation should be considered.
The Biological Activity of Nonsteroidal Vitamin D Hormone Analogs Lacking Both the C- and D-Rings
Authors: Verstuyf Annemieke , Verlinden Lieve , Van Baelen Hugo , Sabbe Katrien , D'Hallewyn Chris , De Clercq Pierre , Vandewalle Maurits , Bouillon Roger
Published: April 2006

1α,25-dihydroxyvitamin D is a key calcium-regulating hormone but also displays potent differentiating and antiproliferative activities on many cell types. The structural requirements of this secosteroid hormone have been extensively studied for the A-ring and side chain, whereas relatively little is known about the requirements of the natural CD-ring structure for the vitamin D–like biological activity. We have embarked on a vast program in which derivatives were synthesized and evaluated characterized by profound structural changes in the central C/D-region. This first series of nonsteroidal analogs consists of (1R,3S)-5-((Z,2E)-4-((1S,3S)-3-(4-hydroxy-4-methylpentyl)-1,2,2,-trimethylcyclopentyl)-2-butenylidene)-4-methylenecyclohexane-1,3-diol (KS 176) and derivatives thereof. These analogs are characterized by the absence of normal C- and D-rings and by the presence of an unnatural five-membered ring which we call the E-ring. KS 176 with the otherwise natural side chain structure of 1α,25(OH)2D3 has between 10 and 30% of the biological activity of 1α,25(OH)2D3 when tested in vitro (prodifferentiating effects on HL-60 and MG-63; antiproliferating activity on MCF-7 and keratinocytes) but has minimal in vivo calcemic effects. Introduction of several side chain modifications created analogs with increased intrinsic noncalcemic biological properties, whereas their calcemic potency remains very low. These data demonstrate that the full CD-rings are not mandatory for the biological activity of 1α,25(OH)2D3 since they can be replaced by a new ring structure which generates an appropriate spacing of the A-seco B-rings in relation to the side chain. The biological activity of these nonsteroidal analogs probably involves a classical genomic activation since they are also active in transfection assays using an osteocalcin vitamin D responsive element coupled to a human growth hormone reporter gene.
Na+-Phosphate Cotransport in Mouse Distal Convoluted Tubule Cells: Evidence for Glvr-1 and Ram-1 Gene Expression
Authors: Tenenhouse Harriet S. , Gauthier Claude , Martel Josée , Gesek Frank A. , Coutermarsh Bonita A. , Friedman Peter A.
Published: April 2006

While there is considerable evidence for phosphate (Pi) reabsorption in the distal tubule, Pi transport and its regulation have not been well characterized in this segment of the nephron. In the present study, we examined Na+-dependent Pi transport in immortalized mouse distal convoluted tubule (MDCT) cells. Pi uptake by MDCT cells is Na+-dependent and, under initial rate conditions, is inhibited by phosphonoformic acid (41 ± 3% of control), a competitive inhibitor of Na+-Pi cotransport. The transport system has a high affinity for Pi (Km = 0.46 mM) and is stimulated by lowering the extracellular pH from 7.4 to 6.4 and inhibited by raising the pH from 7.4 to 8.4. Exposure to Pi-free medium for 21 h increased Na+-Pi cotransport from 2.1 to 5.5 nmol/mg of protein/5 minutes (p < 0.05) while parathyroid hormone, forskolin, and phorbol 12-myristate 13-acetate failed to alter Pi uptake in MDCT cells. Reverse transcriptase polymerase chain reaction of MDCT cell RNA provided evidence for the expression of the Npt1 but not the Npt2 Na+-Pi cotransporter gene. However, preincubation of MDCT cells with Npt1 antisense oligonucleotide led to only 20% inhibition of Na+-Pi cotransport, suggesting that other Na+-Pi cotransporters are operative in MDCT cells. Indeed, we showed, by ribonuclease protection assay, that MDCT cells express the ubiquitous cell surface receptors for gibbon ape leukemia virus (Glvr-1) and amphoteric murine retrovirus (Ram-1) that also function as Na+-Pi cotransporters. In summary, we demonstrate that the pH dependence and regulation of Na+-Pi cotransport in MDCT cells is distinct from that in the proximal tubule and suggest that different gene products mediate Na+-Pi cotransport in the proximal and distal segments of the nephron.
Immortalization and Characterization of Bone Marrow Stromal Fibroblasts from a Patient with a Loss of Function Mutation in the Estrogen Receptor-α Gene
Authors: Dieudonné S. C. , Xu T. , Chou J. Y. , Kuznetsov S. A. , Satomura K. , Mankani M. , Fedarko N. S. , Smith E. P. , Robey P. Gehron , Young M. F.
Published: April 2006

A male patient with abnormal postpubertal bone elongation was shown earlier to have a mutation in both alleles of the estrogen receptor, resulting in a nonfunctional gene. Marrow stromal fibroblasts (MSFs) derived from this patient were called HERKOs (human estrogen receptor knock outs), and in order to obtain continuous HERKO cell lines, they were immortalized using a recombinant adenovirus-origin-minus SV40 virus. MSFs are unique cells because they support hematopoesis and contain a mixed population of precursor cells for bone, cartilage, and fat. Three established cell lines (HERKO2, HERKO4, and HERKO7) were characterized and compared with the heterogeneous population of nonimmortalized HERKOs for their osteogenic potential. We performed Northern analysis of matrix genes implicated in bone development and metabolism and an in vivo bone formation assay by transplanting the cells subcutaneously into immunodeficient mice. All three HERKO lines expressed high amounts of collagen 1A1, osteopontin, osteonectin, fibronectin, decorin, biglycan, and alkaline phosphatase. Except for osteopontin, expression of these genes was slightly lower compared with nonimmortalized HERKOs. In the in vivo bone formation assay, the heterogeneous population of nonimmortalized HERKOs formed bone with high efficiency, while the HERKO lines induced a high-density, bone-like matrix. Finally, all HERKO cell types secreted high levels of insulin-like growth factor I and interleukin-6 into the culture medium relative to cells of normal human subjects. In summary, these lines of HERKO cells retain several of the phenotypic traits of MSFs after immortalization, including matrix and cytokine production, and provide a valuable source of a unique human material for future studies involving estrogen action in bone and bone marrow metabolism.
Reciprocal Temporospatial Patterns of Msx2 and Osteocalcin Gene Expression During Murine Odontogenesis
Authors: Bidder Miri , Latifi Tammy , Dr. Towler Dwight A.
Published: April 2006

Msx2 is a homeodomain transcription factor that regulates craniofacial development in vivo and osteocalcin (Osc) promoter activity in vitro. Msx2 is expressed in many craniofacial structures prior to embryonic day (E) E14 but is expressed at later stages in a restricted pattern, primarily in developing teeth and the calvarium. We examine Osc expression by in situ hybridization during murine development, detailing temporospatial relationships with Msx2 expression during preappositional and appositional odontogenesis and calvarial osteogenesis. Osc expression at E14–14.5 is very low, limited to a few perichondrial osteoblasts in the dorsal aspect of developing ribs. At E16.5 and E18.5, Osc expression is much higher, widely expressed in skeletal osteoblasts, including calvarial osteoblasts that do not express Msx2. No Osc is detected in early preappositional teeth that express Msx2. In incisors studied at an early appositional phase, Msx2 is widely expressed in the tooth, primarily in ovoid preodontoblasts and subjacent dental papilla cells. Osc is detected only in a small number of maturing odontoblasts that also express α1(I) collagen(Col1a1) and that are postproliferative (do not express histone H4). Msx2 expression greatly overlaps both histone H4 and Col1a1 expression in ovoid preodontoblasts and dental papilla cells. By the late appositional phases of E18.5 and neonatal teeth, Osc mRNA is highly expressed in mature columnar odontoblasts adjacent to accumulating dentin. In appositional bell-stage molars, reciprocal patterns of Msx2 and Osc are observed in adjacent preodontoblasts and odontoblasts within the same tooth. Osc is expressed in mature columnar odontoblasts, while Msx2 is expressed in adjacent immature ovoid preodontoblasts. In less mature teeth populated only by immature ovoid preodontoblasts, only Msx2 is expressed-–no Osc is detected. Thus, Msx2 and Osc are expressed in reciprocal patterns during craniofacial development in vivo, and Msx2 expression in preodontoblasts clearly preceeds Osc expression in odontoblasts. In functional studies using MC3T3-E1 calvarial osteoblasts, Msx2 suppresses endogeneous Osc, but not osteopontin, mRNA accumulation. In toto, these data suggest that Msx2 suppresses Osc expression in the craniofacial skeleton at stages immediately preceeding odontoblast and osteoblast terminal differentiation.
Spatiotemporal Assessment of Fetal Bovine Osteoblast Culture Differentiation Indicates a Role for BSP in Promoting Differentiation
Authors: Cooper L. F. , Yliheikkilä P. K. , Felton D. A. , Whitson S. W.
Published: April 2006

Fetal bovine mandible-derived osteoblasts were cultured for the purpose of obtaining a spatiotemporal assessment of bone matrix protein expression during in vitro differentiation. The results obtained from electron microscopic, immunohistological, biochemical, and molecular biological analyses indicated that these primary cultured osteoblasts produce an abundant extracellular matrix which mineralizes during a 14-day culture period. During this process, a restricted, spatiotemporal pattern of bone sialoprotein expression was indicated by immunohistological and molecular evaluations. To test the possibility that bone sialoprotein promoted the continued morphodifferentiation of osteoblastic cells, cultures were grown in the presence of anti-bone sialoprotein antibodies known to interfere with cell–bone sialoprotein attachment. Compared with cultures grown in the presence of normal rabbit serum (1:150), cultures grown in the media containing anti-bone sialoprotein antibody (1:150) failed to mineralize as demonstrated by von Kossa staining and failed to express osteocalcin and osteopontin as shown by the reverse transcription polymerase chain reaction. These results contribute to the growing evidence that bone sialoprotein is an important determinant of osteoblast differentiation and bone formation. Matrix protein–cell interactions may be examined using this spatiotemporally defined model.
Human Osteoblasts Express a Repertoire of Cadherins, Which Are Critical for BMP-2–Induced Osteogenic Differentiation
Authors: Cheng Su-Li , Lecanda Fernando , Davidson Mari K. , Warlow Pamela M. , Zhang Shu-Fang , Zhang Liming , Suzuki Shintaro , St. John Tom , Civitelli Roberto
Published: April 2006

Direct cell–cell interactions are fundamental for tissue development and differentiation. We have studied the expression and function of cadherins in human osteoblasts during in vitro differentiation. Using reverse transcription-polymerase chain reaction and mRNA hybridization, we found that human trabecular bone osteoblasts (HOBs), osteoprogenitor marrow stromal cells (BMCs), and the osteogenic sarcoma lines, SaOS-2 and MG-63, expressed mRNA for cadherin-11 (C11) and N-cadherin (N-cad). HOBs and BMCs also expressed low levels of cadherin-4 (C4) mRNA. C11 was the most abundant cadherin protein present in human osteoblasts, and its expression was unaffected by bone morphogenetic protein-2 (BMP-2) treatment of either BMCs or HOBs. Likewise, N-cad mRNA did not change during BMP-2 incubation. Conversely, C4 protein, undetectable in transformed cell lines, was down-regulated by BMP-2 treatment of normal cells. Both C11 and C4 were localized to sites of cell–cell contact in both HOBs and BMCs, colocalized with β-catenin, and bands corresponding to cadherins were coimmunoprecipitated by a β-catenin antibody, findings indicative of functional cadherins. A decapeptide containing the HAV motif of human N-cad partially inhibited Ca2+-dependent cell–cell adhesion and completely prevented BMP-2–induced stimulation of alkaline phosphatase activity by BMCs. Thus, human osteoblasts and their progenitor cells express a repertoire of multiple cadherins. Cadherin-mediated cell-to-cell adhesion is critical for normal human osteoblast differentiation.