bluuebunny/crossref_metadata_2025 huggingface dataset:
Fractal Analysis of Radiographic Trabecular Bone Texture and Bone Mineral Density: Two Complementary Parameters Related to Osteoporotic Fractures
Authors: Benhamou C. L. , Poupon S. , Lespessailles E. , Loiseau S. , Jennane R. , Siroux V. , Ohley W. , Pothuaud L.
Published: April 2006

Trabecular bone microarchitecture and bone mineral density (BMD) are two main factors related to osteoporotic fractures. Currently, however, microarchitecture is not evaluated. We have developed and validated a trabecular bone texture analysis from radiographic images. The objective was to determine if the fractal analysis of texture was able to distinguish osteoporotic fracture groups from control groups, either in vertebrae, hip, or wrist fractures, and to determine if this indicator and BMD were independent and complementary. In this cross-sectional unicenter case-control population study in postmenopausal women, 107 fracture cases were enrolled and age-matched with 197 control cases. This population comprised 40 vertebral fractures (with 70 controls), 30 hip fractures (55 controls), and 37 wrist fractures (62 controls). Hip and lumbar spine BMD were measured by double-energy X-ray absorptiometry. Fractal analysis of texture was performed on calcaneus radiographs and the result was expressed as the H parameter (H = 2-fractal dimension). The H parameter showed a lower value (0.679 ± 0.053 SD) in fracture cases versus control cases (0.696 ± 0.030; p = 0.007), the statistical significance persisting after adjustment for age and for lumbar spine (LS) or hip BMD. This result was confirmed in vertebral fractures (p = 0.0001) and hip fractures (p = 0.003) but not wrist fractures (p = 0.07). We determined the threshold between high and low H values and then the odds ratios (OR) of fracture for low H for BMD ≤ −2.5 SD in T score and for the combinations of both parameters. The OR of fracture for low H was 1.6 (95% CI, 1.1–2.6). For LS BMD ≤ −2.5 SD the OR of 6.1 (3.4–10.8) shifted to 9.0 (4.0–20.4) when we added low H and for hip BMD it shifted from 5.6 (3.3–9.4) to 8.1 (4.0–16.8). In vertebral, hip, and wrist fracture cases the results were also significant. These data have shown that the fractal analysis of texture on calcaneus radiographs can distinguish osteoporotic fracture groups from control groups. This analysis and BMD provide independent and complementary information. These data suggest that we can improve the fracture risk evaluation by adding information related to microarchitecture, derived from analysis of conventional radiographic images.
Mechanical Strain Effect on Bone-Resorbing Activity and Messenger RNA Expressions of Marker Enzymes in Isolated Osteoclast Culture
Authors: Kurata Kosaku , Uemura Toshimasa , Nemoto Atsuko , Tateishi Tetsuya , Murakami Teruo , Higaki Hidehiko , Miura Hiromasa , Iwamoto Yukihide
Published: April 2006

Adaptive modeling and remodeling are controlled by the activities of osteoblasts and osteoclasts, which are capable of sensing their mechanical environments and regulating deposition or resorption of bone matrix. The effects of mechanical stimuli on isolated osteoclasts have been scarcely examined because it has proven to be difficult to prepare a number of pure osteoclasts and to cultivate them on mineralized substratum during mechanical stimulation. Recently, we developed an apparatus for applying mechanical stretching to the ivory slice/plastic plate component on which cells could be cultured. The loading frequency, strain rate, and generated strain over an ivory surface could be controlled by a personal computer. Using this apparatus, we examined the role of mechanical stretching on the bone-resorbing activity of the osteoclasts. Mature and highly enriched osteoclasts were cultured for 2, 12, and 24 h on the ivory/plate component while being subjected to intermittent tensile strain. The stretched osteoclasts showed enhanced messenger RNA (mRNA) expression levels of osteoclast marker enzymes, tartrate-resistant acid phosphatase (TRAP), and cathepsin K and increases of resorbed-pit formation, suggesting that the mechanical stretching up-regulated the bone-resorbing activity of the osteoclasts. A stretch-activated cation (SA-cat) channel blocker significantly inhibited the increases of the mRNA level and pit formation after 24 h of stretching. This study suggested the possibility that the mature osteoclasts responded to mechanical stretching through a mechanism involving a SA-cat channel in the absence of mesenchymal cells and, as a result, up-regulated their bone-resorbing activity.
Study of the Maturation of the Organic (Type I Collagen) and Mineral (Nonstoichiometric Apatite) Constituents of a Calcified Tissue (Dentin) as a Function of Location: A Fourier Transform Infrared Microspectroscopic Investigation
Authors: Magne David , Weiss Pierre , Bouler Jean-Michel , Laboux Olivier , Daculsi Guy
Published: April 2006

Fourier transform infrared microspectroscopy (FTIRM) was used to investigate the organic and mineral phases of a calcified tissue (dentin) as a function of its location from predentin toward enamel. Thin dentin slices (decalcified or not) were fixed in formaldehyde and embedded in glycolmethylmethacrylate (GMA). Fixation did not denature collagen, and GMA did not interact with organic or mineral constituents of dentin. The ν1ν3 PO4 domain was studied in particular in order to estimate mineral maturity and amide I, II, A, and B to obtain data on protein conformation. The results showed that dentin apatite became increasingly mature (stoichiometric) from the mineralization front toward the enamel, especially through loss of HPO42− groups and vacancies. Moreover, collagen fibrils became less and less hydrated, suggesting that intrafibrillar mineralization partially dehydrated the collagen. Combined study of the organic and mineral fractions of calcified tissues may help clarify their relationships in physiological and pathological tissues.
Pubertal Maturation Characteristics and the Rate of Bone Mass Development Longitudinally Toward Menarche
Authors: Van Coeverden Silvia C. C. M. , De Ridder Christine M. , Roos Jan C. , Van'T Hof Martin A. , Netelenbos J. Coen , Delemarre-Van De Waal Henriette A.
Published: April 2006

To assess risks for osteoporosis and to compare bone mass in different groups of healthy children or children with diseases, it is important to have knowledge of their sexual maturation status during puberty. The aim of our study was to evaluate bone mass formation longitudinally in relation to pubertal maturation characteristics in healthy white girls. We investigated the bone mineral content (BMC) and the bone mineral density (BMD) at different skeletal sites in 151 girls with increasing pubertal stages in relation with their chronological age and with an early or late onset of puberty or menarche and with a slow or fast maturation. Bone mass was measured at the onset of puberty, during puberty, and at menarche. We conclude the following: (1) from midpuberty to menarche, the increase in bone mass formation is highest at all skeletal sites in white girls; (2) early mature girls at the onset of puberty have slightly but definitely lower bone masses at all skeletal sites and at all pubertal stages than late mature girls, whereas the average bone mass formation from the onset of puberty to menarche is similar in both groups; (3) girls with a slow rate of pubertal maturation have lower bone mass values 2 years after the onset of puberty, but at menarche bone mass is similar compared with fast maturers; and (4) it cannot be confirmed that there is an effect of menarcheal age on bone mass values at menarche.
Inhaled Corticosteroids Reduce Bone Mineral Density in Early Postmenopausal but Not Premenopausal Asthmatic Women
Authors: Fujita K. , Kasayama S. , Hashimoto J. , Nagasaka Y. , Nakano N. , Morimoto Y. , Barnes P. J. , Miyatake A.
Published: April 2006

Inhaled corticosteroids are widely used in the treatment of bronchial asthma, but it is still uncertain whether long-term use of the inhaled corticosteroids affects bone metabolism in asthmatic patients. In this study, we examined the effect of inhaled beclomethasone dipropionate (BDP) on bone mineral density (BMD) and biochemical markers of bone metabolism in pre- and early postmenopausal asthmatic women. Thirty-six (17 premenopausal and 19 early postmenopausal) asthmatic women and 45 healthy control (24 premenopausal and 21 early postmenopausal) women were investigated. All the asthmatic patients were treated with BDP (542 ± 298 μg/day; 100–1200 μg/day) without any systemic administration of corticosteroids for at least 1 year. In premenopausal women, BMD as well as the biochemical markers of bone metabolism did not differ between control subjects and BDP-treated asthmatic patients. By contrast, in early postmenopausal women, BMD was significantly lower in BDP-treated asthmatic patients than in control subjects. In these early postmenopausal women, serum intact osteocalcin concentration was lower in the BDP-treated asthmatic patients than in the control subjects whereas urinary free pyridinoline (F-PYD) and free deoxypyridinoline (F-DPD) concentrations did not differ between the groups. Thus, early postmenopausal, but not premenopausal, asthmatic patients who were treated with inhaled BDP had reduced BMD, which was associated with a decreased level of the bone formation marker. Ovarian hormones may be protective against the adverse effect of inhaled BDP on bone metabolism in the premenopausal patients.
Naphthol-ASBI Phosphate as a Preferred Substrate for Tartrate-Resistant Acid Phosphatase Isoform 5b
Authors: Janckila Anthony J. , Takahashi Karen , Sun Susan Z. , Yam Lung T.
Published: April 2006

Tartrate-resistant acid phosphatase (TRAP) isoform 5b is a potential serum marker for osteoclastic activity. Biochemical assays for serum TRAP activity with para-nitrophenylphosphate (pNPP) have low specificity for bone because of hydrolysis by unrelated nontype 5 TRAPs of blood cells and by related isoform 5a. Our purpose was to increase the specificity of TRAP assay for osteoclastic activity by using naphthol-ASBI phosphate (N-ASBI-P) as a substrate for serum type 5 TRAP activity and heparin as an inhibitor of isoform 5a. TRAP activity in individual and pooled sera of normal subjects and patients with endstage renal disease (ESRD) and rheumatologic diseases was quantitated using pNPP and N-ASBI-P as substrate at pH 5.5 and 6.1. For some experiments, heparin (23U/ml) was added as a specific inhibitor of isoform 5a activity. Isoforms 5a and 5b were separated from serum pools by cation exchange chromatography and identified by nondenaturing polyacrylamide gel electrophoresis (PAGE). N-ASBI-P was selectively hydrolyzed by TRAP isoform 5b. TRAP assays with pNPP and N-ASBI-P correlated only in ESRD sera, which contained primarily isoform 5b. The two assays did not correlate in normal or rheumatic sera with significant amounts of 5a. Heparin inhibited isoform 5a activity approximately 50% but had little effect on isoform 5b activity. Biochemical assay of serum TRAP activity can be made specific for isoform 5b by using N-ASBI-P and heparin. This method can be adapted to simple microplate biochemical or immunochemical assays. This simplified method for assessment of osteoclastic TRAP 5b activity warrants a detailed investigation in diseases of bone metabolism.
Expression of Osteoprotegerin, Receptor Activator of NF-κB Ligand (Osteoprotegerin Ligand) and Related Proinflammatory Cytokines During Fracture Healing
Authors: Kon Tamiyo , Cho Tae-Joon , Aizawa Toshimi , Yamazaki Masashi , Nooh Nasser , Graves Dana , Gerstenfeld Louis C. , Einhorn Thomas A.
Published: April 2006

Fracture healing is a unique biological process regulated by a complex array of signaling molecules and proinflammatory cytokines. Recent evidence for the role of tumor necrosis family members in the coupling of cellular functions during skeletal homeostasis suggests that they also may be involved in the regulation of skeletal repair. The expression of a number of cytokines and receptors that are of functional importance to bone remodeling (osteoprotegerin [OPG], macrophage colony-stimulating factor [M-CSF], and osteoprotegerin ligand [receptor activator of NF-κB ligand (RANKL)]), as well as inflammation (tumor necrosis factor α [TNF-α] and its receptors, and interleukin-1α [IL-1α] and -β and their receptors) were analyzed over a 28-day period after the generation of simple transverse fractures in mouse tibias. OPG was expressed constitutively in unfractured bones and elevated levels of expression were detected throughout the repair process. It showed two distinct peaks of expression: the first occurring within 24 h after fracture and the second at the time of peak cartilage formation on day 7. In contrast, the expression of RANKL was nearly undetectable in unfractured bones but strongly induced throughout the period of fracture healing. The peak in expression of RANKL did not correlate with that of OPG, because maximal levels of expression were seen on day 3 and day 14, when OPG levels were decreasing. M-CSF expression followed the temporal profile of RANKL but was expressed at relatively high basal levels in unfractured bones. TNF-α, lymphotoxin-β (LT-β), IL-1α, and IL-1β showed peaks in expression within the first 24 h after fracture, depressed levels during the period of cartilage formation, and increased levels of expression on day 21 and day 28 when bone remodeling was initiated. Both TNF-α receptors (p55 and p75) and the IL-1RII receptor showed identical patterns of expression to their ligands, while the IL-1R1 was expressed only during the initial period of inflammation on day 1 and day 3 postfracture. Both TNF-α and IL-1α expression were localized primarily in macrophages and inflammatory cells during the early periods of inflammation and seen in mesenchymal and osteoblastic cells later during healing. TNF-α expression also was detected at very high levels in hypertrophic chondrocytes. These data imply that the expression profiles for OPG, RANKL, and M-CSF are tightly coupled during fracture healing and involved in the regulation of both endochondral resorption and bone remodeling. TNF-α and IL-1 are expressed at both very early and late phases in the repair process, which suggests that these cytokines are important in the initiation of the repair process and play important functional roles in intramembraneous bone formation and trabecular bone remodeling.
The Ratio of Messenger RNA Levels of Receptor Activator of Nuclear Factor κB Ligand to Osteoprotegerin Correlates with Bone Remodeling Indices in Normal Human Cancellous Bone but Not in Osteoarthritis
Authors: Fazzalari Nicola L. , Kuliwaba Julia S. , Atkins Gerald J. , Forwood Mark R. , Findlay David M.
Published: April 2006

The determinants of cancellous bone turnover and trabecular structure are not understood in normal bone or skeletal disease. Bone remodeling is initiated by osteoclastic resorption followed by osteoblastic formation of new bone. Receptor activator of nuclear factor κB ligand (RANKL) is a newly described regulator of osteoclast formation and function, the activity of which appears to be a balance between interaction with its receptor RANK and with an antagonist binding protein osteoprotegerin (OPG). Therefore, we have examined the relationship between the expression of RANKL, RANK, and OPG and indices of bone structure and turnover in human cancellous bone from the proximal femur. Bone samples were obtained from individuals with osteoarthritis (OA) at joint replacement surgery and from autopsy controls. Histomorphometric analysis of these samples showed that eroded surface (ES/BS) and osteoid surface (OS/BS) were positively associated in both control (p < 0.001) and OA (p < 0.02), indicating that the processes of bone resorption and bone formation remain coupled in OA, as they are in controls. RANKL, OPG, and RANK messenger RNA (mRNA) were abundant in human cancellous bone, with significant differences between control and OA individuals. In coplotting the molecular and histomorphometric data, strong associations were found between the ratio of RANKL/OPG mRNA and the indices of bone turnover (RANKL/OPG vs. ES/BS: r = 0.93, p < 0.001; RANKL/OPG vs. OS/BS: r = 0.80, p < 0.001). These relationships were not evident in trabecular bone from severe OA, suggesting that bone turnover may be regulated differently in this disease. We propose that the effective concentration of RANKL is related causally to bone turnover.
Mechanical Strain and Estrogen Activate Estrogen Receptor α in Bone Cells
Authors: Jessop H. L. , Sjöberg M. , Cheng M. Z. , Zaman G. , Wheeler-Jones C. P. D. , Lanyon L. E.
Published: April 2006

Bone cells' early responses to estrogen and mechanical strain were investigated in the ROS 17/2.8 cell line. Immunoblotting with antiphosphorylated estrogen receptor α (ER-α) antibody showed that when these cells were exposed for 10 minutes to estrogen (10−8 M) or a single period of cyclic dynamic strain (peak 3400 μϵ, 1Hz, 600 cycles), there was an increase in the intensity of a 66-kDa band, indicating phosphorylation of ser122 in the amino terminus of ER-α. Increased phosphorylation was detected within 5 minutes of exposure to estrogen and 5 minutes after the end of the period of strain. Estrogen and strain also activated the mitogen-activated protein kinase (MAPK) family member extracellular regulated kinase-1 (ERK-1). Increases in ERK activation coincided with increased ER-α phosphorylation. Activation of ERK-1 and the phosphorylation of ER-α, by both estrogen and strain, were prevented by the MAP kinase kinase (MEK) inhibitor U0126 and the protein kinase A (PKA) inhibitor (PKI). These data support previous suggestions that resident bone cells' early responses to strain and estrogen share a common pathway, which involves ER-α. This pathway also appears to involve PKA and ERK-mediated phosphorylation of ser122 within the amino terminus of ER-α. Reduced availability of this pathway when estrogen levels are reduced could explain diminished effectiveness of mechanically related control of bone architecture after the menopause.
Role of D1 and E Cyclins in Cell Cycle Progression of Human Fibroblasts Adhering to Cementum Attachment Protein
Authors: Yokokoji Takayoshi , Narayanan A. S.
Published: April 2006

Cementum attachment protein (CAP) is a collagenous protein present in the matrix of tooth cementum that mediates preferential attachment of some mesenchymal cell types, and CAP binding capacity is related to mineralizing tissue-forming capacity in culture. We have examined if adhesion to surfaces containing CAP as the only attachment protein permits human fibroblasts to escape G1 arrest and synthesize DNA, and if adhesion to CAP modulates the levels of cyclins D1 and E. Human gingival fibroblasts (HGFs) were serum-starved, trypsinized, and added to plates coated with CAP or bovine serum albumin (BSA). Cells were then exposed to either 10% fetal bovine serum (FBS) or to cementum-derived growth factor (CGF), an insulin-like growth factor I (IGF-I)-like molecule sequestered in tooth cementum, plus epidermal growth factor (EGF). DNA synthesis was measured as [3H]thymidine uptake, and cyclin D1 and E levels were determined by Western analysis. Cyclin E-dependent kinase (Cdk) activity was assessed in terms of H1 kinase activity in immunoprecipitates of cyclin E. Cells adhering to CAP synthesized DNA, whereas on BSA they remained unattached and did not synthesize DNA. Protein levels of cyclin D1 were higher in cells adhering to CAP in the absence and presence of growth factors. Cyclin E levels were not affected by adhesion alone, but they increased in the presence of growth factors. Cyclin E-associated kinase activity was higher in cells adherent on CAP, and it increased further in the presence of growth factors. Our results indicate that adhesion to CAP increases cyclin D1 levels and cyclin E-associated Cdk activity, and that these increases contribute to cell cycle progression. We previously observed that the signaling reactions induced during adhesion are characteristic of the CAP; together these observations indicate that specific matrix components present in the local environment can contribute to recruitment and differentiation of specific cell types for normal homeostasis and wound healing.
Growth Hormone Induces Bone Morphogenetic Proteins and Bone-Related Proteins in the Developing Rat Periodontium
Authors: Li Huika , Bartold P. Mark , Young William G. , Xiao Yin , Waters Michael J.
Published: April 2006

The hypothesis that growth hormone (GH) up-regulates the expression of enzymes, matrix proteins, and differentiation markers involved in mineralization of tooth and bone matrices was tested by the treatment of Lewis dwarf rats with GH over 5 days. The molar teeth and associated alveolar bone were processed for immunohistochemical demonstration of bone morphogenetic proteins 2 and 4 (BMP-2 and -4), bone morphogenetic protein type IA receptor (BMPR-IA), bone alkaline phosphatase (ALP), osteocalcin (OC), osteopontin (OPN), bone sialoprotein (BSP), and E11 protein (E11). The cementoblasts, osteoblasts, and periodontal ligament (PDL) cells responded to GH by expressing BMP-2 and -4, BMPR-IA, ALP, OC, and OPN and increasing the numbers of these cells. No changes were found in patterns of expression of the late differentiation markers BSP and E11 in response to GH. Thus, GH evokes expression of bone markers of early differentiation in cementoblasts, PDL cells, and osteoblasts of the periodontium. We propose that the induction of BMP-2 and -4 and their receptor by GH compliments the role of GH-induced insulin-like growth factor 1 (IGF-1) in promoting bone and tooth root formation.
The Contribution of Reduced Peak Accrual of Bone and Age-Related Bone Loss to Osteoporosis at the Spine and Hip: Insights from the Daughters of Women with Vertebral or Hip Fractures
Authors: Tabensky Aaron , Duan Yunbo , Edmonds Jan , Seeman Ego
Published: April 2006

The genetic hypothesis states that a daughter will resemble her mother by about 50% in a given trait because she shares, on average, half her genes. We used this trait resemblance in mothers and daughters to determine whether abnormalities in volumetric bone mineral density (vBMD) or bone size in women with fractures originate in growth or aging. vBMD and volume of the third lumbar vertebra and femoral neck were estimated using posteroanterior (PA) scanning by dual-energy X-ray absorptiometry (DXA). Vertebral volume was estimated as (scan area)3/2 and femoral neck volume was π ∗︁ (width/2)2 ∗︁ height. vBMD was bone mineral content (BMC)/volume. The data were expressed as age-specific SD or Z scores (mean ± SEM). Vertebral vBMD was reduced by −0.98 ± 0.14 SD (p < 0.001) in 34 women with vertebral fractures, and by −0.36 ± 0.13 SD (p < 0.05) in their 44 premenopausal daughters. The vBMD deficit in the daughters (relative to age-matched controls) was no different from one-half their mothers' deficit (relative to their age-matched controls). Vertebral volume was reduced in the women with vertebral fractures relative to age-matched controls (−0.77 ± 0.15 SD; p < 0.001), but not in their daughters (−0.17 ± 0.13 SD, NS). The 31 women with hip fractures and their 41 premenopausal daughters had no deficits in vertebral volume or vBMD. Femoral neck vBMD was reduced in the women with hip fractures (−1.24 ± 0.12 SD; p < 0.001) but not in their daughters (−0.17 ± 0.13 SD, NS). Femoral neck volume was increased by 0.98 ± 0.30 SD (p < 0.05) in women with hip fractures (relative to age-matched controls) and by 0.54 ± 0.14 SD (p < 0.001) in their daughters (relative to age-matched controls); that is, about one-half that of their mothers. We propose that women with vertebral fractures have reduced vertebral vBMD because of, in large part, reduced accrual of bone during growth (because the deficit in their daughters was almost one-half their mothers' deficit); reduced vertebral volume in women with vertebral fractures is caused by reduced periosteal apposition during aging (because their daughters have no deficit in vertebral volume). Women with hip fractures have reduced vBMD because of age-related bone loss (because their daughters have no deficit in vBMD) but the increased femoral neck volume is growth related (because their daughters' femoral neck size is increased by one-half as much). The pathogenesis of bone fragility at the axial and appendicular skeleton is heterogeneous and has its origins in growth and aging.
Structural Adaptation to Changing Skeletal Load in the Progression Toward Hip Fragility: The Study of Osteoporotic Fractures
Authors: Beck Thomas J. , Oreskovic Tammy L. , Stone Katie L. , Ruff Christopher B. , Ensrud Kristine , Nevitt Michael C. , Genant Harry K. , Cummings Steven R.
Published: April 2006

Longitudinal, dual-energy X-ray absorptiometry (DXA) hip data from 4187 mostly white, elderly women from the Study of Osteoporotic Fractures were studied with a structural analysis program. Cross-sectional geometry and bone mineral density (BMD) were measured in narrow regions across the femoral neck and proximal shaft. We hypothesized that altered skeletal load should stimulate adaptive increases or decreases in the section modulus (bending strength index) and that dimensional details would provide insight into hip fragility. Weight change in the ∼3.5 years between scan time points was used as the primary indicator of altered skeletal load. “Static” weight was defined as within 5% of baseline weight, whereas “gain” and “loss” were those who gained or lost >5%, respectively. In addition, we used a frailty index to better identify those subjects undergoing changing in skeletal loading. Subjects were classified as frail if unable to rise from a chair five times without using arm support. Subjects who were both frail and lost weight (reduced loading) were compared with those who were not frail and either maintained weight (unchanged loading) or gained weight (increased loading). Sixty percent of subjects (n = 2559) with unchanged loads lost BMD at the neck but not at the shaft, while section moduli increased slightly at both regions. Subjects with increasing load (n = 580) lost neck BMD but gained shaft BMD; section moduli increased markedly at both locations. Those with declining skeletal loads (n = 105) showed the greatest loss of BMD at both neck and shaft; loss at the neck was caused by both increased loss of bone mass and greater subperiosteal expansion; loss in shaft BMD decline was only caused by greater loss of bone mass. This group also showed significant declines in section modulus at both sites. These results support the contention that mechanical homeostasis in the hip is evident in section moduli but not in bone mass or density. The adaptive response to declining skeletal loads, with greater rates of subperiosteal expansion and cortical thinning, may increase fragility beyond that expected from the reduction in section modulus or bone mass alone.
Canine COL1A2 Mutation Resulting in C-Terminal Truncation of Pro-α2(I) and Severe Osteogenesis Imperfecta
Authors: Campbell Bonnie G. , Wootton Joyce A. M. , Macleod James N. , Minor Ronald R.
Published: April 2006

RNA and type I collagen were analyzed from cultured skin fibroblasts of a Beagle puppy with fractures consistent with type III osteogenesis imperfecta (OI). In a nonisotopic RNAse cleavage assay (NIRCA), the proband's RNA had a unique cleavage pattern in the region of COL1A2 encoding the C-propeptide. DNA sequence analyses identified a mutation in which nucleotides 3991-3994 (“CTAG”) were replaced with “TGTCATTGG.” The first seven bases of the inserted sequence were identical to nucleotides 4002-4008 of the normal canine COL1A2 sequence. The resulting frameshift changed 30 amino acids and introduced a premature stop codon. Reverse-transcription polymerase chain reaction (RT-PCR) with primers flanking the mutation site amplified two complementary DNA (cDNA) fragments for the proband and a single product for the control. Restriction enzyme digestions also were consistent with a heterozygous mutation in the proband. Type I procollagen labeled with [3H]proline was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Increased density of pC-α2(I) suggested comigration with the similarly sized pro-α2(I) derived from the mutant allele. Furthermore, α-chains were overhydroxylated and the ratio of α1(I):α2(I) was 3.2:1, consistent with the presence of α1(I) homotrimers. Analyses of COL1A2 and type I collagen were both consistent with the described heterozygous mutation affecting the pro-α2(I) C-propeptide and confirmed a diagnosis of OI.
Osteoblast Apoptosis and Bone Turnover
Authors: Hock J. M. , Krishnan V. , Onyia J. E. , Bidwell J. P. , Milas J. , Stanislaus D.
Published: April 2006

With the discoveries of different death mechanisms, an emerging definition of apoptosis is the process of cell death associated with caspase activation or caspase-mediated cell death. This definition accepts that caspases represent the final common mechanistic pathway in apoptosis. Apoptosis may be triggered either by activation events that target mitochondria or endoplasmic reticulum or by activation of cell surface “death receptors,” for example, those in the tumor necrosis factor (TNF) superfamily. In the postnatal and adult skeleton, apoptosis is integral to physiological bone turnover, repair, and regeneration. The balance of osteoblast proliferation, differentiation, and apoptosis determines the size of the osteoblast population at any given time. Although apoptosis has been recorded in many studies of bone, the selective mechanisms invoked in the different models studied rarely have been identified. This review offers a broad overview of the current general concepts and controversies in apoptosis research and then considers specific examples of osteoblast apoptosis pertinent to skeletal development and to the regulation of bone turnover. In reviewing selected work on interdigital apoptosis in the developing skeleton, we discuss the putative roles of the bone morphogenetic proteins (BMPs), Msx2, RAR-γ, and death inducer obliterator 1 (DIO-1). In reviewing factors regulating apoptosis in the postnatal skeleton, we discuss roles of cytokines, growth factors, members of the TNF pathway, and the extracellular matrix (ECM). Finally, the paradoxical effects of parathyroid hormone (PTH) on osteoblast apoptosis in vivo are considered in the perspective of a recent hypothesis speculating that this may be a key mechanism to explain the anabolic effects of the hormone. An improved understanding of the apoptotic pathways and their functional outcomes in bone turnover and fracture healing may facilitate development of more targeted therapeutics to control bone balance in patients with osteoporosis and other skeletal diseases.
Quantitative Trait Loci for Femoral and Lumbar Vertebral Bone Mineral Density in C57BL/6J and C3H/HeJ Inbred Strains of Mice
Authors: Beamer Wesley G. , Shultz Kathryn L. , Donahue Leah Rae , Churchill Gary A. , Sen Saunak , Wergedal Jon R. , Baylink David J. , Rosen Clifford J.
Published: April 2006

Significant differences in vertebral (9%) and femoral (50%) adult bone mineral density (BMD) between the C57BL/6J (B6) and C3H/HeJ (C3H) inbred strains of mice have been subjected to genetic analyses for quantitative trait loci (QTL). Nine hundred eighty-six B6C3F2 females were analyzed to gain insight into the number of genes that regulate peak BMD and their locations. Femurs and lumbar vertebrae were isolated from 4-month-old B6C3F2 females at skeletal maturity and then BMD was determined by peripheral quantitative computed tomography (pQCT). Estimates of BMD heritability were 83% for femurs and 72% for vertebrae. Genomic DNA from F2 progeny was screened for 107 polymerase chain reaction (PCR)-based markers discriminating B6 and C3H alleles on all 19 autosomes. The regression analyses of markers on BMD revealed ten chromosomes (1, 2, 4, 6, 11, 12, 13, 14, 16, and 18) carrying QTLs for femurs and seven chromosomes (1, 4, 7, 9, 11, 14, and 18) carrying QTLs for vertebrae, each with log10 of the odds ratio (LOD) scores of 2.8 or better. The QTLs on chromosomes (Chrs) 2, 6, 12, 13, and 16 were unique to femurs, whereas the QTLs on Chrs 7 and 9 were unique to vertebrae. When the two bone sites had a QTL on the same chromosome, the same marker had the highest, although different, LOD score. A pairwise comparison by analysis of variance (ANOVA) did not reveal significant gene × gene interactions between QTLs for either bone site. BMD variance accounted for by individual QTLs ranged from 1% to 10%. Collectively, the BMD QTLs for femurs accounted for 35.1% and for vertebrae accounted for 23.7% of the F2 population variances in these bones. When mice were homozygous c3/c3 in the QTL region, 8 of the 10 QTLs increased, while the remaining two QTLs on Chrs 6 and 12 decreased, femoral BMD. Similarly, when mice were homozygous c3/c3 in the QTL region for the vertebrae, five of the seven QTLs increased, while two QTLs on Chrs 7 and 9 decreased, BMD. These findings show the genetic complexity of BMD with multiple genes participating in its regulation. Although 5 of the 12 QTLs are considered to be skeleton-wide loci and commonly affect both femurs and vertebrae, each of the bone sites also exhibited unique QTLs. Thus, the BMD phenotype can be partitioned into its genetic components and the effects of these loci on normal bone biology can be determined. Importantly, the BMD QTLs that we have identified are in regions of the mouse genome that have known human homology, and the QTLs will become useful experimental tools for mechanistic and therapeutic analyses of bone regulatory genes.
Discordance Between Changes in Bone Mineral Density Measured at Different Skeletal Sites in Perimenopausal Women—Implications for Assessment of Bone Loss and Response to Therapy: The Danish Osteoporosis Prevention Study
Authors: Abrahamsen B. , Stilgren L. S. , Hermann A. P. , Tofteng C. L. , Bärenholdt O. , Vestergaard P. , Brot C. , Nielsen S. P.
Published: April 2006

Assessing bone loss and gain is important in clinical decision-making, both in evaluating treatment and in following untreated patients. The aim of this study was to correlate changes in bone mineral density (BMD) at different skeletal sites during the first 5 years after menopause and determine if forearm measurements can substitute for dual-energy X-ray absorptiometry (DXA) of the spine and hip. BMD was measured at 0, 1, 2, 3, and 5 years using Hologic 1000/W and 2000 densitometers in 2016 perimenopausal women participating in a national cohort study. This analysis comprises 1422 women remaining in the study after 5 years without changes to their initial treatment (hormone-replacement therapy [HRT], n = 497, or none, n = 925). Despite correlated rates of change between forearm and spine (r2 = 0.11; p < 0.01), one-half of those who experienced a significant decrease in spine BMD at 5 years showed no significant fall in forearm BMD (sensitivity, 50%; specificity, 85%; κ = 0.25). The total hip had significant better agreement with spine (sensitivity, 63%; specificity, 85%; κ = 0.37; p < 0.01). Analysis of quartiles of change also showed significant better agreement with spine and whole body for the total hip than for the femoral neck or ultradistal (UD) forearm. In a logistic regression analysis for identification of group (HRT or control), the prediction was best for whole body (82.6%) and spine (80.9%), followed by total hip (78.5%) and forearm (74.7%). In conclusion, changes at the commonly measured sites are discordant, and DXA of the forearm is less useful than DXA of the hip or spine in determining the overall skeletal response to therapy or assessing bone loss in untreated women.
Stimulation of Interleukin-6 Promoter by Parathyroid Hormone, Tumor Necrosis Factor α, and Interleukin-1β in UMR-106 Osteoblastic Cells Is Inhibited by Protein Kinase C Antagonists
Authors: Nagy Zsolt , Radeff Julie , Stern Paula H.
Published: April 2006

To investigate the level at which protein kinase C (PKC) regulates expression of interleukin-6 (IL-6) in osteoblastic cells, effects of several PKC antagonists and PKC down-regulation by phorbol ester were studied in UMR-106 osteoblastic cells that had been transiently transfected with a −224/+11-base pair (bp) IL-6 promoter coupled to a luciferase reporter. Parathyroid hormone (PTH) elicited a dose-dependent stimulation of the IL-6 promoter expression, with significant increases produced by 5 h of treatment with concentrations of PTH as low as 10−14 M. The increase in IL-6 promoter expression was inhibited by the PKC antagonists GF109203X, 30 nM to 1 μM, and calphostin C, 250 nM. Prior down-regulation of PKC with 100 nM phorbol-12,13-dibutyrate (PDBU) for 48 h inhibited the PTH effect as well as the smaller stimulatory effects elicited by tumor necrosis factor α (TNF-α), 10−9-10−8 M, and by IL-1β, 1-10 ng/ml. In contrast to these findings, the stimulatory effects of PTH, TNF-α, and IL-1β on the IL-6 promoter expression were enhanced by staurosporine. Treatment with GF109203X or down-regulation of PKC with PDBU prevented the stimulatory effects of staurosporine. PKC activity was increased by staurosporine. The findings with staurosporine are consistent with our earlier observations that this agent enhances the calcium signaling and bone resorption elicited by PTH. The studies support the role of PKC in the stimulatory effects of PTH, TNF-α, and IL-1β on IL-6 expression.
The Polymorphism in the Caudal-Related Homeodomain Protein Cdx-2 Binding Element in the Human Vitamin D Receptor Gene
Authors: Arai Hidekazu , Miyamoto Ken-Ichi , Yoshida Michiko , Yamamoto Hironori , Taketani Yutaka , Morita Kyoko , Kubota Megumi , Yoshida Shigeko , Ikeda Mikiko , Watabe Fumiko , Kanemasa Yasuhiro , Takeda Eiji
Published: April 2006

The major physiological activity of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is the regulation of calcium absorption in the small intestine, and the level of vitamin D receptor (VDR) is an important factor in this regulation. In a previous study, we indicated that the caudal-related homeodomain Cdx-2 played an important role in the intestine-specific transcription of the human VDR gene. In this study, the polymorphism was identified in the core sequence 5′-ATAAAAACTTAT-3′ in the Cdx-2 binding site in the VDR gene promoter. In 261 Japanese women with genotyped VDR polymorphisms, 48 were genotype Cdx-A (adenine at −3731 nucleotides [nt] relative to the transcription start site of human VDR gene 5-ATAAAAACTTAT), 82 were genotype Cdx-G (guanine at −3731 nt, 5′-GTAAAAACTTAT-3′), and 131 were genotype Cdx-A/G (heterozygote). In postmenopausal Japanese women, the bone mineral density (BMD) in the lumbar spine (L2-L4) with the Cdx-G homozygote was 12% lower than that with the Cdx-A homozygote (p < 0.05). In electrophoretic gel mobility shift assay (EMSA), the oligonucleotide with Cdx-G allele markedly decreased the binding to Cdx-2 compared with that in the Cdx-A allele. The transcriptional activity of the VDR promoter with Cdx-G allele was decreased to 70% of the Cdx-A allele. In addition, in the herpes simplex virus thymidine kinase promoter, the Cdx-2 binding element with the G allele showed significantly lower transcriptional activity than that of the A allele. Thus, the polymorphism in the Cdx-2 binding site of the VDR gene (Cdx-polymorphism) would affect the expression of VDR in the small intestine. In addition, this polymorphism may modulate BMD in postmenopausal Japanese women.
Distribution of Intracortical Porosity in Human Midfemoral Cortex by Age and Gender
Authors: Bousson Valérie , Meunier Alain , Bergot Catherine , Vicaut Éric , Rocha Maria Augusta , Morais Maria Helena , Laval-Jeantet Anne-Marie , Laredo Jean-Denis
Published: April 2006

The purpose of this study was to describe the age-specific distribution of midfemoral intracortical porosity throughout the cortical width in males and females. Microradiography and an automated image analysis system were used to study midfemoral cortical bone specimens from 163 white people, including 77 males and 86 females, in a recent anthropological collection covering a broad age range. In each specimen, porosity (percentage of the cortical bone area occupied by pores), pore number, and pore size were measured throughout the entire cortex and in three cortical subregions of equal width labeled the periosteal, midcortical, and endosteal subregions. For each gender, relationships linking age to porosity, pore number, and mean pore size were assessed using regression analysis. In addition, age- and site-related changes in these three variables were tested for significance using two-way analysis of variance (ANOVA). Age explained 52% of the porosity variance in females and 13.5% in males. In each gender, there were significant age- and site-related differences in porosity, pore number, and pore size. In adults aged 60 years or younger, both pore size and pore number increased with increasing age, whereas in adults older than 60 years, pore size continued to increase but pore number decreased. In males, the age-related changes in pore size and pore number were proportionally similar in the three cortical subregions. In females, in contrast, the changes predominated in the endosteal subregion and resulted in significant cortical thinning.
Intracapsular Hip Fracture and the Region-Specific Loss of Cortical Bone: Analysis by Peripheral Quantitative Computed Tomography
Authors: Crabtree N. , Loveridge N. , Parker M. , Rushton N. , Power J. , Bell K. L. , Beck T. J. , Reeve J.
Published: April 2006

Generalized bone loss within the femoral neck accounts for only 15% of the increase in intracapsular hip fracture risk between the ages of 60 and 80 years. Conventional histology has shown that there is no difference in cancellous bone area between cases of intracapsular fracture and age and sex-matched controls. Rather, a loss of cortical bone thickness and increased porosity is the key feature with the greatest change occurring in those regions maximally loaded during a fall (the inferoanterior [IA] to superoposterior [SP] axis). We have now reexamined this finding using peripheral quantitative computed tomography (pQCT) to analyze cortical and cancellous bone areas, density, and mass in a different set of ex vivo biopsy specimens from cases of intracapsular hip fracture (female, n = 16, aged 69-92 years) and postmortem specimens (female, n = 15, aged 58-95 years; male, n = 11, aged 56-86 years). Within-neck location was standardized by using locations at which the ratio of maximum to minimum external diameters was 1.4 and at more proximal locations. Cortical widths were analyzed using 72 radial profiles from the center of area of each of the gray level images using a full-width/half-maximum algorithm. In both male and female controls, cancellous bone mass increased toward the femoral head and the rate of change was gender independent. Cancellous bone mass was similar in cases and controls at all locations. Overall, cortical bone mass was significantly lower in the fracture cases (by 25%; p < 0.001) because of significant reductions in both estimated cortical area and density. These differences persisted at locations that are more proximal. The mean cortical width in the cases was significantly lower in the IA (22.2%; p = 0.002) and inferior regions (19%; p < 0.001). The SP region was the thinnest in both cases and controls. These data confirm that a key feature in the etiology of intracapsular hip fracture is the site-specific loss of cortical bone, which is concentrated in those regions maximally loaded during a fall on the greater trochanter. An important implication of this work is that the pathogenesis of bone loss leading to hip fracture must be by a mechanism that varies in its effect according to location within the femoral neck. Key candidate mechanisms would include those involving locally reduced mechanical loading. This study also suggests that the development of noninvasive methodologies for analyzing the thickness and estimated densities of critical cortical regions of the femoral neck could improve detection of those at risk of hip fracture.
Bone Turnover and Density in Obese Premenopausal Women During Moderate Weight Loss and Calcium Supplementation
Authors: Shapses Sue A. , Von Thun Nancy L. , Heymsfield Steven B. , Ricci Trina A. , Ospina Margaret , Pierson Richard N. , Stahl Theodore
Published: April 2006

Bone turnover is increased during weight loss in postmenopausal women and can be suppressed with calcium supplementation. In this study, we assessed the influence of energy restriction with and without calcium supplementation (1 g/day) in premenopausal women. Thirty-eight obese premenopausal women (body mass index [BMI] of 35.0 ± 3.9 kg/m2) completed a 6-month study of either moderate weight loss or weight maintenance. During weight loss, women were randomly assigned to either a calcium supplementation (n = 14) or placebo group (n = 14) and lost 7.5 ± 2.5% of their body weight. The control group of women (n = 10) maintained their body weight. Total body and lumbar bone mineral density (LBMD) and content were measured by dual-energy X-ray absorptiometry (DXA) at baseline and after weight loss. Throughout the study, blood and urine samples were collected to measure bone turnover markers and hormones. During moderate energy restriction, dietary calcium intake decreased (p < 0.05) and the bone resorption marker deoxypyridinoline (DPD) increased slightly (p ≤ 0.05) without evidence of bone loss. Calcium supplementation during weight loss tended to increase lumbar BMD by 1.7% (p = 0.05) compared with the placebo or weight maintenance groups. In contrast to our previous findings in postmenopausal women, premenopausal obese women who consume a low calcium diet do not lose bone over a 6-month period, whether their weight is stable or decreasing moderately.