bluuebunny/crossref_metadata_2025 huggingface dataset:
Smad3-Deficient Chondrocytes Have Enhanced BMP Signaling and Accelerated Differentiation
Authors: Li Tian-Fang , Darowish Michael , Zuscik Michael J , Chen Di , Schwarz Edward M , Rosier Randy N , Drissi Hicham , O'Keefe Regis J
Published: April 2006

Smad3 deficiency accelerates chondrocyte maturation and leads to osteoarthritis. Primary chondrocytes without Smad3 lack compensatory increases of TGF-β signaling factors, but BMP-related gene expression is increased. Smad2 or Smad3 overexpression and BMP blockade abrogate accelerated maturation in Smad3−/− chondrocytes. BMP signaling is increased in TGF-β deficiency and is required for accelerated chondrocyte maturation.

Introduction: Disruption of TGF-β signaling results in accelerated chondrocyte maturation and leads to postnatal dwarfism and premature osteoarthritis. The mechanisms involved in this process were studied using in vitro murine chondrocyte cultures.

Materials and Methods: Primary chondrocytes were isolated from the sterna of neonatal wildtype and Smad3−/− mice. Expressions of maturational markers, as well as genes involved in TGF-β and BMP signaling were examined. Chondrocytes were treated with TGF-β and BMP-2, and effects on maturation-related genes and BMP/TGF-β responsive reporters were examined. Recombinant noggin or retroviral vectors expressing Smad2 or Smad3 were added to the cultures.

Results: Expression of colX and other maturational markers was markedly increased in Smad3−/− chondrocytes. Smad3−/− chondrocytes lacked compensatory increases in Smad2, Smad4, TGFRII, Sno, or Smurf2 and had reduced expression of TGF-β1 and TGFRI. In contrast, Smad1, Smad5, BMP2, and BMP6 expression was increased, suggesting a shift from TGF-β toward BMP signaling. In Smad3−/− chondrocytes, alternative TGF-β signaling pathways remained responsive, as shown by luciferase assays. These non-Smad3-dependent TGF-β pathways reduced colX expression and alkaline phosphatase activity in TGF-β-treated Smad3−/− cultures, but only partially. In contrast, Smad3−/− chondrocytes were more responsive to BMP-2 treatment and had increased colX expression, phosphoSmads 1, 5, and 8 levels, and luciferase reporter activity. Overexpression of both Smad2 and Smad3 blocked spontaneous maturation in Smad3-deficient chondrocytes. Maturation was also abrogated by the addition of noggin, an extracellular BMP inhibitor.

Conclusions: These findings show a key role for BMP signaling during the chondrocyte maturation, occurring with loss of TGF-β signaling with important implications for osteoarthritis and cartilage diseases.
Loop Diuretics Increase Bone Turnover and Decrease BMD in Osteopenic Postmenopausal Women: Results From a Randomized Controlled Study With Bumetanide
Authors: Rejnmark Lars , Vestergaard Peter , Heickendorff Lene , Andreasen Frederik , Mosekilde Leif
Published: April 2006

To study effects of loop diuretics on bone, 87 women were randomized to 1 year of treatment with bumetanide or placebo. Compared with placebo, bumetanide decreased BMD by 2% at the total hip and by 1.4% at the whole body. Levels of biochemical bone markers were lower in the placebo than in the bumetanide group. Thus, treatment with loop diuretics affects bone metabolism.

Introduction: Loop diuretics may affect bone because they increase the renal calcium excretion and alters the diurnal rhythm of plasma PTH levels. We studied the effects of 1 year of treatment with the loop diuretic bumetanide on bone metabolism.

Materials and Methods: In a double-blinded design, 87 healthy postmenopausal women with osteopenia were randomized to 1-year bumetanide treatment 2 mg/day or placebo. BMD, plasma levels of calcitropic hormones, and biochemical bone markers were measured at baseline, after 1 year of treatment (week 52), and 6 months after withdrawal of treatment (week 78). Calcium (800 mg/day) and vitamin D (10 μg/day) were administered to all participants during the entire 1.5-year study period.

Results: Compared with placebo, urinary calcium (+17%) and plasma PTH levels (+9%) increased significantly in response to bumetanide. After 1 year of treatment, BMD in the bumetanide compared with the placebo group was significantly decreased by 2% at the total hip and ultradistal forearm and by 1.4% at the whole body. In addition, levels of biochemical markers of bone turnover differed significantly (∼20%) between groups, with lower levels in the placebo than in the bumetanide group. Six months after the end of treatment, the effects of bumetanide were weakening.

Conclusions: Treatment with loop diuretics affects bone turnover and decreases BMD. Further studies may reveal whether loop diuretics should be considered as a risk factor for fracture.
Two-Year Clinical Trial of Oral Alendronate Versus Intravenous Pamidronate in Children With Osteogenesis Imperfecta
Authors: DiMeglio Linda A , Peacock Munro
Published: April 2006

A 2-year prospective, partially randomized open-label trial comparing oral alendronate with intravenous pamidronate therapy in children with OI showed equivalence in increasing total body BMD, spine BMD, and linear growth, and decreasing bone turnover and fracture incidence. Children with mild OI had greater responses than severe OI in BMD and growth.

Introduction: Bisphosphonate therapies increase BMD and may reduce fractures in children with osteogenesis imperfecta (OI). A study directly comparing oral with intravenous bisphosphonate has not been published. This clinical trial compares oral alendronate with intravenous pamidronate in children with OI using an open-label, prospective, 2-year, randomized design.

Materials and Methods: Children over the age of 3 years were stratified by bone age, pubertal stage, and type of OI and then randomized to receive oral alendronate 1 mg/kg/day in tablet form or intravenous pamidronate, 3 mg/kg/4 months. One child was assigned to pamidronate. One child randomized to intravenous pamidronate changed to oral alendronate. Eighteen children completed 12 months of therapy: nine on oral alendronate and nine on intravenous pamidronate. Primary outcome efficacy was increase in BMD. Secondary outcomes included changes in bone turnover biomarkers, fracture incidence, and growth.

Results: Total body and lumbar spine BMD increased, turnover markers decreased, and linear growth increased equivalently with oral and intravenous therapy. Fracture incidence showed a trend to decrease in both groups, with a significant decrease in fracture rates when the oral and intravenous groups were pooled. There were greater responses in BMD and growth in children with milder OI (type I) than those with more severe disease (types III and IV), but there were no significant effects of age or pubertal stage.

Conclusions: Oral and intravenous bisphosphonate therapies are equally effective in children with OI and are particularly effective in milder forms. The oral route is highly acceptable in children and has practical advantages over the intravenous route.
Association of Functionally Different RUNX2 P2 Promoter Alleles With BMD
Authors: Doecke James D , Day Christopher J , Stephens Alexandre SJ , Carter Shea L , van Daal Angela , Kotowicz Mark A , Nicholson Geoff C , Morrison Nigel A
Published: April 2006

RUNX2 gene SNPs were genotyped in subjects from the upper and lower deciles of age- and weight-adjusted femoral neck BMD. Of 16 SNPs in RUNX2 and its two promoters (P1 and P2), only SNPs in the P2 promoter were significantly associated with BMD. These P2 promoter SNPs were functionally different in gel-shift and promoter activity assays.

Introduction: Specific osteoblast genes are induced by Runx2, a cell-specific transcription factor that is a candidate gene for controlling BMD. We tested the hypothesis that RUNX2 genetic variation is associated with BMD.

Materials and Methods: From a population repository of normal subjects, the age- and weight-adjusted femoral neck BMD was ranked, and the upper and lower deciles (n = 132 each) were taken to represent the adjusted extremes of the population distribution. In these 264 subjects, we identified 16 allelic variations within the RUNX2 gene and promoters (P1 and P2) through DNA sequencing and denaturing high-performance liquid chromatography. Characterization of these alleles was performed through allele-specific cloning, transfection into ROS 17/2.8 cells, luciferase reporter analysis, and electrophoretic mobility shift assays.

Results: Within the P2 promoter were three polymorphic nucleotides for which the minor alleles were over-represented in the upper decile of BMD (0.117 and 0.064 in the upper and lower deciles, respectively). These alleles are in near complete linkage disequilibrium with each other and represent a haplotype block that is significantly associated with increased BMD. The common and rare P2 promoter alleles were cloned upstream of luciferase, and when transfected into osteoblast-like cells, the construct representing the rare haplotype showed significantly greater P2 promoter activity than the common haplotype.

Conclusions: Because the high BMD allele had higher P2 promoter activity, the data suggest that greater RUNX2 P2 promoter activity is associated with higher BMD.
Effects of Oral Alendronate on BMD in Adult Patients With Osteogenesis Imperfecta: A 3-Year Randomized Placebo-Controlled Trial
Authors: Chevrel Guillaume , Schott Anne-Marie , Fontanges Elisabeth , Charrin Jeanne E , Lina-Granade Geneviève , Duboeuf François , Garnero Patrick , Arlot Monique , Raynal Claude , Meunier Pierre J
Published: April 2006

A 3-year, randomized, double-blind, placebo-controlled trial evaluated the effect of oral alendronate on the BMD of 64 adult patients with osteogenesis imperfecta. The mean increases in the lumbar spine BMD were 10.1 ± 9.8% (p < 0.001) and 0.7 ± 5.7% in the alendronate and placebo groups, respectively. Oral alendronate increases BMD in adult patients with osteogenesis imperfecta.

Introduction: This study evaluated the effect of oral alendronate on the BMD of adult patients with osteogenesis imperfecta.

Materials and Methods: We carried out a 3-year, randomized, double-blind, placebo-controlled trial of oral alendronate in 64 adult patients with osteogenesis imperfecta. The primary endpoint was the difference between the groups in the mean percent change in lumbar spine BMD at 3 years. Secondary outcomes included changes in BMD of total hip, vertebral and peripheral fracture incidence, pain, hearing loss, and bone turnover biochemical markers. Patients were treated daily with either placebo or 10 mg alendronate. All received 1 g of calcium and 800 IU of vitamin D daily.

Results: The mean ± SD increases in the lumbar spine BMD were 10.1 ± 9.8% (p < 0.001) and 0.7 ± 5.7% in the alendronate and placebo groups, respectively. Hip BMD increased in the alendronate group by 3.3 ± 0.5% (p = 0.001) and decreased in the placebo group by 0.3 ± 0.6%. The sample size was not sufficient to determine an effect of alendronate on fracture rate. A significant increase of the pain score was noted in the alendronate group (p = 0.04) in the intent-to-treat analysis but not in the per protocol analysis. There was no change in hearing in either group. Bone resorption and formation biochemical markers were significantly decreased in the alendronate group (p < 0.001). There were no differences in severe adverse effects between the groups, but there was an increase in nonsevere upper gastrointestinal effects in the alendronate group (p = 0.003).

Conclusions: Oral alendronate increases BMD and increase nonsevere gastrointestinal adverse effects but does not modify the hearing loss in adult patients with osteogenesis imperfecta. More studies are needed to evaluate an effect on the fracture rate.
Reversibility of Calcitriol-Induced Medial Artery Calcification in Rats With Intact Renal Function
Authors: Bas Alicia , Lopez Ignacio , Perez Jose , Rodriguez Mariano , Aguilera-Tejero Escolastico
Published: April 2006

VC is an important clinical entity; however, very little information is available on its resolution. Induction and regression of calcitriol-induced VC was studied in 47 rats. After calcitriol withdrawal, there was a relatively rapid regression of VC mediated by an active cellular process.

Introduction: Vascular calcifications (VCs) represent an important risk factor for cardiovascular death. Although VCs are prevalent in relevant diseases (e.g., chronic kidney disease, osteoporosis, diabetes), the reversibility of extraskeletal calcifications is an unresolved issue. This study was conducted to investigate (1) the in vivo effect of calcitriol on VC and (2) whether calcitriol-induced VC would regress after suppression of calcitriol treatment.

Materials and Methods: The calcifying effect of calcitriol was studied in four groups of rats (n = 8) that received calcitriol (1 μg/kg, IP) for 2, 4, 6, and 8 days. The reversibility of VC was studied in three additional groups (n = 5) treated with 1 μg/kg of calcitriol for 8 days that were subsequently killed 1, 2, and 9 weeks after the last calcitriol dose. Aortic VC was assessed by histology and by quantification of aortic calcium and phosphorus content. The aortic wall was studied by histology and immunohistochemistry. Statistical analysis was performed by ANOVA and t-tests.

Results: Calcitriol administration resulted in a time-dependent induction of VC, with aortic calcium and phosphorus being significantly increased at 6 and 8 days. Treatment with calcitriol for 8 days resulted in massive medial calcification of the aorta with a 10- to 30-fold increase in the aortic Ca and P content. After suppressing calcitriol administration, a progressive decrease in von Kossa staining and aortic Ca (from 32.8 ± 2.5 to 9.3 ± 1.8 mg/g of tissue, p < 0.001) and P (from 11.9 ± 1.2 to 2.7 ± 1.8 mg/g of tissue, p = 0.001) content was evidenced. Histology of the aortic wall showed monocytes adhered to the aortic endothelium and macrophages involved in the reabsorption of calcium deposits.

Conclusions: Our results show that calcitriol treatment induces time-dependent VC. After calcitriol withdrawal, VC regress rapidly with aortic calcium and phosphorus decreasing by 75% in the course of 9 weeks. An active cellular process seems to be involved in regression of VC.
Correcting the Magnification Error of Fan Beam Densitometers
Authors: Griffiths M. R. , Noakes K. A. , Pocock N. A.
Published: April 2006

Dual-energy X-ray absorptiometry (DXA), using a narrow pencil-shaped X-ray beam coupled to a single detector, has been used extensively. More recently, DXA using a fan-shaped X-ray beam coupled to an array of detectors has been introduced. This new generation of scanners causes an inherent magnification of scanned structures as the distance from the X-ray source decreases. This magnification, which occurs in the medial-lateral direction but not in the craniocaudal direction, does not affect bone mineral density (BMD). There are, however, significant changes of bone mineral content (BMC), bone area, and parameters of hip geometry, with varying distance of the bone scanned from the X-ray source. Variability of soft tissue thickness in vivo, by altering the distance of the skeleton from the scanning table and X-ray source, may cause clinically significant errors of BMC, bone area, and proximal femur geometry when measured using fan-beam densitometers. We analyzed the geometry of Lunar and Hologic fan beam scanners to derive equations expressing the true width of scanned structures in terms of the apparent width and machine dimensions. We also showed mathematically that performing an additional scan, at a different distance from the X-ray source than the first scan, provides simultaneous equations that can be solved to derive the real width of a scanned bone. This hypothesis was tested on the Lunar Expert using aluminium phantoms scanned at different table heights. There was an excellent correlation, r = 0.99 (p < 0.001), between the predicted phantom width and the measured phantom width. In conclusion, this study shows that the magnification error of fan beam DXA can be corrected using a dual scanning technique. This has important implications in the clinical usefulness of BMC and geometrical measurements obtained from these scanners.
Fat or Lean Tissue Mass: Which One Is the Major Determinant of Bone Mineral Mass in Healthy Postmenopausal Women?
Authors: Chen Zhao , Lohman Timothy G. , Stini William A. , Ritenbaugh Cheryl , Aickin Mikel
Published: April 2006

The relative importance of fat and lean tissue mass in determining bone mineral mass among postmenopausal women was examined in this 1-year longitudinal study. Fifty postmenopausal Caucasian women entered the study; 45 of them completed a 1-year follow-up. Dual-energy X-ray absorptiometry was employed for measuring total and regional bone mineral density (BMD) and bone mineral content (BMC), fat tissue mass (FTM), lean tissue mass (LTM), and body weight. Results from linear regression analysis using the cross-sectional data (n = 50) of the study indicated that LTM explained a larger percentage of variation in bone mineral mass than did FTM. FTM and LTM were found to be moderately correlated (r = 0.55); when FTM was entered in the same predicting regression models, LTM was a significant predictor (p < 0.05) of the total and regional BMC, but not BMD. The percent FTM (and inversely %LTM) was correlated with BMD and BMC, but significant correlation was primarily found only for total body BMD (or BMC). Weight was the best predictor of total body BMD and BMC. Longitudinally (n = 45), annual changes in both FTM and weight were significantly associated with annual changes in regional BMD after adjustment for initial bone mineral values (p < 0.05). We conclude that bone mineral mass is more closely related to LTM than to FTM, while annual changes in regional BMD are more closely correlated with changes in FTM in healthy postmenopausal women. Meanwhile, increased body weight is significantly associated with increased bone mineral mass.
[3H]Bradykinin Receptor-Binding, Receptor-Recycling, and Receptor-Internalization of the B2 Bradykinin Receptor in the Murine Osteoblast-like Cell Line MC3T3-E1
Authors: Windischhofer Werner , Leis Hans J.
Published: April 2006

Bradykinin (BK) has been demonstrated to induce inositol phosphate production, release of intracellular Ca2+, and prostaglandin E2 (PGE2) synthesis in the murine osteoblast-like cell line MC3T3-E1. Because cellular response to BK is a function of receptor affinity, receptor coupling, and receptor recycling, we investigated kinetic properties, specificity, and regulation at the BK-receptor level on intact, BK-sensitive MC3T3-E1 cells. Our results clearly demonstrate the existence of a single category of binding sites for [3H]BK (kD = 366 ± 98 pM; Bmax = 45.3 ± 6.6 fmol/mg of protein). Displacement studies with various BK analogs gave a rank order compatible with a B2 BK-receptor type (BK > Lys-BK > [Hyp3]-BK > Met-Lys-BK > HOE140 > Tyr-BK > Tyr8-BK > D-Arg, [Hyp3, Thi5,8, D-Phe7]-BK > [D-Phe7]-BK > des-Arg9-BK > des-Arg9, [Leu8]-BK = angiotensin II). No atypic high-affinity binding sites for the B1 receptor agonist des-Arg9-BK could be observed. Prestimulation of MC3T3-E1 cells with BK resulted in the disappearance of accessible B2 receptors at the cell surface by internalization. Postexposure of BK-pretreated cells to ligand-free medium resulted in almost complete receptor restoration within 30 minutes, exhibiting an intermediate state of two categories of binding sites (kD1 = 444 ± 37 pM, Bmax1 = 9.2 ± 0.3 fmol/mg of protein and kD2 = 2.7 ± 0.28 pM, Bmax2 = 24.2 ± 0.2 fmol/mg of protein), probably representing coupled and uncoupled B2 receptors. Prolonged stimulation with BK (2.5–5 h) also revealed the temporal occurrence of two categories of binding sites after 2.5 h (kD1 = 228 ± 3.5 pM; Bmax1 = 15.6 ± 0.6 fmol/mg of protein; kD2 = 2.7 ± 0.25 nM; Bmax2 = 40.7 ± 1.5 fmol/mg of protein), whereas low-affinity binding sites disappeared after 5 h.
Signal Transduction of Mechanical Stimuli Is Dependent on Microfilament Integrity: Identification of Osteopontin as a Mechanically Induced Gene in Osteoblasts
Authors: Toma C. D. , Ashkar S. , Gray M. L. , Schaffer J. L. , Gerstenfeld L. C.
Published: April 2006

Mechanical perturbation has been shown to modulate a wide variety of changes in second message signals and patterns of gene expression in osteoblasts. Embryonic chick osteoblasts were subjected to a dynamic spatially uniform biaxial strain (1.3% applied strain) at 0.25 Hz for a single 2-h period, and osteopontin (OPN), an Arg-Gly-Asp (RGD)-containing protein, was shown to be a mechanoresponsive gene. Expression of opn mRNA reached a maximal 4-fold increase 9 h after the end of the mechanical perturbation that was not inhibited by cycloheximide, thus demonstrating that mechanoinduction of opn expression is a primary response through the activation of pre-existing transcriptional factors. The signal transduction pathways, which mediated the increased expression of opn in response to mechanical stimuli, were shown to be dependent on the activation of a tyrosine kinase(s) and protein kinase A (PKA) or a PKA-like kinase. Selective inhibition of protein kinase C (PKC) had no effect on the mechanoinduction of osteopontin even though opn has been demonstrated to be an early response gene to phorbol 12-myristate 13-acetate (PMA) stimulation. Mechanotransduction was dependent on microfilament integrity since cytochalasin-D blocked the up-regulation of the opn expression; however, microfilament disruption had no effect on the PMA induction of the gene. The microtubule component of the cytoskeleton was not related to the mechanism of signal transduction involved in controlling opn expression in response to mechanical stimulation since colchicine did not block opn expression. Mechanical stimulus was shown to activate focal adhesion kinase (FAK), which specifically became associated with the cytoskeleton after mechanical perturbation, and its association with the cytoskeleton was dependent on tyrosine kinase activity. In conclusion, these results demonstrate that the signal transduction pathway for mechanical activation of opn is uniquely dependent on the structural integrity of the microfilament component of the cytoskeleton. In contrast, the PKC pathway, which also activates this gene in osteoblasts, acts independently of the cytoskeleton in the transduction of its activity.
Fas and Fas Ligand Interaction Is Necessary for Human Osteoblast Apoptosis
Authors: Kawakami Atsushi , Eguchi Katsumi , Matsuoka Naoki , Tsuboi Masahiko , Koji Takehiko , Urayama Satoshi , Fujiyama Kaoru , Kiriyama Takeshi , Nakashima Tomoki , Nakane Paul K. , Nagataki Shigenobu
Published: April 2006

We investigated the cellular and humoral interactions between peripheral blood mononuclear cells (PBMCs) and human osteoblasts, leading to apoptosis of osteoblasts. Human osteoblastic cell line MG63 and human primary osteoblast-like cells obtained from biopsy specimens were used in this study. PBMCs were isolated from healthy donors and cultured with or without stimulation by recombinant interleukin-2 followed by 12-o-tetradecanoyl-phorbol 13-acetate with ionomycin. Fas was functionally expressed on MG63 and primary osteoblast-like cells. Activated PBMCs expressed Fas ligand (FasL) strongly on their surface and killed MG63 and primary osteoblast-like cells. Cultured supernatants of activated PBMCs also induced apoptotic cell death of MG63 and primary osteoblast-like cells. In contrast, both unstimulated PBMCs and cultured supernatants of unstimulated PBMCs did not induce apoptosis of these cells. Furthermore, the cytotoxic effect and induction of apoptosis against MG63 and primary osteoblast-like cells by activated PBMCs and cultured supernatants were inhibited significantly by human Fas chimeric protein. Our data showed that human osteoblasts expressed Fas fuctionally and both membrane-type and soluble form FasL from activated PBMCs induced apoptosis of these cells, providing the one possible mechanism of bone loss in inflammatory diseases such as rheumatoid arthritis.
Phosphorylation of the Human Calcitonin Receptor by Multiple Kinases Is Localized to the C-Terminus
Authors: Nygaard Sean C. , Kuestner Rolf E. , Moore Emma E. , Stroop Steven D.
Published: April 2006

The calcitonin receptor is a seven-transmembrane G-protein coupled receptor which is located on osteoclasts, in kidney, and in brain. The receptor signals through multiple pathways, including activation of adenylate cyclase, leading to inhibition of bone resorption. In the present study, we used antibodies raised against the C-terminus of the human calcitonin (CT) receptor to study receptor phosphorylation. In baby hamster kidney cells transfected with the human CT receptor, phosphorylation of the receptor increased approximately 2.5-fold after cells were treated with calcitonin, phorbol ester, forskolin, or calcitonin plus phorbol ester. Phosphorylation reached a maximum 20 minutes after treatment with sCT and half-maximal phosphorylation was observed at 0.1 nM sCT, a hormone concentration related to receptor occupancy. Digestion of the immunoprecipitated receptor with cyanogen bromide (CNBr) yielded a single32P-labeled fragment which migrates at Mr 14 kD on gel electrophoresis. This corresponds to the predicted size of the CNBr fragment containing the C-terminal domain of the receptor. No32P-labeled bands were observed for CNBr fragments predicted to contain the first, second, or third intracellular loops. An identical labeling pattern was seen with cells expressing an alternatively spliced isoform of the human receptor (insert-positive isoform). Phosphorylation of the receptor by phorbol ester and forskolin was further localized to a Mr 6 kD proteolytic fragment within the C-terminus. The protein kinase A and C inhibitors staurosporine, chelerythrine, and H-89 had little effect on CT-induced phosphorylation, suggesting that nonsecond messenger-activated kinases are involved in hormone-dependent CT receptor phosphorylation.
A Collagenous Cementum-Derived Attachment Protein Is a Marker for Progenitors of the Mineralized Tissue-Forming Cell Lineage of the Periodontal Ligament
Authors: Liu Hong Wei , Yacobi Rinat , Savion Naphtali , Narayanan A. Sampath , Pitaru Sandu
Published: April 2006

The periodontal ligament (PDL) is a fibrous and cellular connective tissue that mediates tooth attachment to bone, and it comprises fibroblastic and mineralized tissue-forming (MTF) progenitors. The MTF progenitors are believed to give rise to the cementoblastic and osteoblastic lineages. Cementum attachment protein (CAP) is a collagenous cementum-derived protein which binds strongly to osteoblasts, moderately to PDL cells, and weakly to gingival fibroblasts. The aim of the present study was to determine the relationship between the capacity of PDL progenitors to bind CAP and their potential to express alkaline phosphatase (ALP) and form mineralized-like tissue in culture. Cloned human PDL progenitor populations obtained from nine human donors were assayed for their constitutive capacity to bind CAP and express ALP, and for the dexamethasone-induced potential to form mineralized-like tissue in culture in the presence of ascorbic acid and β-glycerophosphate. Forty percent of the progenitor clones produced mineralized-like tissue. Two patterns of mineralization were observed: a spread and flat pattern similar to that produced by human bone cells in culture and a nodular ridge–like type resembling that formed by human cementoma-derived cells. A direct correlation was found between the percentage of ALP positive cells in each progenitor clone and the amount of mineralized-like tissue formed (r = 0.565). Similar correlations were found between the number of ALP positive cells and the binding capacity of each clone (r = 0.392) and between the CAP binding capacity and mineralized-like tissue formation (r = 0.584). Multiple regression analysis indicated that the constitutive capacity of a clone to bind CAP and express ALP is directly correlated to its dexamethasone-induced potential to form mineralized tissue (r = 0.675). These results indicate that CAP binding and ALP expression can serve as markers for the identification of MTF progenitors in the heterogeneous cultured population of the human periodontal ligament. These data show for the first time that binding capacity to extracellular components of mineralized tissues can be a marker for mineralized tissue-forming progenitors.
Evidence for Increased Bone Formation Following a Brief Endurance-Type Training Intervention in Adolescent Males
Authors: Eliakim Alon , Raisz Lawrence G. , Brasel Jo Anne , Cooper Dan M.
Published: April 2006

The effect of exercise training, particularly relatively brief periods, on bone turnover markers in adolescents has been poorly studied. Thirty-eight healthy males (16 ± 0.7 years) participated in a 5-week summer school program in which 20 subjects were randomly assigned to a training group consisting of 2 h/day, 5 days/week of endurance exercise, and 18 subjects were assigned to a control group. Bone formation was assessed by measurements of circulating osteocalcin, bone-specific alkaline phosphatase (BSAP), and the C-terminal procollagen peptide (PICP). Bone resorption was assessed by urinary levels of free deoxypyridinoline cross-links (dPYR) and the C-(CTX) and N-terminal (NTX) telopeptide cross-links. Prior to training, there was a weak positive correlation between fitness and PICP (r = 0.27, p < 0.05), but no correlations were observed between fitness and either the other markers of bone formation or bone resorption. Training led to a significant increase in (1) osteocalcin (15 ± 4%, p < 0.03), (2) BSAP (21 ± 6%, p < 0.02), and (3) PICP (30 ± 11%, p < 0.03) and to a significant decrease in NTX (−21 ± 3%, p < 0.05). These bone turnover markers did not change in the control subjects (osteocalcin, 0 ± 4%; BSAP, 2 ± 4%; PICP, −4 ± 6%; NTX, −6 ± 4%). There was no change in urinary dPYR and CTX in either control or trained subjects. Fitness is only weakly, if at all, correlated with bone formation, but a relatively brief period of endurance training leads to a substantial increase in bone formation markers in adolescent males. School-based, short-term exercise training programs could play a role in enhancing bone formation in adolescents.
Long-Term Effects of Intravenous Pamidronate in Fibrous Dysplasia of Bone
Authors: Chapurlat Roland D. , Delmas Pierre D. , Liens Daniel , Meunier Pierre J.
Published: April 2006

Fibrous dysplasia of bone (FD) is a rare disorder characterized by proliferation of fibrous tissue in bone marrow leading to osteolytic lesions. It causes bone pain and fractures. To date the only treatment is orthopedic. Histological and biochemical similarities between FD and Paget's bone disease related to increased osteoclastic resorption led us to propose treatment with the bisphosphonate pamidronate. The aim of the study was to assess the long-term effects of intravenous pamidronate in FD. In this open label phase III study, 20 patients with FD (11 males and 9 females; mean age 31 years) received courses of 180 mg of intravenous pamidronate every 6 months (60 mg/day during 3 days by infusion). The mean duration of follow-up was 39 months (range 18–64). Severity of bone pain, number of painful skeletal sites per patient, X-rays of all involved areas, serum alkaline phosphatase, fasting urinary hydroxyproline, and urinary type I collagen C-telopeptide were assessed every 6 months. The severity of bone pain and the number of painful sites appeared to be significantly reduced. All biochemical markers of bone remodeling were substantially lowered. We observed a radiographic response in nine patients with refilling of osteolytic lesions. A mineralization defect proven by bone biopsy was observed in one case. Four patients sustained bone stress lines, but no fracture occurred. We suggest that intravenous pamidronate alleviates bone pain, reduces the rate of bone turnover assessed by biochemical markers, and improves radiological lesions of FD. Few side effects were observed.
Appendicular Bone Mass in Children with a High Prevalence of Low Dietary Calcium Intakes
Authors: Pettifor John M. , Moodley Gopal P.
Published: April 2006

We have previously documented evidence of dietary calcium deficiency in black children living in a rural community in the eastern part of South Africa. The present study determined the bone mass of the distal one-third of the radius in a random sample of children living in the same community and compared their bone mass measurements with those of black children living in a similar rural community but without evidence of dietary calcium deficiency. Further, factors (weight, height, serum corrected total calcium, phosphorus, and alkaline phosphatase [ALP]) that might influence appendicular bone mass were assessed and correlated with the bone mass measurements. A random sample of 306 boys and 345 girls between the ages of 1 and 20 years were included in the study. Hypocalcemia was found in 6.5% of the boys and 5% of the girls, while elevated ALP values were recorded in 20 and 26% of the boys and girls, respectively. After adjusting for differences in age, weight, and height, bone mineral density (BMD) and bone mineral apparent density (BMAD) were significantly lower and bone width (BW) greater in study than control children. In a stepwise regression analysis, weight and/or height accounted for the majority of the observed variance in BMC, BW, and BMD; however, a significant effect of serum calcium (positively) and ALP (negatively) on BMC and BMD was also found. In boys, but not girls, serum ALP also had a positive effect on BW. BMAD was negatively correlated to ALP and positively correlated to serum calcium in both boys and girls. Those children with hypocalcemia or elevated ALP levels had significantly lower BMC, BMD, and BMAD and a trend toward greater BW than children with normal biochemistry. The findings suggest that low dietary calcium intake may have a detrimental effect on appendicular bone density in rural black children. Whether or not these effects are disadvantageous in the long-term is not known.
The Short-Term Effects of Cisplatin Chemotherapy on Bone Turnover
Authors: Young David R. , Virolainen Petri , Inoue Nozomu , Frassica Frank J. , Chao Edmund Y. S.
Published: April 2006

Cisplatin is an effective agent in the treatment of osteosarcoma of bone but little is known of its effects on normal bone turnover. Twenty-four dogs divided into three study groups were used to study the effect of cisplatin on normal bone turnover at the distant site of surgery. Group 1 served as the control group, group 2 received four cycles of cisplatin every 3 weeks before the surgery, and group 3 received four cycles postoperatively. The bone turnover rate was evaluated by measuring levels of systemic bone markers, osteocalcin, alkaline phospohatase, urine pyridinoline cross-links, and by determination histomorphometric indices. Histomorphological analysis showed poor correlation on bone formation with systemic bone markers at distant sites of surgery. Histomorphometrically normal bone turnover was affected by administration of cisplatin, but the effect was temporary, late, and less significant than what occurred at the surgical site. Our data showed that significant effects of cisplatin are observed at the site of active cellular induction and proliferation, such as implant-host interface, and less effects are seen at the sites of normal bone turnover.
Anomalies in the Measurement of Changes in Total-Body Bone Mineral by Dual-Energy X-Ray Absorptiometry During Weight Change
Authors: Tothill P. , Hannan W. J. , Cowen S. , Freeman C. P.
Published: April 2006

For an eating disorder study over a period of 1 year, we measured total-body bone mineral using a Hologic QDR 1000W in a total of 157 subjects and observed anomalies that questioned the accuracy of such measurements. Using the recommended Enhanced software, a change in total bone mineral content (ΔBMC) correlated positively with a change in weight (ΔW; r = 0.66), but a loss of weight was associated with an increase in bone mineral areal density (BMD; r = 0.58), arising from a reduction in bone area (AREA). Both regressions were highly significant. The dominant factor in this relationship was a strong correlation between ΔAREA and ΔBMC, for all parts of the skeleton, r > 0.9, with a slope close to 1. This is implausible because bone area would not be expected to change. When Standard software was used, the slope of the ΔBMC/ΔW correlation was steeper, but the ΔBMD/ΔW regression became positive. An artefact of dual-energy X-ray absorptiometry processing was suspected, and phantom measurements were made. The phantom consisted of tissue-equivalent hardboard cut and stacked to form cylinders corresponding to the head, trunk, arms, and legs of a standard man. The skeleton was constructed from layers of aluminium sheet as an approximation of the average shape, BMD, BMC, and AREA in each region. When aluminium thickness was varied, BMD thresholds were found, approximately 0.4 g/cm2 for the legs and 0.2 g/cm2 for the arms. Above these, bone area rose fairly rapidly toward a plateau. At higher skeletal densities, the relationships between measured and true BMDs were close to linear, but slopes were less than unity, so that changes would be underestimated by 10–30%. Increases of thickness of the soft tissue of the phantom lowered AREA slightly. Uniform fat proportion increases led to decreases in BMC and AREA, but lard wrapped in an annulus around the limbs led to spurious increases in BMC and AREA of a similar magnitude to those observed in vivo, while BMD fell slightly, although there had been no true change of bone variables. Similar results were obtained with lard around the limbs of a volunteer. Reanalysis of phantom scans using Standard software confirmed the software differences noted in vivo. The phantom measurements offer an explanation of the anomaly in vivo and demonstrate that, under different circumstances, change in both BMC and BMD can be wrongly recorded. We believe that no valid conclusions can be drawn from measurements by the Holgic QDR 1000W of bone changes during weight change.
Bone Mass and Hip Axis Length in Healthy Asian, Black, Hispanic, and White American Youths
Authors: Wang May-Choo , Aguirre Melissa , Bhudhikanok Grace S. , Kendall Carmen G. , Kirsch Susannah , Marcus Robert , Bachrach Laura K.
Published: April 2006

The primary objective of this study was to examine the associations of ethnicity, diet (calcium, protein, energy), and weight-bearing activity with dual-energy X-ray absorptiometry (DXA)-measured bone mass and hip axis length (HAL) in 423 Asians, blacks, Hispanics, and non-Hispanic Caucasians, aged 9–25 years. Bone mass was expressed as bone mineral content (BMC), bone mineral density (BMD), and bone mineral apparent density (BMAD). The data were analyzed using multiple linear regression, after stratifying for gender and pubertal stage and adjusting for height and weight. With few exceptions, Asians and Hispanics had comparable bone mass to whites at all pubertal stages. Greater femoral neck BMAD in black than white females was observed at all pubertal stages. Black males displayed greater BMD and BMAD than white males at all sites in early puberty and at the femoral neck in maturity. Calcium was positively and protein negatively related to BMAD at the femoral neck in early pubertal females. Among males, calcium was negatively associated with whole body BMC and BMD and spine BMD and BMAD in midpuberty. Weight-bearing activity was not associated with bone mass in females; in males, it was positively related only to femoral neck BMC in early puberty. There was an absence of evidence for ethnic differences in HAL among females. In males, we observed shorter HAL in mature Asians and blacks than whites. Neither diet nor activity was associated with HAL.
Pharmacological and Biochemical Evidence for the Regulation of Osteocalcin Secretion by Potassium Channels in Human Osteoblast-like MG–63 Cells
Authors: Moreau R. , Aubin R. , Lapointe J. Y. , Lajeunesse D.
Published: April 2006

Previous reports have suggested the involvement of voltage-activated calcium (Ca2+) channels in bone metabolism and in particular on the secretion of osteocalcin by osteoblast-like cells. 1 We now report that potassium (K+) channels can also modulate the secretion of osteocalcin by MG–63 cells, a human osteosarcoma cell line. When 1,25-dihydroxyvitamin D3(1,25(OH)2D3)-treated MG–63 cells were depolarized by step increases of the extracellular K+ concentration ([K+]out) from 5–30 mM, osteocalcin (OC) secretion increased from a control value of 218 ± 13 to 369 ± 18 ng/mg of protein/48 h (p < 0.005 by analysis of variance). In contrast, in the absence of 1,25(OH)2D3, there is no osteocalcin secretion nor any effect of cell depolarization on this activity. The depolarization-induced increase in 1,25(OH)2D3-dependent osteocalcin secretion was totally inhibited in the presence of 10 μM Nitrendipine (a Ca2+ channel blocker, p < 0.005) without affecting cellular alkaline phosphatase nor cell growth. Charybdotoxin, a selective blocker of Ca2+-dependent K+ channels (maxi-K) present in MG–63 cells, 2 stimulated 1,25(OH)2D3-induced osteocalcin synthesis about 2-fold (p < 0.005) after either 30, 60, or 120 minutes of treatment. However, Charybdotoxin was without effect on basal release of osteocalcin in the absence of 1,25(OH)2D3 pretreatment. Using patch clamp technique, we occasionally observed the presence of a small conductance K+ channel, compatible with an ATP-dependent K+ channel (GKATP) in nonstimulated cells, whereas multiple channel openings were observed when cells were treated with Diazoxide, a sulfonamide derivative which opens GKATP. Western blot analysis revealed the presence of the N-terminal peptide of GKATP in MG–63 cells, and its expression was regulated with the proliferation rate of these cells, maximal detection by Western blots being observed during the logarithmic phase of the cycle. Glipizide and Glybenclamide, selective sulfonylureas which can block GKATP, dose-dependently enhanced 1,25(OH)2D3-induced OC secretion (p < 0.005). Reducing the extracellular calcium concentration with EGTA (μM range) totally inhibited the effect of Glipizide and Glybenclamide on osteocalcin secretion (p < 0.005), which remained at the same levels as controls. Diazoxide totally prevented the effect of these sulfonylureas. These results suggest that voltage-activated Ca2+ channels triggered via cell depolarization can enhance 1,25(OH)2D3-induced OC release by MG–63 cells. In addition, OC secretion is increased by blocking two types of K+ channels: maxi-K channels, which normally hyperpolarize cells and close Ca2+ channels, and GKATP channels. The role of these channels is closely linked to the extracellular Ca2+ concentration.
Trabecular Bone Formation in the Healing of the Rodent Molar Tooth Extraction Socket
Authors: DeVlin H. , Hoyland J. , Newall J. F. , Ayad S.
Published: April 2006

The aim of this study was to investigate the nature of the template structure on which trabecular bone formation occurs during healing of the rodent tooth extraction socket, a well studied bone healing system. The presence of collagen type II mRNA has previously been described in the healing socket, although the formation of the protein or cartilage has not been observed. However, recent evidence from developmental and other bone healing studies indicates that collagen type III may be important in forming the preliminary scaffold on which bone trabeculae are formed. The maxillary right molar teeth were removed from rats under general anaesthesia and the animals killed at various times afterward. The tissues were examined using histological, in situ hybridization, and immunohistochemical staining techniques. It was concluded that collagen type IIA mRNA was produced by osteoblast cells of the socket, but that collagen type II, if present, would account for less than 0.01% of the total proteins extracted. During bone formation, Sharpey's fibers were seen radiating from the peripheral bone toward the center of the socket. These optically active collagen fibers were inserted into the forming bone trabeculae and were recognized by antibodies raised against collagen type III. The arrangement and composition of these fibers therefore suggest that they form a preliminary framework on which deposition of woven bone trabeculae occurs.
Effects of Thyroid Hormone Administration and Estrogen Deficiency on Bone Mass of Female Rats
Authors: Gouveia Cecilia H. A. , Jorgetti Vanda , Bianco Antonio C.
Published: April 2006

To investigate the effects of thyroxine (T4) administration on bone mass, five 81-day-old female rats were treated with T4 (25 μg of T4/100 g of body weight [bw]/day), and bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DXA) 28 days later. The BMD values for the total skeleton, femoral, and tibial subsegments were lower than in controls (p ≤ 0.05). The lumbar spine (L2–L5) was not significantly affected by T4 treatment. Next, thirty-seven 211 ± 1.5 (mean ± SEM)-day-old female rats were divided into six groups as follows: (1) control; (2) ovariectomized (OVX); (3) 1xT4 (∼1.0 μg of T4/100 g of bw/day; approximately physiological replacement dose); (4) OVX+1xT4; (5) 2xT4 (∼2.0 μg of T4/100 g of bw/day); (6) OVX+2xT4. DXA scans were performed at days 0 and 85. Control rats showed a generalized BMD increase, as opposed to a decrease in OVX rats. The trabecular bone volume of the fifth lumbar vertebra was also lower in OVX rats than in controls (p < 0.05). The 1xT4 treatment had no effect on BMD of intact rats, while treatment with 2xT4 impaired the expected BMD increase. Unexpectedly, the OVX+1xT4 group presented a generalized BMD increase that was significant for the total skeleton, L2–L5, and femoral subsegments (p < 0.05), comparable to controls. Treating OVX animals with 2xT4 did not potentiate the osteopenic effects of estrogen deficiency, nor did it reverse the osteopenic effects of OVX. In conclusion, treatment with high doses of T4 caused BMD to decrease substantially, particularly at the femur, whereas near physiological doses of T4 prevented bone loss associated with OVX, and regardless of bone type (trabecular or cortical), the skeleton site seems to be a more important determinant of the effects of thyroid hormone on bone mass.