bluuebunny/crossref_metadata_2025 huggingface dataset:
Early Changes in Biochemical Markers of Bone Formation Predict BMD Response to Teriparatide in Postmenopausal Women With Osteoporosis
Authors: Chen Peiqi , Satterwhite Julie H , Licata Angelo A , Lewiecki E Michael , Sipos Adrien A , Misurski Derek M , Wagman Rachel B
Published: April 2006

The relationship between early changes in biochemical markers of bone turnover and the subsequent BMD response to daily teriparatide therapy in women with postmenopausal osteoporosis was studied. Changes in five biochemical markers, obtained from a subset of women enrolled in the Fracture Prevention Trial, were examined. Early increases in the PICP and the PINP were the best predictors of BMD response to teriparatide in this analysis.

Introduction: Early reductions in biochemical markers of bone turnover with antiresorptive therapy negatively correlate with subsequent increases in BMD. We undertook this analysis to determine if early changes in biochemical markers with teriparatide therapy predict subsequent increases in BMD.

Materials and Methods: In the Fracture Prevention Trial, 1637 postmenopausal women with osteoporosis were randomized to receive daily, self-administered, subcutaneous injections of placebo, teriparatide 20 μg/day, or teriparatide 40 μg/day. Serum concentrations of two bone formation markers (bone-specific alkaline phosphatase [bone ALP] and the carboxy-terminal extension peptide of procollagen type 1 [PICP]) and urinary concentrations of two bone resorption markers (free deoxypyridinoline [DPD] and N-terminal telopeptide [NTX]) were assessed in a trial population subset (n = 520) at baseline and at 1, 3, 6, and 12 months. We also assessed serum concentrations of another bone formation marker, the amino-terminal extension peptide of procollagen type 1 (PINP), in a subset of 771 women at baseline and 3 months. Lumbar spine (LS) BMD was measured by DXA at baseline and 18 months. Femoral neck BMD was measured at baseline and 12 months.

Results and Conclusion: Baseline bone turnover status correlated positively and significantly with BMD response. The highest correlations occurred for the LS BMD response to teriparatide 20 μg/day. Among all studied biochemical markers, increases in PICP at 1 month and PINP at 3 months correlated best with increases in LS BMD at 18 months (0.65 and 0.61, respectively; p < 0.05). The relationships between these two biochemical markers and the LS BMD response were stronger than the corresponding relationships for the femoral neck BMD response. Using receiver operator curve analysis, we determined that the increases in PICP at 1 month and PINP at 3 months were the most sensitive and accurate predictors of the LS BMD response.
Growth Patterns at Distal Radius and Tibial Shaft in Pubertal Girls: A 2-Year Longitudinal Study
Authors: Wang Qingju , Alén Markku , Nicholson Patrick , Lyytikäinen Arja , Suuriniemi Miia , Helkala Erkki , Suominen Harri , Cheng Sulin
Published: April 2006

Bone changes, in terms of both size and BMD, were assessed longitudinally in pubertal girls. Before puberty, BMD at the distal radius declined, whereas bone size increased, suggesting that normal growing girls experience a transient period of increased bone fragility. This could explain the elevated low-trauma forearm fracture rates reported in earlier studies.

Introduction: Longitudinal data on bone growth during puberty are sparse. Such information is needed to understand the sequence of biological changes, the physical and mechanical consequences for the growing skeleton, and the implications for later life.

Materials and Methods: The geometric properties and volumetric BMD (vBMD) of the distal radius and tibial shaft were measured using pQCT in 258 pubertal girls followed over 2 years. A new hierarchical linear statistical modeling approach was used to determine true longitudinal trends.

Results: The growth rates of cross-sectional area (CSA) and BMC of the distal radius peaked at 16 and 9 months before menarche, respectively. This growth asynchrony between bone size and mass meant that total vBMD of the distal radius declined until 1 year before menarche. At the tibial shaft, total vBMD and cortical vBMD increased monotonically without any such transient reduction. Cortical thickness increased linearly, which was accounted for mainly by bone formation at the periosteal surface before menarche, but by both periosteal and endocortical apposition after menarche. During puberty, the ratio of cortical-to-total CSA of the tibial shaft increased and that of marrow-to-total CSA decreased.

Conclusions: The temporal pattern of bone growth during puberty differs at the distal radius and tibial shaft. A transient decrease in vBMD, arising from asynchronous bone size and mass growth, occurs only at the radius. In the tibia, the mechanism of cortical thickening changes from periosteal apposition premenarche to both periosteal and endocortical apposition postmenarche.
Enamel Matrix Protein Interactions
Authors: Wang HongJun , Tannukit Sissada , Zhu DanHong , Snead Malcolm L , Paine Michael L
Published: April 2006

The recognized structural proteins of the enamel matrix are amelogenin, ameloblastin, and enamelin. While a large volume of data exists showing that amelogenin self-assembles into multimeric units referred to as nanospheres, other reports of enamel matrix protein-protein interactions are scant. We believe that each of these enamel matrix proteins must interact with other organic components of ameloblasts and the enamel matrix. Likely protein partners would include integral membrane proteins and additional secreted proteins.

Introduction: The purpose of this study was to identify and catalog additional proteins that play a significant role in enamel formation.

Materials and Methods: We used the yeast two-hybrid assay to identify protein partners for amelogenin, ameloblastin, and enamelin. Once identified, RT-PCR was used to assess gene transcription of these newly identified and potential “enamel” proteins in ameloblast-like LS8 cells.

Results: In the context of this yeast assay, we identified a number of secreted proteins and integral membrane proteins that interact with amelogenin, ameloblastin, and enamelin. Additionally, proteins whose functions range from the inhibition of soft tissue mineralization, calcium ion transport, and phosphorylation events have been identified as protein partners to these enamel matrix proteins. For each protein identified using this screening strategy, future studies are planned to confirm this physiological relationship to biomineralization in vivo.

Conclusion: Identifying integral membrane proteins of the secretory surface of ameloblast cells (Tomes' processes) and additional enamel matrix proteins, based on their abilities to interact with the most abundant enamel matrix proteins, will better define the molecular mechanisms of enamel formation at its most rudimentary level.
Serum Retinoids and β-Carotene as Predictors of Hip and Other Fractures in Elderly Women
Authors: Barker Margo E , McCloskey Eugene , Saha Shikha , Gossiel Fatma , Charlesworth Diane , Powers Hilary J , Blumsohn Aubrey
Published: April 2006

There is debate about the possible deleterious effect of excessive vitamin A exposure on fracture risk. In this nested case control study in older women (312 cases and 934 controls), serum retinol, retinyl palmitate, and β-carotene were not associated with fracture risk, and there was no evidence of excess risk with multivitamin or cod liver oil supplementation.

Introduction: Recent studies have suggested that higher vitamin A intake may account for a component of fracture risk within the general population and that supplemental vitamin A may be harmful even within recommended limits. No studies have examined the relationship between biochemical retinol status and fracture in older women.

Materials and Methods: We examined serum retinol, retinyl palmitate, and β-carotene as predictors of incident hip and other fractures in a large prospective study of British women over the age of 75 years (n = 2606, 312 incident osteoporotic fractures, 92 incident hip fractures; mean follow-up duration, 3.7 years). Fasting blood samples (9:00-11:00 a.m.) were collected at baseline. Using a case-control design (three controls per case), serum retinol, retinyl palmitate, and β-carotene were assessed as univariate predictors of incident osteoporotic fracture or hip fracture. Baseline BMD at the total hip, age, 25(OH)D, serum β Crosslaps, bone-specific alkaline phosphatase, weight, height, and smoking were considered as covariates in a multivariate model.

Results: Serum retinol, retinyl palmitate, and β-carotene were not significant univariate predictors of either hip fracture or any fracture (all p > 0.05; Cox proportional hazards regression). For all osteoporotic fractures, the hazard ratio (HR) was 0.92 (95% CI, 0.81-1.05) per 1 SD increase in serum retinol. Risk of any osteoporotic fracture was slightly less in the highest quartile of serum retinol compared with the lowest quartile (HR, 0.85; 95% CI, 0.69-1.05; p = 0.132) There was a tendency for increased serum retinol to predict benefit rather than harm in terms of BMD (r = 0.09, p = 0.002). Multivitamin or cod liver oil supplementation was associated with a significantly lower risk of any fracture (HR, 0.76; 95% CI, 0.60-0.96; p = 0.021). In multivariate analysis, only age, total hip BMD, and weight were associated with fracture risk (p < 0.05).

Conclusions: We found no evidence to support any skeletal harm associated with increased serum indices of retinol exposure or modest retinol supplementation in this population.
A Frequent Regulatory Variant of the Estrogen-Related Receptor α Gene Associated With BMD in French-Canadian Premenopausal Women
Authors: Laflamme Nathalie , Giroux Sylvie , Loredo-Osti J Concepción , Elfassihi Latifa , Dodin Sylvie , Blanchet Claudine , Morgan Kenneth , Giguère Vincent , Rousseau François
Published: April 2006

Genes are important BMD determinants. We studied the association of an ESRRA gene functional variant with BMD in 1335 premenopausal women. The ESRRA genotype was an independent predictor of L2-L4 BMD, with an effect similar to smoking and equivalent to a 10-kg difference in weight.

Introduction: Several genetic polymorphisms have been associated with osteoporosis or osteoporosis fractures, but no functional effect has been shown for most of these gene variants. Because functional studies have implicated estrogen-related receptor α (ESRRA) in bone metabolism, we evaluated whether a recently described regulatory variant of the ESRRA gene is associated with lumbar and hip BMD as measured by DXA and with heel bone parameters as measured by quantitative ultrasound (QUS).

Materials and Methods: Heel bone parameters were measured by right calcaneal QUS in 1335 healthy French-Canadian premenopausal women, and one-half of these women also had their BMD evaluated at two sites: femoral neck and lumbar spine (L2-L4) by DXA. All bone measures were tested separately for association with the ESRRA genotype by analysis of covariance. The significance of the ESRRA contribution to the model was also assessed by two different permutation tests.

Results: A statistically significant association between ESRRA genotype and lumbar spine BMD was observed: women carrying the long ESRRA genotype had a 3.9% (0.045 g/cm2) higher lumbar spine BMD than those carrying the short ESRRA genotype (p = 0.004), independently of other risk factors measured. This effect of ESRRA genotype is similar to the effect of smoking and equivalent to a 10-kg difference in weight. This association was confirmed by permutation tests (p = 0.004). The same trend was observed for femoral neck BMD (2.6%, p = 0.07). However, no association was observed between ESRRA and QUS heel bone measures.

Conclusion: These results support the genetic influence of this ESRRA regulatory variant on BMD.
What Proportion of Incident Radiographic Vertebral Deformities Is Clinically Diagnosed and Vice Versa?
Authors: Fink Howard A , Milavetz Donna L , Palermo Lisa , Nevitt Michael C , Cauley Jane A , Genant Harry K , Black Dennis M , Ensrud Kristine E
Published: April 2006

We prospectively examined, in a large cohort of older women, the proportion of incident radiographic vertebral deformities diagnosed as incident clinical vertebral fractures in the same women at the same vertebral level. The proportion of deformities clinically diagnosed ranged from <15% for milder deformities to nearly 30% for more severe deformities.

Introduction: The relationship between radiographic and clinical vertebral fractures is incompletely understood. No previous study has prospectively compared the agreement between incident radiographic vertebral deformities and incident community-recognized, radiographically confirmed vertebral fractures in the same women at the same vertebral level(s).

Materials and Methods: This analysis of data from the Fracture Intervention Trial included all participants who completed both baseline and at least one scheduled follow-up lateral spinal radiograph (n = 6084). Incident vertebral deformities were defined at a given vertebral level as a reduction between baseline and closeout radiographs of ≥20% and 4 mm in any vertebral height and subdivided into two severity categories. Incident clinical vertebral fractures were those reported to clinical centers by participants and confirmed by the study radiologist, who compared the community spinal radiograph with the participant's baseline study radiograph using semiquantitative methods.

Results: A total of 446 incident radiographic vertebral deformities were identified in 330 women, whereas 121 women experienced one or more confirmed incident clinical vertebral fracture. Of incident radiograpic vertebral deformities, 22.6% were also clinically diagnosed as incident vertebral fractures, with clinical diagnoses made for 28.4% of the deformities that exceeded 30% and 4 mm height loss (severe deformity) compared with 14.3% for deformities that involved ≥20% and 4 mm but <30% height loss (milder deformity). Of incident clinical vertebral fractures, 72.7% were morphometrically identified as incident deformities, most of them as severe deformities. More than 20% of incident clinical fractures were not identified as incident deformities by even the most liberal morphometric criterion used in this study.

Conclusions: Approximately one-fourth of incident radiographic vertebral deformities were clinically diagnosed as new vertebral fractures, although the proportion clinically diagnosed was increased for more severe deformities. Whereas most incident clinical vertebral fractures were identified as severe morphometric deformities, approximately one-fourth did not meet even the most liberal study criterion for morphometric deformity. Further study of factors that may explain the discordance between incident vertebral deformities and incident clinical vertebral fractures is important.
Long-Term Strontium Ranelate Administration in Monkeys Preserves Characteristics of Bone Mineral Crystals and Degree of Mineralization of Bone
Authors: Farlay Delphine , Boivin Georges , Panczer Gérard , Lalande Agnès , Meunier Pierre Jean
Published: April 2006

In monkeys, long-term strontium ranelate administration results in a dose-dependent bone strontium uptake (mainly into newly formed bone) that preserves the degree of mineralization of bone and the bone mineral at the crystal level, showing its safety at bone mineral level.

Introduction: Strontium ranelate simultaneously increases bone formation and decreases bone resorption, leading to prevention of bone loss and increase in bone mass and bone strength in normal and ovariectomized rats. This study investigated the interactions of stable strontium (Sr) with bone mineral in monkeys after long-term strontium ranelate treatment and after a period of treatment withdrawal.

Materials and Methods: Iliac bone was obtained from untreated monkeys, monkeys at the end of a 52-week strontium ranelate administration (200, 500, 1250 mg/kg/day orally), and in parallel groups 10 weeks after the end of strontium ranelate administration (same three doses; n = 3–7). Sr uptake and distribution in bone mineral were quantified by X-ray microanalysis, changes at the crystal level by X-ray diffraction, and the degree of mineralization of bone (DMB) by quantitative microradiography.

Results: After strontium ranelate administration, dose-dependent Sr uptake occurred into cortical and cancellous bone, with higher content (1.6 times) in new than in old bone. This Sr uptake decreased (50%) 10 weeks after treatment withdrawal; the decrease occurred almost exclusively in new bone. At the end of strontium ranelate treatment and after its withdrawal, a preservation of crystal characteristics was observed, suggesting that Sr was only faintly linked to crystals by ionic substitution and of DMB.

Conclusions: These results show the absence of a deleterious effect of long-term strontium ranelate treatment on bone mineralization, confirming the histomorphometric observations made in postmenopausal osteoporotic women treated with strontium ranelate.
Differential Effect of BMP4 on NIH/3T3 and C2C12 Cells: Implications for Endochondral Bone Formation
Authors: Li Guangheng , Peng Hairong , Corsi Karin , Usas Arvydas , Olshanski Anne , Huard Johnny
Published: April 2006

After intramuscular implantation, BMP4-expressing NIH/3T3 fibroblasts and BMP4-expressing C2C12 myoblasts can promote ectopic cartilage and bone formation. Fibroblasts tend to undergo chondrogenesis, whereas myoblasts primarily undergo osteogenesis. These results suggest that endochondral bone formation may involve different cell types, a finding that could have major implications for the tissue engineering of bone and cartilage.

Introduction: The delivery of BMP4 through cell-based gene therapy can trigger ectopic endochondral bone formation in skeletal muscle. We hypothesized that, when stimulated with or transduced to express BMP4, different types of cells residing within skeletal muscle might participate in different stages of endochondral bone formation.

Materials and Methods: We compared the responses of a fibroblast cell line (NIH/3T3), a myoblast cell line (C2C12), primary fibroblasts, and primary myoblasts to BMP4 stimulation in vitro. We then transduced the four cell populations to express BMP4 and compared their ability to promote ectopic endochondral bone formation in skeletal muscle.

Results: Under the influence of BMP4 in vitro and in vivo, NIH/3T3 cells differentiated toward both chondrogenic and osteogenic lineages, whereas most C2C12 cells underwent primarily osteogenic differentiation. NIH/3T3 cells genetically modified to express BMP4 induced delayed but more robust cartilage formation than did genetically modified C2C12 cells, which promoted rapid ossification. These differences in terms of the timing and amount of cartilage and bone formation persisted even after we introduced a retrovirus encoding dominant negative Runx2 (DNRunx2) into the C2C12 cells, which interferes with the function of Runx2. Superior osteogenic potential was also displayed by the primary myoblasts in vitro and in vivo compared with the primary fibroblasts. The different proliferation abilities and differentiation potentials exhibited by these cells when influenced by BMP4 may at least partially explain the differing roles that BMP4-expressing myogenic cells and BMP4-expressing fibroblastic cells play in endochondral bone formation.

Conclusions: Our findings suggest that the process of endochondral bone formation in skeletal muscle after delivery of BMP4 involves different cell types, including fibroblastic cells, which are more involved in the chondrogenic phases, and myoblastic cells, which are primarily involved in osteogenesis. These findings could have important implications for the development of tissue engineering applications focused on bone and cartilage repair.
Hypothalamic Control of Bone Formation: Distinct Actions of Leptin and Y2 Receptor Pathways
Authors: Baldock Paul A , Sainsbury Amanda , Allison Susan , Lin En-Ju D , Couzens Michelle , Boey Dana , Enriquez Ronaldo , During Matthew , Herzog Herbert , Gardiner Edith M
Published: April 2006

Leptin and Y2 receptors on hypothalamic NPY neurons mediate leptin effects on energy homeostasis; however, their interaction in modulating osteoblast activity is not established. Here, direct testing of this possibility indicates distinct mechanisms of action for leptin anti-osteogenic and Y2−/− anabolic pathways in modulating bone formation.

Introduction: Central enhancement of bone formation by hypothalamic neurons is observed in leptin-deficient ob/ob and Y2 receptor null mice. Similar elevation in central neuropeptide Y (NPY) expression and effects on osteoblast activity in these two models suggest a shared pathway between leptin and Y2 receptors in the central control of bone physiology. The aim of this study was to test whether the leptin and Y2 receptor pathways regulate bone by the same or distinct mechanisms.

Materials and Methods: The interaction of concomitant leptin and Y2 receptor deficiency in controlling bone was examined in Y2−/−ob/ob double mutant mice, to determine whether leptin and Y2 receptor deficiency have additive effects. Interaction between leptin excess and Y2 receptor deletion was examined using recombinant adeno-associated viral vector overproduction of NPY (AAV-NPY) to produce weight gain and thus leptin excess in adult Y2−/− mice. Cancellous bone volume and bone cell function were assessed.

Results: Osteoblast activity was comparably elevated in ob/ob, Y2−/−, and Y2−/−ob/ob mice. However, greater bone resorption in ob/ob and Y2−/−ob/ob mice reduced cancellous bone volume compared with Y2−/−. Both wildtype and Y2−/− AAV-NPY mice exhibited marked elevation of white adipose tissue accumulation and hence leptin expression, thereby reducing osteoblast activity. Despite this anti-osteogenic leptin effect in the obese AAV-NPY model, osteoblast activity in Y2−/− AAV-NPY mice remained significantly greater than in wildtype AAV-NPY mice.

Conclusions: This study suggests that NPY is not a key regulator of the leptin-dependent osteoblast activity, because both the leptin-deficient stimulation of bone formation and the excess leptin inhibition of bone formation can occur in the presence of high hypothalamic NPY. The Y2−/− pathway acts consistently to stimulate bone formation; in contrast, leptin continues to suppress bone formation as circulating levels increase. As a result, they act increasingly in opposition as obesity becomes more marked. Thus, in the absence of leptin, the cancellous bone response to loss of Y2 receptor and leptin activity can not be distinguished. However, as leptin levels increase to physiological levels, distinct signaling pathways are revealed.
Continuous PTH and PTHrP Infusion Causes Suppression of Bone Formation and Discordant Effects on 1,25(OH)2Vitamin D
Authors: Horwitz Mara J , Tedesco Mary Beth , Sereika Susan M , Syed Mushtaq A , Garcia-Ocaña Adolfo , Bisello Alessandro , Hollis Bruce W , Rosen Clifford J , Wysolmerski John J , Dann Pamela , Gundberg Caren , Stewart Andrew F
Published: April 2006

Osteoblast activity and plasma 1,25(OH)2vitamin D are increased in HPT but suppressed in HHM. To model HPT and HHM, we directly compared multiday continuous infusions of PTH versus PTHrP in humans. Continuous infusion of both PTH and PTHrP results in marked and prolonged suppression of bone formation; renal 1,25(OH)2D synthesis was stimulated effectively by PTH but poorly by PTHrP.

Introduction: PTH and PTH-related protein (PTHrP) cause primary hyperparathyroidism (HPT) and humoral hypercalcemia of malignancy (HHM), respectively. Whereas HHM and HPT resemble one another in many respects, osteoblastic bone formation and plasma 1,25(OH)2vitamin D are increased in HPT but reduced in HHM.

Materials and Methods: We performed 2- to 4-day continuous infusions of escalating doses of PTH and PTHrP in 61 healthy young adults, comparing the effects on serum calcium and phosphorus, renal calcium and phosphorus handling, 1,25(OH)2vitamin D, endogenous PTH(1-84) concentrations, and plasma IGF-1 and markers of bone turnover.

Results: PTH and PTHrP induced comparable effects on renal calcium and phosphorus handling, and both stimulated IGF-1 and bone resorption similarly. Surprisingly, PTH was consistently more calcemic, reflecting a selectively greater increase in renal 1,25(OH)2 vitamin D production by PTH. Equally surprisingly, continuous infusion of both peptides markedly, continuously, and equivalently suppressed bone formation.

Conclusions: PTHrP and PTH produce markedly different effects on 1,25(OH)2vitamin D homeostasis in humans, leading to different calcemic responses. Moreover, both peptides produce profound suppression of bone formation over multiple days, contrasting with events in HPT, but mimicking HHM. These findings underscore the facts that the mechanisms underlying the anabolic skeletal response to PTH and PTHrP in humans is poorly understood, as are the signal transduction mechanisms that link the renal PTH receptor to 1,25(OH)2vitamin D synthesis. These studies emphasize that much remains to be learned regarding the normal regulation of vitamin D metabolism and bone formation in response to PTH and PTHrP in humans.
Effect of Testosterone Replacement on Trabecular Architecture in Hypogonadal Men
Authors: Benito Maria , Vasilic Branimir , Wehrli Felix W , Bunker Benjamin , Wald Michael , Gomberg Bryon , Wright Alexander C , Zemel Babette , Cucchiara Andrew , Snyder Peter J
Published: April 2006

We evaluated the effect of testosterone treatment on trabecular architecture by μMRI in 10 untreated severely hypogonadal men. After 2 years, μMRI parameters of trabecular connectivity improved significantly, suggesting the possibility that testosterone improves trabecular architecture.

Introduction: Osteoporosis, characterized by low BMD and diminished bone quality, is a significant public health problem in men. Hypogonadal men have decreased BMD and deteriorated trabecular architecture compared with eugonadal men, and testosterone treatment improves their BMD. We tested the hypothesis that testosterone replacement in hypogonadal men would also improve their trabecular architecture.

Materials and Methods: We selected 10 untreated severely hypogonadal men and treated them with a testosterone gel for 24 months to maintain their serum testosterone concentrations within the normal range. Each subject was assessed before and after 6, 12, and 24 months of testosterone treatment by magnetic resonance microimaging (μMRI) of the distal tibia and by DXA of the spine and hip. The μMRI parameters reflect the integrity of the trabecular network and include the ratio of all surface voxels (representing plates) to curve voxels (representing rods) and the topological erosion index, a ratio of topological parameters expected to increase on trabecular deterioration to those expected to decrease. The higher the surface-to-curve ratio and the lower the topological erosion index, the more intact the trabecular network.

Results: Serum testosterone concentrations increased to midnormal after 3 months of treatment and remained normal thereafter. After 24 months of testosterone treatment, BMD of the spine increased 7.4% (p < 0.001), and of the total hip increased 3.8% (p = 0.008). Architectural parameters assessed by μMRI also changed: the surface-to-curve ratio increased 11% (p = 0.004) and the topological erosion index decreased 7.5% (p = 0.004).

Conclusions: These results suggest the possibility that testosterone replacement of hypogonadal men improves trabecular architecture.
Identification of Osteopenic Women at High Risk of Fracture: The OFELY Study
Authors: Sornay-Rendu Elisabeth , Munoz Françoise , Garnero Patrick , Duboeuf François , Delmas Pierre D
Published: April 2006

About one-half of women with incident fractures have BMD above the WHO diagnostic threshold of osteoporosis. In the OFELY study, low BMD, increased markers of bone turnover, and prior fracture could be used to identify, within osteopenic women, those at high risk of fracture.

Introduction: Recent data suggest that about one-half of women with incident fractures have BMD above the World Health Organization (WHO) diagnostic threshold of osteoporosis (T score ≤ −2.5). We aimed to identify, within osteopenic women, those at high risk of fracture.

Materials and Methods: In the 671 postmenopausal women (mean age: 62 years) belonging to the Os des Femmes de Lyon (OFELY) population-based prospective cohort, we measured at baseline BMD by DXA at the spine and total hip, bone turnover markers (BTM) and clinical risk factors for osteoporosis. All fragility vertebral or nonvertebral fractures, confirmed by radiographs, were assessed during a median follow-up of 9.1 years (IQ: 2.3).

Results: 158 incident fractures were recorded in 116 women: 8% in normal, 48% in osteopenic, and 44% in osteoporotic women. Among osteopenic women, low BMD (−2.5 < T score ≤ −2.0) was associated with an increased fracture risk with an age-adjusted hazard ratio (HR) of 2.5 (1.3-4.6). In addition, age, prior fracture, and high BTM—but not other risk factors—were independently associated with an increased fracture risk with an age-adjusted HR of 2.2 (1.2-4.3) for prior fractures and 2.2 (1.4-3.8) for bone alkaline phosphatase (BALP) in the highest quartile. In the whole group of osteopenic women, a large majority of incident fractures occurred in those with a low BMD, prior fractures, or BALP in the highest quartile, with an age-adjusted HR of 5.3 (2.3-11.8). The 10-year probability of fracture in osteopenic women was 26% if at least one predictor was present, contrasting with 6% in those without any of the three risk factors.

Conclusions: In postmenopausal women with osteopenia, low BMD, increased BTM, and prior fracture are associated with an increased risk of fracture in the subsequent 10 years. Their assessment may play an important role in identifying women at high risk of fracture who could not be adequately detected by BMD measurement alone and who may benefit from a therapeutic intervention.
Biglycan Modulates Osteoblast Differentiation and Matrix Mineralization
Authors: Parisuthiman Duenpim , Mochida Yoshiyuki , Duarte Wagner R , Yamauchi Mitsuo
Published: April 2006

MC3T3-E1 cell-derived clones expressing higher (S) or lower (AS) levels of biglycan were generated and characterized. The processes of cell differentiation and matrix mineralization were accelerated in S but delayed in AS, indicating that BGN modulates osteoblastic cell differentiation.

Introduction: Biglycan (BGN), a member of the small leucine-rich proteoglycan family, is one of the major proteoglycans found in bone and has been implicated in bone formation. In this study, the effects of over- or underexpression of BGN on osteoblastic cell phenotypes and matrix mineralization were studied.

Materials and Methods: MC3T3-E1 cells were transfected with vectors containing the BGN cDNA in a sense or antisense orientation to generate clones expressing higher (S clones) or lower (AS clones) levels of BGN. MC3T3-E1 cells and those transfected with an empty vector (EV) were used as controls. The levels of BGN synthesized by these clones were evaluated by Western blot analysis. Cell growth was analyzed by cell counting and cell differentiation by the gene expression patterns of several osteoblastic markers using quantitative real-time PCR. The abilities of these clones to form mineralized matrices were evaluated by in vitro and in vivo mineralization assays. Furthermore, the clones were treated with BMP-4 and their responsiveness was assessed.

Results: The cell growth in these clones was unaffected; however, osteoblast differentiation was significantly accelerated in S clones and suppressed in AS clones. The in vitro matrix mineralization in S clones was significantly enhanced but severely impaired in AS clones. When transplanted into immunodeficient mice, S clone transplants exhibited larger areas of lamellar bonelike matrices, whereas only minute amounts of woven bone-type structure was found in AS transplants. The response to BMP-4 was higher in S clones but poorer in AS clones compared with that of controls.

Conclusions: BGN modulates osteoblast differentiation, possibly by regulating BMP signaling, and consequently matrix mineralization.
Critical Role of β3 Integrin in Experimental Postmenopausal Osteoporosis
Authors: Zhao Haibo , Kitaura Hideki , Sands Mark S , Ross F Patrick , Teitelbaum Steven L , Novack Deborah Veis
Published: April 2006

We show that mice lacking β3 integrin are protected from OVX-induced bone loss. Using a lentiviral-based strategy to express β3 mutants in β3−/− mice, we also show that β3S752, but not β3Y747/Y759, is important for osteoclastic bone resorption in vivo.

Introduction: Mice lacking the β3 integrin have dysfunctional osteoclasts and therefore accumulate bone mass with age. Thus, the αvβ3 integrin is a potential anti-osteoporosis target. Identifying components of the β3 integrin that determine its function in vivo is essential for therapeutically exploiting the antiresorptive properties of αvβ3.

Materials and Methods: We used DXA and histomorphometry to assess bone loss after ovariectomy in wildtype and β3 integrin null mice. We used lentiviral vectors carrying various human β3 (hβ3) integrin constructs to transduce β3−/− bone marrow and reconstituted lethally irradiated β3−/− mice with the transduced marrow. The expressed constructs include the intact integrin and two mutants, namely hβ3Y747F/Y759F and hβ3S752P, each of which induces the bleeding dyscrasia, Glanzmann's thrombasthenia, in humans. Two months after transplantation, the expression of hβ3 was measured by flow cytometry of marrow-derived macrophages. Osteoclast differentiation and function were assessed ex vivo by TRACP and actin-ring staining, respectively. Reconstituted mice were ovariectomized, and bone loss was assessed by DXA, histomorphometry, and serum TRACP5b assay.

Results: β3−/− mice are protected from ovariectomy-induced bone loss, showing no difference in BMD compared with sham-operated controls. We successfully expressed hβ3 integrins in β3−/− hosts using lentiviral transduction of bone marrow. Two months after transplantation, 25–35% of marrow-derived macrophages expressed the hβ3 constructs. Similar to its effect in vitro, hβ3WT completely rescued the osteoclast and platelet phenotype of β3−/− mice. Whereas platelet function remained deranged in β3−/− mice overexpressing hβ3Y747F/Y759F, osteoclast function was fully restored. In contrast, β3−/− mice expressing hβ3S752P continued to exhibit prolonged bleeding times and dysfunctional osteoclasts in vitro and ex vivo. Most importantly, hβ3WT and hβ3Y747F/Y759F transplanted mice underwent equivalent ovariectomy-induced bone loss, whereas, like those bearing the control vector, hβ3S752P transplanted mice were protected.

Conclusions: Functional β3 integrin is required for ovariectomy-induced bone loss. β3S752, but not β3Y747/Y759, is critical for osteoclast function in vivo.
Relationship Between Changes in BMD and Nonvertebral Fracture Incidence Associated With Risedronate: Reduction in Risk of Nonvertebral Fracture Is Not Related to Change in BMD
Authors: Watts Nelson B , Geusens Piet , Barton Ian P , Felsenberg Dieter
Published: April 2006

Whether greater treatment-related changes in BMD result in greater decreases in fracture risk is controversial. We analyzed the relationship between BMD change and nonvertebral fracture risk in postmenopausal osteoporotic women from the risedronate fracture program. Change in BMD did not influence the magnitude of risedronate's effect on nonvertebral fractures; the incidence of nonvertebral fractures was equally low in treated patients whose BMD increased or decreased.

Introduction: In untreated patients, low BMD correlates with increased fracture risk. Whether greater increases in BMD induced by anti-osteoporosis drugs are related to greater decreases in vertebral fracture risk is controversial, and little has been written about the relationship between change in BMD and nonvertebral fracture risk. We analyzed the relationship between BMD change and nonvertebral fracture incidence using individual patient data from postmenopausal osteoporotic women receiving antiresorptive treatment with risedronate.

Materials and Methods: This posthoc analysis combined data from three pivotal risedronate fracture endpoint trials. Women received risedronate 2.5 or 5 mg (n = 2561) or placebo (n = 1418) daily for up to 3 years. BMD and nonvertebral fractures confirmed by radiograph (hip, wrist, pelvis, humerus, clavicle, and leg) were assessed periodically over 3 years.

Results: The incidence of nonvertebral fractures in risedronate-treated patients was not different between patients whose spine BMD decreased (7.8%) and those whose spine BMD increased (6.4%; hazard ratio to subgroup of patients who lost BMD [HR], 0.79; 95% CI, 0.50, 1.25) or between those whose femoral neck BMD decreased (7.6%) and those whose femoral neck BMD increased (7.5%; HR, 0.93; 95% CI, 0.68, 1.28). The changes in lumbar spine and femoral neck BMD explained only 12% (95% CI, 2%, 21%; p = 0.014) and 7% (95% CI, 2%, 13%; p = 0.005), respectively, of risedronate's nonvertebral fracture efficacy.

Conclusions: For patients treated with risedronate, changes in BMD as measured by DXA do not predict the degree of reduction in nonvertebral fractures.
DMP1 Depletion Decreases Bone Mineralization In Vivo: An FTIR Imaging Analysis
Authors: Ling Yunfeng , Rios Hector F , Myers Elizabeth R , Lu Yongbo , Feng Jian Q , Boskey Adele L
Published: April 2006

The role of DMP1 in mineralization was analyzed by comparing bone mineral and matrix properties in dmp1-null female mice to heterozygous and wildtype controls by FTIR imaging spectroscopy. The observed decreased mineral content in dmp1 null mice indicates a key role for dmp1 in bone mineralization. Indirect effects of DMP1 on other systems also determine the KO phenotype.

Introduction: Dentin matrix protein 1 (DMP1), an acidic phosphorylated extracellular matrix protein, is highly expressed in mineralized tissues. In vitro, DMP1 peptides can promote or inhibit mineralization depending on the extent of phosphorylation, the peptide size, and concentration. To clarify the biological function of DMP1 protein on in vivo mineralization, this study analyzed bone properties of dmp1 knockout (KO) mice compared with heterozygous (HET) and wildtype (WT) controls.

Materials and Methods: Tibias from dmp1 KO and age-, sex-, and background-matched HET and WT mice at 4 and 16 weeks (Ntotal = 60) were examined by Fourier transform infrared imaging (FTIRI), histology (n = 6 per genotype and age; N = 36), and geometry by μCT (n = 4 per genotype and age; N = 24). Serum ionic calcium and phosphate concentrations were also determined.

Results: The mineral-to-matrix ratios (spectroscopic parameter of relative mineral content) were significantly lower in dmp1 KO mice tibias compared with WT and HET at 4 and 16 weeks. The mineral crystallinity (crystal size/perfection) was significantly increased in dmp1 KO and HET mice relative to WT. Collagen cross-link ratios (a spectroscopic parameter related to the relative amounts of nonreducible/reducible collagen cross-links) in dmp1 KO were not significantly different from WT and HET. Based on μCT, cortical bone cross-sectional areas at 16 but not 4 weeks were significantly reduced in the KO compared with controls. Maximum, minimum, and polar cross-sectional moments of inertia were significantly lower in dmp1 KO than in HET at 16 weeks but not at 4 weeks. Histological analysis and μCT 3-D images suggested that dmp1 KO mice had osteomalacia. Dmp1 KO mice had significantly lower ionic calcium and phosphate concentrations relative to WT, whereas in the HET, values for phosphate were equivalent, and calcium values were decreased relative to WT values.

Conclusions: The findings of decreased mineral-to-matrix ratio and increased crystal size in bones of dmp1 KO mice suggest that DMP1 has multiple roles (both direct and indirect) in the regulation of postnatal mineralization. We suggest that direct effects on mineral formation, crystal growth, and indirect effects on regulation of Ca × P concentrations and matrix turnover all contribute to the dominant phenotype in the dmp1 KO mouse.
New Target Genes for NOV/CCN3 in Chondrocytes: TGF-β2 and Type X Collagen
Authors: Lafont Jerome , Jacques Claire , Le Dreau Gwenvael , Calhabeu Frederico , Thibout Helene , Dubois Catherine , Berenbaum Francis , Laurent Maryvonne , Martinerie Cecile
Published: April 2006

We studied the involvement of NOV/CCN3, whose function is poorly understood, in chondrocyte differentiation. NOV was found to upregulate TGF-β2 and type X collagen and to act as a downstream effector of TGF-β1 in ATDC5 and primary chondrocytes. Thus, NOV is a positive modulator of chondrogenesis.

Introduction: NOV/CCN3 is a matricellular protein that belongs to the CCN family. A growing body of evidence indicates that NOV could play a role in cell differentiation, particularly in chondrogenesis. During chick embryo development, NOV expression is tightly regulated in cartilage, and a high expression of NOV has been associated with cartilage differentiation in Wilms' tumors. However, a precise role for NOV and potential target genes of NOV in chondrogenesis are unknown.

Materials and Methods: ATDC5 cells and primary chondrocytes were either treated with NOV recombinant protein or transfected with a NOV-specific siRNA to determine, using quantitative RT-PCR, the effect of NOV on the expression of several molecules involved in chondrocyte differentiation. Stable ATDC5 clones expressing NOV were also established to show that NOV was a downstream effector of TGF-β1.

Results: We established that NOV/CCN3 expression increases in ATDC5 cells at early stages of chondrogenic differentiation and precedes the appearance of TGF-β2 and of several chondrocytic markers such as SOX9 or type X collagen. When exogenously administered, NOV recombinant protein up-regulates TGF-β2 and type X collagen mRNA levels both in ATDC5 cells and in primary mouse chondrocytes but does not influence SOX9 expression. This regulation also occurs at the endogenous level because downregulation of NOV expression is correlated with an inhibition of TGF-β2 and type X collagen in primary chondrocytes. Furthermore, we found that NOV expression is downregulated when chondrocytes are exposed to TGF-β1-dedifferentiating treatment in chondrocytes, further providing evidence that NOV may counteract TGF-β1 effects on chondrocytes.

Conclusions: This study provides the first characterization of two new targets of NOV involved in chondrocyte differentiation, shows that NOV acts with TGF-β1 in a cascade of gene regulation, and indicates that NOV is a positive modulator of chondrogenesis.
Development and Characterization of Conditionally Immortalized Osteoblast Precursor Cell Lines from Human Bone Marrow Stroma
Authors: Hicok Kevin C. , Thomas Thierry , Gori Francesca , Rickard David J. , Spelsberg Thomas C. , Riggs B. Lawrence
Published: April 2006

Although the differentiation of mature osteoblasts has been well studied, there is still a need for a convenient way to study preosteoblast differentiation. Our laboratory has recently described a method for isolating small numbers of authentic osteoblast precursor cells from human bone marrow (Rickard et al., J Bone Miner Res 11:312–324, 1996). Here we describe the conditional immortalization of these cells by retroviral transfection with the amphotrophic vector, pZipSV40tsa58, which encodes for a temperature-sensitive mutant form of the simian virus large T-antigen. At the permissive temperature of 34°C, the cell lines proliferated, but differentiation was arrested, whereas at the restrictive temperature of 39.5°C, proliferation was decreased and differentiation was induced. As assessed by semiquantitative reverse transcriptase PCR after 4 days of culture at 39.5°C, the six cell lines expressed similar mRNA levels both constitutively and in response to dexamethasone (Dex) and 1α,25-dihydroxyvitamin D3 (1,25(OH2)D3) for osteoblast (alkaline phosphatase [ALP], type I collagen [Col I], osteocalcin [OC], and parathyroid hormone receptor [PTH-R] and adipocyte (lipoprotein lipase [LPL]) genes. In the presence of 10−8 M Dex, gene expression for ALP, PTH-R, and LPL increased, but that for OC decreased. Stimulation with 10−8 M 1,25(OH2)D3 increased gene expression for ALP, OC, and Col I. Changes in protein production for ALP, OC, and type I procollagen in response to Dex and 1,25(OH2)D3 were similar to changes in mRNA levels. When cultured at 39.5°C with ascorbate and β-glycerolphosphate for 21 days, mineralization of matrix occurred, whereas culture with Dex plus 1,25(OH2)D3, or rabbit serum led to enhanced formation of cytoplasmic lipid droplets within 6 days. Thus, these cell lines are capable of bipotential differentiation and should serve as an excellent tool to study the molecular mechanisms that regulate and select for osteoblast and adipocyte differentiation in humans.
Cyclic Stretch Enhances Gap Junctional Communication Between Osteoblastic Cells
Authors: Ziambaras Konstantinos , Lecanda Fernando , Steinberg Thomas H. , Civitelli Roberto
Published: April 2006

Mechanical loading is essential to maintain skeletal integrity. Because gap junctions in bone are affected by mechanical factors, we studied whether stretch, an anabolic stimulus for osteoblasts, modulates direct intercellular communication in these cells. Gap junctional communication during stretch was assessed using a newly developed method, the “parachute assay,” which allows monitoring of dye diffusion without disruption of the plasma membrane. Application of cyclic stretch for 2 or 24 h to well-coupled ROS 17/2.8 cells resulted in a 56.5% and 30.4% increase in dye coupling, respectively, compared with resting conditions. Stretch increased dye diffusion less dramatically (12.4% compared with unstimulated cells) in the poorly coupled UMR 106-01 cells. The stretch-induced increase of cell coupling was abolished in the presence of the gap junctional inhibitor, heptanol. Steady-state mRNA levels of connexin43 (Cx43), the gap junction protein that mediates cell-to-cell diffusion of negatively charged dyes between osteoblasts, were not different between control and stretched ROS 17/2.8 or UMR 106-01 cultures after various periods of cyclic stretch. However, phosphorylated forms of Cx43 protein were more abundant in stretched ROS 17/2.8 than in controls. This was associated with increased punctate Cx43-specific immunostain at appositional membranes of stretched cells. Thus, cyclic stretch increases gap junctional communication between osteoblastic cells by modulating intracellular localization of Cx43.