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We have previously obtained compelling proof-of-principle evidence for COX2 gene therapy for fracture repair using integrating retroviral vectors. For this therapy to be suitable for patient uses, a suitable vector with high safety profile must be used. Accordingly, this study sought to evaluate the feasibility of AAV as the vector for this COX2 gene therapy, because AAV raises less safety issues than the retroviral vectors used previously. However, an appropriate AAV serotype is required to provide early increase in and adequate level of COX2 expression that is needed for fracture repair. Herein, we reported that AAV-DJ, an artificial AAV pseudoserotype, is highly effective in delivering COX2 gene to fracture sites in a mouse femoral fracture model. Compared with AAV-2, the use of AAV-DJ led to ~5-fold increase in infectivity in mesenchymal stem cells (MSCs) and provided an earlier and significantly higher level of transgene expression at the fracture site. Injection of this vector at a dose of 7.5 × 10(11) genomic copies led to high COX2 level at the fracture site on day 3 after injections and significantly promoted fracture union at 21 days, as analyzed by radiography and µ-CT. The therapeutic effect appears to involve enhanced osteoblastic differentiation of MSCs and remodeling of callus tissues to laminar bone. This interpretation is supported by the enhanced expression of several key genes participating in the fracture repair process. In conclusion, AAV-DJ is a promising serotype for the AAV-based COX2 gene therapy of fracture repair in humans.

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The findings that sex-specific effects on femoral structure and peak bone mineral density (BMD) are linked to quantitative trait loci (QTL) provide evidence for the involvement of specific genes that contribute to gender variation in skeletal phenotype. Based on previous findings that the BMD QTL in chromosome 1 (Chr 1) exerts a sex-specific effect on femoral structure, we predicted that congenic sublines of mice that carry one or more of the Chr 1 BMD loci would exhibit gender difference in the volumetric BMD (vBMD) phenotype. To test this hypothesis, we compared skeletal parameters of male and female of five C57BL/6J (B6).CAST/EiJ (CAST)-1 congenic sublines of mice that carry overlapping CAST chromosomal segments from the vBMD loci in Chr 1. Femur vBMD measurements were performed by the peripheral quantitative computed tomography in male and female mice at 16 weeks of age. The skeletal phenotype of the C175-185 and C178-185 congenic sublines of mice provided evidence for the presence of the BMD1-4 locus at 178-180 Mb from the centromere. This QTL affects femur vBMD only in female mice. In contrast, CAST chromosomal region carrying BMD1-1 locus increased femur vBMD both in male and female mice. Furthermore, a gender specific effect on BMD of femur mid-shaft region (mid-BMD) was identified at 168-176 Mb in Chr 1 (F=16.49, P=0.0002), while no significant effect was found on total femur BMD (F=2.67, P=0.11). Moreover, this study allowed us to locate a body weight QTL at 168-172 Mb of Chr 1, the effect of this locus was altered in female mice that carry CAST chromosomal segment 168-176 Mb of Chr 1. Based on this study, we conclude that Chr 1 carries at least two vBMD gender-dependent loci; one genetic locus at 178-180 Mb (BMD1-4 locus) which affects both mid-shaft and total femur vBMD in female mice only, and another gender-dependent locus at 168-176 Mb (BMD1-2 locus) which affects femur mid-shaft vBMD in female but not male mice.

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Most previous studies to identify loci involved in bone mineral density (BMD) regulation have used inbred strains with high and low BMD in generating F(2) mice. However, differences in BMD may not be a requirement in selecting parental strains for BMD quantitative trait loci (QTL) studies. In this study, we intended to identify novel QTL using a cross of two strains, MRL/MpJ (MRL) and CAST/EiJ (CAST), both of which exhibit relatively high BMD when compared to previously used strains. In addition, CAST was genetically distinct. We generated 328 MRL x CAST F(2) mice of both sexes and measured femur BMD and periosteal circumference (PC) using peripheral quantitative computed tomography. Whole-genome genotyping was performed with 86 microsatellite markers. A new BMD QTL on chromosome 10 and another suggestive one on chromosome 15 were identified. A significant femur PC QTL identified on chromosome 9 and a suggestive one on chromosome 2 were similar to those detected in MRL x SJL. QTL were also identified for other femur and forearm bone density and bone size phenotypes, some of which were colocalized within the same chromosomal positions as those for femur BMD and femur PC. This study demonstrates the utility of crosses involving inbred strains of mice which exhibit a similar phenotype in QTL identification.

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Four-and-a-half LIM 2 (FHL2) is a member of a family of LIM domain proteins which mediate protein-protein interactions. FHL2 acts as a coactivator and binds to important regulators of bone formation such as insulin-like growth factor binding protein (IGFBP)-5, androgen receptor, and beta-catenin. We hypothesized that FHL2 is an important regulator of bone formation. We evaluated growth and skeletal parameters in FHL2 knockout (KO) and wild-type (WT) mice at 4, 8, and 12 weeks of age. At 4 weeks of age, lack of FHL2 reduced femur, tibia, and total bone mineral content (BMC) and body weight in all mice. A gender-by-treatment interaction (P

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This study sought to confirm that osteoblasts of C3H/HeJ (C3H) mice, which have higher differentiation status and bone-forming ability compared to C57BL/6J (B6) osteoblasts, also have a lower apoptosis level and to test whether the higher differentiation status and bone-forming ability of C3H osteoblasts were related to the lower apoptosis. C3H mice had 50% fewer (P < 0.01) apoptotic osteoblasts on the endocortical bone surface than B6 mice as determined by the TUNEL assay. Primary C3H osteoblasts in cultures also showed a 50% (P < 0.05) lower apoptosis level than B6 osteoblasts assayed by acridine orange/ethidium bromide staining of apoptotic osteoblasts. The lower apoptosis in C3H osteoblasts was accompanied by 22% (P < 0.05) and 56% (P < 0.001) reduction in the activity of total caspases and caspases 3/7, respectively. C3H osteoblasts also displayed greater alkaline phosphatase (ALP) activity (P < 0.001) and higher expression of Cbfa1, type-1 collagen, osteopontin, and osteocalcin genes (P < 0.05 for each). To assess if an association existed between population apoptosis and the differentiation status (ALP-specific activity) and/or bone-forming activity (insoluble collagen synthesis), C3H and B6 osteoblasts were treated with several apoptosis enhancers (tumor necrosis factor-alpha, dexamethasone, lipopolysaccharide, etoposide) and inhibitors (parathyroid hormone, insulin-like growth factor I, transforming growth factor beta1, estradiol). Both ALP (r = -0.61, P < 0.001) and insoluble collagen synthesis (r = -0.61, P < 0.001) were inversely correlated with apoptosis, suggesting that differentiation (maturation) and/or bone-forming activity of these mouse osteoblasts were inversely associated with apoptosis. In conclusion, these studies support the premise that higher bone density and bone formation rate in C3H mice could be due in part to lower apoptosis in C3H osteoblasts.

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We previously found that serum levels of insulin-like growth factor I (IGF-I) and IGF-binding protein (IGFBP)-3, but not IFGBP-2, were associated with bone mineral density (BMD) and the risk of vertebral fractures. The aim of the present study was to investigate the roles of IGFBP-4 and -5 in age-dependent bone loss and vertebral fracture risk in postmenopausal Japanese women and to compare them with those of IGF-I and IGFBP-3. One hundred and ninety-three Japanese women aged 46-88 years (mean 62.5) were enrolled in the cross-sectional study. BMD was measured at the lumbar spine, femoral neck, ultradistal radius (UDR), and total body by dual-energy X-ray absorptiometry. Serum levels of IGFBP-4 and -5 as well as IGF-I and IGFBP-3 were measured by radioimmunoassay. Serum levels of IGF-I, IGFBP-3, and IGFBP-5 declined with age, while serum IGFBP-4 increased with age. Multiple regression analysis was performed between BMD at each skeletal site and serum levels of IGF-I and IGFBPs adjusted for age, body weight, height, and serum creatinine. BMD at the UDR was significantly and positively correlated with all serum levels of IGF-I and IGFBPs measured (P < 0.01), while BMD at the femoral neck was correlated with none of them. Serum IGF-I level was significantly and positively correlated with BMD at all sites except the femoral neck (P < 0.01), while serum IGFBP-3 and -4 levels were significantly and positively correlated with only radial BMD (P < 0.01). Serum IGFBP-5 level was positively correlated with UDR BMD (P < 0.001) and negatively correlated with total BMD (P < 0.05). Serum IGF-I, IGFBP-3, and IFGBP-5 levels were significantly lower in women with vertebral fractures than in those without fractures (mean +/- SD: 97.1 +/- 32.1 vs. 143.9 +/- 40.9 ng/dl, P < 0.0001; 2.18 +/- 1.02 vs. 3.23 +/- 1.07 microg/ml, P < 0.0001; 223.6 +/- 63.3 vs. 246.5 +/- 71.5 ng/ml, P = 0.0330, respectively). When multivariate logistic regression analysis was performed with the presence of vertebral fractures as a dependent variable and serum levels of IGF-I and IGFBPs adjusted for age, body weight, height, serum creatinine, and serum alubumin as independent variables, IGF-I and IGFBP-3 were selected as indices affecting the presence of vertebral fractures [odds ratio (OR) = 0.29, 95% confidential interval (CI) 0.15-0.57 per SD increase, P = 0.0003 and OR = 0.31, 95% CI 0.16-0.61 per SD increase, P = 0.0007, respectively]. To compare the significance values, IGF-I, IGFBP-3, and age were simultaneously added as independent variables in the analysis. IGFBP-3 was more strongly associated with the presence of vertebral fractures than IGF-I and age (P = 0.0006, P = 0.0148, and P = 0.0013, respectively). Thus, after comprehensive measurements of serum levels of IGF-I and IGFBPs, it seems that serum IGF-I level is most efficiently associated with bone mass and that serum IGFBP-3 level is most strongly associated with the presence of vertebral fractures in postmenopausal women among the IGF system components examined.

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The roles of insulin-like growth factors (IGFs) in regulating growth and their modulation by six IGF binding proteins (IGFBP) are well established. IGFBP-5, the most abundant IGFBP stored in bone, is an important regulator of bone formation via IGF-dependent and -independent mechanisms. Two new proteins, four and a half lim (FHL)-2, a transcription modulator that interacts with IGFBP-5, and a disintegrin and metalloprotease (ADAM)-9, an IGFBP-5 protease, have been identified as potential regulators of IGFBP-5 action in bone. We tested the hypothesis that agents which modulate bone formation by regulating IGFBP-5 expression would also regulate FHL-2 and ADAM-9 expression in a coordinated manner. We evaluated the expression of IGFBP-5, FHL-2, and ADAM-9 by real-time reverse transcriptase (RT)-PCR during differentiation of mouse bone marrow stromal cells into osteoblasts and in response to treatment with bone formation modulators in the LSaOS human osteosarcoma cell line. IGFBP-5 and FHL-2 increased 4.3- and 3.0-fold (P < or = 0.01), respectively, during osteoblast differentiation. Dexamethasone (Dex), an inhibitor of bone formation, decreased IGFBP-5 and FHL-2 and increased ADAM-9 in LSaOS cells (P < or = 0.05). Bone morphogenic protein (BMP)-7, a stimulator of bone formation, increased IGFBP-5 and decreased ADAM-9 (P<0.01). To determine if BMP-7 would eliminate Dex inhibition of IGFBP-5, cells were treated with Dex+BMP-7. The BMP-7-induced increase in IGFBP-5 was reduced, but not eliminated, in the presence of Dex (P < or = 0.01), indicating that BMP-7 and Dex may regulate IGFBP-5 via different mechanisms. Transforming growth factor (TGF)-beta, a stimulator of bone formation, increased IGFBP-5 and FHL-2 expression (P < or = 0.01). IGF-I and TNF-alpha decreased expression of ADAM-9 (P<0.05). In conclusion, our findings are consistent with the hypothesis that FHL-2 and ADAM-9 are important modulators of IGFBP-5 actions and are, in part, regulated in a coordinated manner in bone.

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Although it is well established that there is considerable inter-individual variation in the circulating levels of IGF-I in normal, healthy individuals and that a genetic component contributes substantially to this variation, the direct evidence that inter-individual variation in IGF-I contributes to differences in peak bone mineral density (BMD) is lacking. To examine if differences in IGF-I expression could contribute to peak BMD differences, we measured skeletal changes at days 23 (prepubertal), 31 (pubertal) and 56 (postpubertal) in mice with haploinsufficiency of IGF-I (+/-) and corresponding control mice (+/+). Mice (MF1/DBA) heterozygous for the IGF-I knockout allele were bred to generate +/+ and +/- mice (n=18-20 per group). Serum IGF-I was decreased by 23% (P<0.001) in mice with IGF-I haploinsufficiency (+/-) group at day 56 compared with the control (+/+) group. Femoral bone mineral content and BMD, as determined by dual energy X-ray absorptiometry, were reduced by 20% (P<0.001) and 12% respectively in the IGF-I (+/-) group at day 56 compared with the control group. The peripheral quantitative computed tomography measurements at the femoral mid-diaphysis revealed that periosteal circumference (7%, P<0.01) and total volumetric BMD (5%, P<0.05) were decreased significantly in the +/- group compared with the +/+ group. Furthermore, serum IGF-I showed significant positive correlations with both areal BMD (r=0.55) and periosteal circumference (r=0.66) in the pooled data from the +/+ and +/- groups. Our findings that haploinsufficiency of IGF-I caused significant reductions in serum IGF-I level, BMD and bone size, together with the previous findings, are consistent with the notion that genetic variations in IGF-I expression could, in part, contribute to inter-individual differences in peak BMD among a normal population.

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To examine the hypothesis that serum alkaline phosphatase (ALP) levels have a heritable component, we analyzed blood from two inbred strains of mice, MRL/MpJ and SJL, which exhibit 90% difference in total serum ALP activity (268+/-26 vs. 140+/-15 U/l, respectively, P<0.001). A genome-wide scan was carried out using 137 polymorphic markers in 518 F2 female mice. Serum ALP activity in the F2 progeny showed a normal distribution with an estimated heritability of 56%. Genome-wide scan for cosegregation of genetic marker data with serum ALP activity revealed three major quantitative trait loci (QTL), one each on chromosomes 2 (LOD score 3.8), chromosome 6 (LOD score 12.0), and chromosome 14 (LOD score 3.7). In addition, there was one suggestive QTL on chromosome 2 (LOD score of 3.3). In aggregate, these QTLs explain 22.5% of variance in serum ALP between these two strains. Serum ALP showed a moderate but significant correlation with body weight adjusted total body bone mineral density (r=0.12, P=0.0108) and periosteal circumference at midshaft tibia (r=0.15, P=0.0006) in F2 mice. The chromosome 6 locus harboring the major serum ALP QTL also contains a major BMD and bone size QTL, identified earlier, between these two strains of mice; in addition, this QTL is also close to the locus that regulates IGF-I levels (LOD score 8-9) in C3HB6 F2 mice. These common QTLs indicate that the observed difference in ALP and BMD or bone size may be regulated by same loci (or genes). Accordingly, the osteoblast cells isolated from femur and tibia of MRL mice showed a significantly higher number of ALP +ve cells/colony and two- to threefold higher ALP activity (P<0.001) as compared to the cells isolated from SJL mice, thus suggesting that differences in serum ALP between MRL and SJL reflect difference in ALP expression from osteoblasts from these strains of mice. These data suggest that serum ALP levels are genetically determined and correlate with cellular mechanisms that differentiate BMD accrual in these two strains of mice. The findings that ALP and BMD traits share the same loci on chromosome 6 suggest a role for genetic determinants of bone formation in overall BMD accretion.

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Chemical mutagenesis followed by screening for abnormal phenotypes in the mouse holds much promise as a method for revealing gene function. We describe a mouse N-ethyl-N-nitrosourea (ENU) mutagenesis program incorporating a genomewide screen of dominant as well as recessive mutations affecting musculoskeletal disorders in C3H/HeJ mice. In a primary screen, progeny of one-generation dominant mutations (F(1)) and three-generation recessive (F(3)) mutations were screened at 10 weeks of age for musculoskeletal disorders using dual-energy X-ray absorptiometery (DEXA) and biochemical markers affecting bone metabolism, such as osteocalcin, type I collagen breakdown product, skeletal alkaline phosphatase, and insulin-like growth factor I (IGF-I). Abnormal phenotypes were identified as +/-3SD units different from baseline data collected from age- and sex-matched nonmutagenized control mice. A secondary screen at 16 weeks of age, which included peripheral quantitative computed tomography (pQCT) in addition to those parameters described in our primary screen, was used to confirm the abnormal phenotypes observed in the primary screen. The phenodeviant or outlier mice were progeny tested to determine whether their abnormality segregates bimodally in their offspring with the expected 1:1 or 1:3 Mendelian ratio, in dominant and recessive screens, respectively. With the above screening strategy, we were able to identify several mice with quantitative abnormalities in BMD, BMC, bone size, and bone metabolism. We have progeny tested and confirmed four outliers with low BMD, low bone size, and growth-related abnormality. Our results indicate that the magnitude of change in quantitative phenotypes in the ENU-mutagenized progeny was between 10 and 15%, and hence, the yield of outliers was dependent on the precision of the methods. So far, this ENU mutagenesis program has identified four outliers that can undergo positional cloning.

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To test the hypothesis that periosteal circumference (PC), which is associated with bone size through cross-sectional moment of inertia (CMI), has heritable components, we performed a linkage analysis using 633 MRL/SJL F(2) mice that have 14% difference in mean PC. PC was determined in femurs by use of peripheral quantitative computerized tomography (pQCT). The genome-wide scan identified nine QTL for PC adjusted by body weight on chromosomes 1 (2 QTL), 2 (2 QTL), 8, 11, 15, 17, and X, which accounted for 38.6% of phenotype variance. QTL on chromosomes 1 (D1Mit33), 8 (D8Mit125), 15 (D15Mit 62), 17 (D17Mit176), and X (DXMit208) were unique for PC adjusted by body weight and femur length, while the remaining PC QTL were shared with body weight but not femur length. Four epistatic interactions were identified which accounted for 37.6% of phenotype variance. There was also evidence of pleiotropic effects on chromosome 11 among four size phenotypes (PC, body length, body weight, bone mineral density, and muscle size), which may represent a common genetic mechanism that may regulate bone size and body size.

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Traditionally, binding proteins are known to regulate the activity of ligands by prolonging their half-life, and insulin-like growth factor (IGF)-binding proteins (IGFBPs) are no exception to this. The IGFBP family contains six high-affinity members with variable functions and mechanisms of actions. In addition to functioning as simple carrier proteins, IGFBPs in serum function to regulate the endocrine actions of IGFs by regulating the amount of IGF available to bind to signaling IGF-I receptors, whereas locally produced IGFBPs act as autocrine/paracrine regulators of IGF action. Furthermore, recent in vitro and in vivo findings that IGFBPs function independently of the IGFs as growth modulators are particularly exciting. Regarding the role of IGFBPs as ligand-independent growth modulators, our recent data that IGFBP-5 stimulates markers of bone formation in osteoblasts lacking functional IGFs provide evidence that IGFBP-5 itself is a growth factor that can act independently of IGFs to regulate bone formation. In terms of the mechanism by which certain IGFBPs mediate their effects in a ligand-independent manner, the binding of IGFBP to its putative receptor on the cell membrane may stimulate the signaling pathway independent of an IGF receptor, to mediate the effects of IGFBPs in certain target cell types. IGFBPs may also exert IGF-independent effects by transcriptional activation of genes by IGFBPs transported into the nucleus via their nuclear localization signal. In conclusion, IGFBPs are unusually pleotrophic molecules with functions ranging from the traditional role of prolonging the half-life of the IGFs to functioning as growth factors independent of the IGFs. In this regard, it was surprising to find that the human genome contains only about 35 000 genes. One mechanism to account for such complexity with a relatively small number of genes is strikingly illustrated by the multifunctional IGFBP class of proteins.

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Bone mineral density variation is a highly heritable trait and is the best predictor of skeletal fragility. Total skeletal density was determined by PIXIMUS, and femur density was determined by pQCT. The data were analyzed for quantitative trait loci (QTL) to determine if bone density at a specific skeletal site (femur) would identify new gene loci or the same gene loci as total body (PIXIMUS). In order to show concordance and differences in QTL for total body bone density versus femur bone density, we performed a genome-wide scan from 633 (MRL x SJL) F2 mice. The bone mineral density (BMD) data from pQCT were used to identify nine QTL on chromosomes 1, 3, 4, 9, 12, 17, and 18, while nine QTL on chromosomes 1, 2, 4, 9, 11, 14, and 15 were identified by PIXIMUSdata, accounting for 32.5% and 30.4% variation in F2 mice, respectively. QTL on chromosomes 1, 2, 3, 9, 11, 12, 14, 15, 17, and 18 are unique to our study, as they have never been described before. Chromosome 1 (D1Mit33 and D1Mit362) had similar QTL between pQCT and PIXIMUS. Several QTL were identified for both femur and total body BMD but only two QTL were common for both of these phenotypes. This suggests that genes regulating bone density differ depending on the skeletal site analyzed.

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Our knowledge of the developmental changes in the concentration of serum and bone osteocalcin (OC) is limited. To investigate the interrelationship between skeletal and circulatory OC during acquisition of peak bone density in mice, we examined the temporal changes in the concentration of serum and bone OC from 3 to 12 weeks of age between C3H/HeJ (C3H) and C57BL/6J (B6), two commonly used inbred strains of mice with a large difference in bone density. We have demonstrated an increase in bone and decrease in serum OC during the acquisition of peak bone density in C3H and B6 mice which parallels an increase in bone mineral density. These two strains exhibited differential changes in the concentration of OC. C3H mice retained more OC in bone and secreted less into serum compared with B6, which coincides with the large differences in bone density between these two strains. These opposite changes of OC levels in bone and serum between C3H and B6 stress the importance of defining the genetic mechanisms underlying the differences in OC metabolism, differences that could be relevant to the acquisition and maintenance of bone mass in mice.

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We isolated and characterized a peptide fragment corresponding to amino acid sequence 14-28 of human osteocalcin in urine from Paget's disease, and developed a polyclonal antibody reactive to this peptide in urine. We used this antibody to measure urinary fragments of osteocalcin and compared to efficacy of the urinary osteocalcin assay with a serum osteocalcin (sOC) assay (ELISA-Osteo, Cis-Bio International) to monitor the short-term changes in bone turnover in response to alendronate treatment. The synthetic peptide-based urinary osteocalcin (uOC) radioimmunoassay (RIA) showed an analytical sensitivity of 6.25 ng/mL, standard curve range of 3.12-400 ng/mL, and mean intra- (n = 20) and interassay (n = 30) coefficient of variation (CV) of <15%. Urine osteocalcin concentrations in postmenopausal osteoporotic patients were approximately 90% higher than in normal premenopausal controls. Series of 24 h urine and matched serum samples were collected at baseline, 30 days, and 90 days after treatment of postmenopausal osteoporotic patients with daily dose of 10 mg alendronate. We measured urinary osteocalcin (uOc) levels and urinary N-telopeptide (uNTx, Ostex) in urine samples and serum N-telopeptide (sNTx), C-telopeptide (sCTx, Osteometer), serum osteocalcin (sOC) as well as bone-specific alkaline phosphatase (sALP) (Alkphose-B, Metra Biosystems) in serum samples. The percent change data obtained between baseline and 30 days (n = 18) posttreatment suggested a rapid decline in uOC concentration (-27%, p < 0.01) in response to alendronate treatment, as compared with a marginal and nonsignificant decrease in sOC (-7.2%, p = 0.417) or sALP (-3.4%, p = 0.689), two specific markers of bone formation. As expected, due to the coupling of bone formation and bone resorption, the concentration of all markers showed a 30%-45% decline compared with baseline values after 90 days (n = 16) of treatment. Correlation of markers after a 30 day treatment with alendronate revealed a higher correlation (r = 0.61, p < 0.01) between uOC and uNTx, as compared with sOC (r = 0.03, p = 0.447) or sALP (r = -0.14, p = 0.295) with uNTx. Similarly, correlation coefficients with r values between 0.48 and 0.55 (p < 0.05) were observed between uOC, sNTx, and sCTx, whereas no significant correlation was observed between sOC and sNTx or sCTx. These results provide indirect evidence that fragments measured by the urine assay probably originated from bone resorption, and suggest that the uOC assay could be used to assess short-term changes in bone metabolism with regard to osteocalcin.

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In order to develop a successful gene therapy system for the healing of bone defects, we developed a murine leukemia virus (MLV)-based retroviral system expressing the human bone morphogenetic protein (BMP) 4 transgene with high transduction efficiency. The bone formation potential of BMP4 transduced cells was tested by embedding 2.5 x 10(6) transduced stromal cells in a gelatin matrix that was then placed in a critical size defect in calvariae of syngenic rats. Gelatin matrix without cells or with untransduced stromal cells were the two control groups. The defect area was completely filled with new bone in experimental rats after 4 weeks, while limited bone formation occurred in either control group. Bone mineral density (BMD) of the defect in the gene therapy group was 67.8 +/- 5.7 mg/cm(2) (mean +/- s.d., n = 4), which was 119 +/- 10% of the control BMD of bone surrounding the defect (57.2 +/- 1.5 mg/cm(2)). In contrast, BMD of rats implanted with untransduced stromal cells was five-fold lower (13.8 +/- 7.4 mg/cm(2), P < 0.001). Time course studies revealed that there was a linear increase in BMD between 2-4 weeks after inoculation of the critical size defect with 2.5 x 10(6) implanted BMP4 cells. In conclusion, the retroviral-based BMP4 gene therapy system that we have developed has the potential for regeneration of large skeletal defects.

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Insulin-like growth factor-1 (IGF-1) increases both bone formation and bone resorption processes. To test the hypothesis that treatment with an antiresorber along with IGF-1, during the pubertal growth phase, would be more effective than IGF-1 alone to increase peak bone mass, we used an IGF-1 MIDI mouse model, which exhibits a >60% reduction in circulating IGF-1 levels. We first determined an optimal IGF-1 delivery by evaluating IGF-1 administration (2 mg/kg body weight/day) by either a single daily injection, three daily injections, or by continuous delivery via a minipump during puberty. Of the three regimens, the three daily IGF-1 injections and IGF-1 through a minipump produced a significant increase in total body bone mineral density (BMD) (6.0% and 4.4%, respectively) and in femoral BMD (4.3% and 6.2%, respectively) compared with the control group. Single subcutaneous (s.c.) administration did not increase BMD. We chose IGF-1 administration three times daily for testing the combined effects of IGF-1 and alendronate (100 microg/kg per day). The treatment of IGF-1 + alendronate for a period of 2 weeks increased total body BMD at 1 week and 3 weeks after treatment (21.1% and 20.5%, respectively) and femoral BMD by 29% at 3 weeks after treatment. These increases were significantly greater than those produced by IGF-1 alone. IGF-1, but not alendronate, increased bone length. IGF-1 and/or alendronate increased both periosteal and endosteal circumference. Combined treatment caused a greater increase in the total body bone mineral content (BMC) and periosteal circumference compared with individual treatment with IGF-1 or alendronate. Our data demonstrate that: (1) inhibition of bone turnover during puberty increases net bone density; and (2) combined treatment with IGF-1 and alendronate is more effective than IGF-1 or alendronate alone in increasing peak bone mass in an IGF-1-deficient MIDI mouse model.

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One QTL and genes and phenotypes have been localized in the region between 92 cM and 95cM of mouse chromosome 1. The QTL locus contributes to approximately 40% of the variation of the peak bone density between C57BL/6J (B6) and CAST/EiJ (CAST) strains. Other loci located in this chromosomal region include a neural tube defect mutant loop-tail (Lp), a lymphocyte-stimulating determinant (Lsd), and the Transgelin 2 (Tagln 2). The human chromosome region homologous to this region is 1q21-23, which also contains a QTL locus for high bone mineral density (BMD). Furthermore, it has been reported that this region may have duplicated several times in the mouse genome. Therefore, genomic sequencing of this region will provide important information for mouse genome structure, for positional cloning of mouse genes, and for the study of human homologous genes. In order to provide a suitable template for genomic sequencing by the NIH-sponsored genomic centers, we have constructed a BAC contig of this region using the RPCI-23 library. We have also identified the currently available mouse genomic sequences localized in our BAC contig. Further analysis of these sequences and BAC clones indicated a high frequency of repetitive sequences within this chromosomal area. This region also contains L1 retrotransposon sequences, providing a potential mechanism for the repetitive sequences described in the literature.

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The fluoroaluminate (AlF(4)(-)) ion and sodium fluoride (NaF) have previously been shown to be bone cell mitogens. This study sought to determine whether the bone cell mitogenic action of AlF(4)(-) and/or NaF would involve the insulin-like growth factor (IGF) regulatory system. We evaluated the effect of mitogenic doses of AlF(4)(-) and NaF on the mRNA levels and the protein level (in conditioned media [CM]) of several components of the IGF system (i.e., IGF-2, IGF binding protein [IGFBP]-4, and IGFBP-5) in human TE85 osteosarcoma cells. Aluminum fluoride (AlF(3)) was included for comparison. NaF, AlF(3), and AlF(4)(-), each at 50-100 micromol/L, increased [3H]thymidine incorporation in TE85 cells. Mitogenic concentrations of AlF(3) and AlF(4)(-): (1) increased the mRNA (up to twofold after 24 h treatment) and protein (in CM) levels (up to 2.5-fold after 48 h treatment) of IGF-2; (2) increased the mRNA level (twofold) and the protein level in CM (up to threefold) of stimulatory IGFBP-5; and (3) either reduced slightly or had no effect on the mRNA and protein (in CM) levels of the inhibitory IGFBP-4. Conversely, mitogenic concentrations of NaF had no significant effects on the protein (in CM) or mRNA level of IGF-2, IGFBP-4, or IGFBP-5. The addition of an inhibitory concentration of IGFBP-4 completely abolished the bone cell mitogenic activity of AlF(3) and AlF(4)(-) but not that of NaF. The findings of this study provide strong evidence that the bone cell mitogenic activity of AlF(4)(-) and AlF(3), but not that of NaF, is mediated by the upregulation of the IGF regulatory system.

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