Opposing effects of Sca-1(+) cell-based systemic FGF2 gene transfer strategy on lumbar versus caudal vertebrae in the mouse.

Our previous work showed that a Sca-1(+) cell-based FGF2 therapy was capable of promoting robust increases in trabecular bone formation and connectivity on the endosteum of long bones. Past work reported that administration of FGF2 protein promoted bone formation in red marrow but not in yellow marrow. The issue as to whether the Sca-1(+) cell-based FGF2 therapy is effective in yellow marrow is highly relevant to its clinical potential for osteoporosis, as most red marrows in a person of an advanced age are converted to yellow marrows. Accordingly, this study sought to compare the osteogenic effects of this stem cell-based FGF2 therapy on red marrow-filled lumbar vertebrae with those on yellow marrow-filled caudal vertebrae of young adult W(41)/W(41) mice. The Sca-1(+) cell-based FGF2 therapy drastically increased trabecular bone formation in lumbar vertebrae, but the therapy not only did not promote bone formation but instead caused substantial loss of trabecular bone in caudal vertebrae. The lack of an osteogenic response was not due to insufficient engraftment of FGF2-expressing Sca-1(+) cells or inadequate FGF2 expression in caudal vertebrae. Previous studies have demonstrated that recipient mice of this stem cell-based FGF2 therapy developed secondary hyperparathyroidism and increased bone resorption. Thus, the loss of bone mass in caudal vertebrae might in part be due to an increase in resorption without a corresponding increase in bone formation. In conclusion, the Sca-1(+) cell-based FGF2 therapy is osteogenic in red marrow but not in yellow marrow.

Chemical mutagenesis induced two high bone density mouse mutants map to a concordant distal chromosome 4 locus.

Phenotype-driven mutagenesis approach in the mouse holds much promise as a method for revealing gene function. Earlier, we have described an N-ethyl-N-nitrosourea (ENU) mutagenesis screen to create genome-wide dominant mutations in the mouse model. Using this approach, we describe identification of two high bone density mutants in C57BL/6J (B6) background. The mutants, named as 12184 and 12137, have been bred more than five generations with wild-type B6 mice, each producing >200 backcross progeny. The average total body areal bone mineral density (aBMD) was 13-17% higher in backcrossed progeny from both mutant lines between 6 and 10 weeks of age, as compared to wild-type (WT) B6 mice (n=60-107). At 3 weeks of age the aBMD of mutant progeny was not significantly affected as compared to WT B6 mice. Data from 10- and 16-week old progeny show that increased aBMD was mainly related to a 14-20% higher bone mineral content, whereas bone size was marginally increased. In addition, the average volumetric BMD (vBMD) was 5-15% higher at the midshaft tibia or femur, as compared to WT mice. Histomorphometric analysis revealed that bone resorption was 23-34% reduced in both mutant mice. Consistent with histomorphometry data, the mRNA expression of genes that regulate osteoclast differentiation and survival were altered in the 12137 mutant mice. To determine the chromosomal location of the ENU mutation, we intercrossed both mutant lines with C3H/HeJ (C3H) mice to generate B6C3H F2 mice (n=164 for line 12137 and n=137 F2 for line 12184). Interval mapping using 60 microsatellite markers and aBMD phenotype revealed only one significant or suggestive linkage on chromosome 4. Since body weight was significantly higher in mutant lines, we also used body weight as additive and interactive covariate for interval mapping; both analyses showed higher LOD scores for both 12137 and 12184 mutants without affecting the chromosomal location. The large phenotype in the mutant mice compared to generally observed QTL effects (<5%) would increase the probability of identifying the mutant gene.

Genetic regulation of femoral bone mineral density: complexity of sex effect in chromosome 1 revealed by congenic sublines of mice.

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.

Detecting novel bone density and bone size quantitative trait loci using a cross of MRL/MpJ and CAST/EiJ inbred mice.

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.

High osteoblastic activity in C3H/HeJ mice compared to C57BL/6J mice is associated with low apoptosis in C3H/HeJ osteoblasts.

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.

A genomewide screening of N-ethyl-N-nitrosourea-mutagenized mice for musculoskeletal phenotypes.

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.

Ex vivo gene therapy with stromal cells transduced with a retroviral vector containing the BMP4 gene completely heals critical size calvarial defect in rats.

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.

Effect of insulin-like growth factor-1 (IGF-1) plus alendronate on bone density during puberty in IGF-1-deficient MIDI mice.

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.

Regulation of bone volume is different in the metaphyses of the femur and vertebra of C3H/HeJ and C57BL/6J mice.

The C3H/HeJ (C3H) mice exhibited a greater bone formation rate (BFR) and a greater mineral apposition rate (MAR) in the cortical bone of the midshafts of the femur and tibia than did C57BL/6J (B6) mice. This study sought to determine if these strain-related differences would also be observed in cancellous bone. Metaphyses of the femur and lumbar vertebra (L5-6) from C3H and B6 mice, 6 and 12 weeks of age, were analyzed by histomorphometry. Similar to cortical bone, the bone volume in the femoral metaphysis of C3H mice was greater (by 54% and 65%, respectively) than that of B6 mice at both 6 and 12 weeks of age. Higher BFR and mineral apposition rate (MAR) contributed to the higher bone volume in the C3H mice compared with the B6 mice. In contrast, bone volume (by 59% and 13%, respectively, p < 0.001) and trabecular number (by 55% and 35%, respectively, p < 0.001) in the vertebrae were lower in the C3H mice than in B6 mice at 6 and 12 weeks of age. At 6 weeks of age, MAR was higher (by 43%, p = 0.004) in C3H mice, but because of a low trabecular number, the BFR (by 37%, p = 0.026) and tetracycline-labeled bone surface (by 52%, p < 0.001) per tissue were lower in the vertebrae of C3H mice than B6 mice. The low bone volume in vertebrae of C3H mice was probably not due to a higher bone resorption, because the osteoclast number (by 55%, p < 0.001) and eroded surface (by 61%, p <0.001) per tissue area in the C3H mice were also lower in B6 mice. At 12 weeks, the trabecular thickness had increased (by 36%, p < 0.001) in the C3H mice and the difference in bone volume between strains was less than that at 6 weeks. These contrasting and apparently opposing strain-related differences in trabecular bone parameters between femur and vertebra in these two mouse strains suggest that the genetic regulation of bone volume in the metaphyses of different skeletal sites is different between C3H and B6 mice.

Mouse genetic model for bone strength and size phenotypes: NZB/B1NJ and RF/J inbred strains.

The relationships of bone size, bone strength, and bone formation were investigated in two strains of mice, NZB/B1NJ and RF/J. Measurement of the femur midshaft size by peripheral quantitative computed tomography (pQCT) showed that the RF/J mice had a 32% greater cross-sectional area than NZB/B1NJ mice at 10 weeks of age, and a 38% greater cross-sectional area at 22 weeks of age. Body weight in the RF/J mice was 10% higher at 10 weeks but 9% lower at 22 weeks. Bone strength was determined by a three-point bending method. In agreement with the difference in bone cross-sectional area, the femurs of the RF/J mice were stronger (80% greater) and stiffer (80% greater) than the bones of the NZB/B1NJ mice. To determine whether periosteal bone formation played a role in the greater size of the RF/J mice, the mice were injected with tetracycline to label areas of new bone formation. Histomorphometrical analysis of the femur diaphysis demonstrated higher rates of periosteal bone formation (131% greater) and of periosteal forming surface (81% greater) in RF/J than in NZB/B1NJ mice. We conclude that a high rate of periosteal bone formation increases bone size and strength in RF/J mice when compared with NZB/B1NJ mice. The NZB/B1NJ and RF/J mice should be an excellent model to investigate the genes that regulate femur size and strength.

Development of an MFG-based retroviral vector system for secretion of high levels of functionally active human BMP4.

We sought to develop a retroviral vector system that would produce secretion of high levels of bone morphogenetic protein (BMP)-4 by optimizing the expression construct and developing an improved retroviral vector. Replacement of the propeptide domain of BMP4 with that of BMP2 increased the secretion level of mature BMP4 protein in transduced cells. The intact BMP2 pro-peptide sequence was essential, as deletion of a small part of the propeptide sequence of BMP2 from the BMP2/4 hybrid construct diminished BMP4 expression and secretion. Addition of a hemaglutinin tag to the carboxy terminus of BMP4 abolished the bioactivity of secreted BMP4. Transduction of rat marrow stromal cells (and fibroblasts) with an MFG-based retroviral vector pseudotyped with VSV-G envelope containing this BMP2/4 hybrid expression construct led to secretion of very high levels of mature BMP4 in conditioned medium (up to 1 microg/10(6) cells/24 hours). The secreted BMP4 was biologically active, as it induced alkaline phosphatase expression in C2C12 cells. The transduced rat marrow stromal cells expressing mature BMP4 induced de novo ectopic bone formation in syngenic immune-competent rats. We have developed an MFG-based retroviral vector system that causes secretion of high levels of functionally active human BMP4 protein.

Postnatal and pubertal skeletal changes contribute predominantly to the differences in peak bone density between C3H/HeJ and C57BL/6J mice.

Previous studies have shown that 60-70% of variance in peak bone density is determined genetically. The higher the peak bone density, the less likely an individual is to eventually develop osteoporosis. Therefore, the amount of bone accrued during postnatal and pubertal growth is an important determining factor in the development of osteoporosis. We evaluated the contribution of skeletal changes before, during, and after puberty to the development of peak bone density in C3H/HeJ (C3H) and C57BL/6J (B6) mice. Volumetric bone density and geometric parameters at the middiaphysis of femora were measured by peripheral quantitative computed tomography (pQCT) from days 7 to 56. Additionally, biochemical markers of bone remodeling in serum and bone extracts were quantified. Both B6 and C3H mice showed similar body and femoral weights. B6 mice had greater middiaphyseal total bone area and thinner cortices than did C3H mice. Within strains, males had thicker cortices than did females. C3H mice accumulated more minerals throughout the study, with the most rapid accumulation occurring postnatally (days 7-23) and during pubertal maturation (days 23-31). C3H mice had higher volumetric bone density as early as day 7, compared with B6 mice. Higher serum insulin-like growth factor I (IGF-I) was present in C3H mice postnatally at day 7 and day 14. Until day 31, B6 male and female mice had significantly higher serum osteocalcin than C3H male and female mice, respectively. Alkaline phosphatase (ALP) was found to be significantly higher in the bone extract of C3H mice compared with B6 mice at day 14. These data are consistent with and support the hypothesis that the greater amount of bone accrued during postnatal and pubertal growth in C3H mice compared with B6 mice may be caused by increased cortical thickness, increased endosteal bone formation, and decreased endosteal bone resorption.

Comparison of bone formation responses to parathyroid hormone(1-34), (1-31), and (2-34) in mice.

In this study we used a mouse model system to compare the in vivo effects of parathyroid hormone(1-34) [PTH(1-34)] with that of PTH(1-31) or PTH(2-34) analogs. Daily subcutaneous administration of PTH(1-34) for 15 days caused a dose-dependent increase in the serum osteocalcin level and bone extract alkaline phosphatase activity, markers of bone formation. PTH(2-34) was much less potent, whereas PTH(1-31) was equipotent in stimulating bone formation parameters in mice. PTH(1-34) caused significant increases in serum calcium (after 4 h) and tartrate-resistant acid phosphatase activity in bone extract (after 4 h), whereas PTH(2-34) and PTH(1-31) were less potent. Because PTH(1-31) caused a smaller increase in bone resorption parameters compared to PTH(1-34), despite similar effects on bone formation parameters, we evaluated the long-term anabolic effects of PTH(1-31) and PTH(1-34) in mice. Weekly evaluations of serum osteocalcin levels demonstrated that daily injections of PTH(1-34) and PTH(1-31) at 80 microg/kg body weight increased serum osteocalcin levels within 1 week of the start of treatment, which were maintained during the entire 22 week treatment. Assessment of bone density at the end of the treatment period with peripheral quantitated computed tomography (pQCT) revealed that PTH(1-34) caused a significantly greater increase in femoral bone density compared to PTH(1-31) at the middiaphysis (18% vs. 9% over vehicle control; p < 0.001). Both PTH(1-34) and PTH(1-31) increased periosteal circumference compared to vehicle (p < 0.01) without a significant difference between the two treatments. In contrast, PTH(1-34) caused a significantly greater reduction in endosteal circumference than PTH(1-31) (p < 0.001). Both analogs significantly increased maximum load and area of moment of inertia over the vehicle group. In conclusion, our findings suggest that PTH(1-34) and PTH(1-31) may exhibit different anabolic effects at the periosteum vs. endosteum in the long bones of mice.

Development and application of a synthetic peptide-based osteocalcin assay for the measurement of bone formation in mouse serum.

The mouse is frequently used as an animal model to study skeletal mechanisms relevant to humans. Biochemical markers of bone formation and resorption provide one of the key parameters for assessing skeletal metabolism. One biochemical marker that has proven to be useful in the studies of mouse skeletal metabolism is osteocalcin. Assay for osteocalcin is available in the mouse. The present study describes development of an osteocalcin radioimmunoassay (RIA) using a synthetic peptide. Intact osteocalcin purified from mouse bone extracts shows parallel displacement with synthetic peptide. Sensitivity of the RIA was 19 ng/ml. The average (n = 9) intra- and interassay coefficient of variation for two controls was less than 10%; the averaged recoveries were 106%. The osteocalcin concentration measured by peptide RIA shows a high correlation (r = 0.88, n = 117, P < 0.0001) with an intact osteocalcin assay. In addition, when the intact assay and peptide assays were applied to evaluate skeletal perturbation, similar results were obtained. Accordingly, osteocalcin levels measured by both intact and peptide-based RIA in 8-week C57BL/6J (n = 8) mice treated with PTH 1-34 were twofold higher compared with the vehicle-treated control group. Further studies of the application of the peptide-based RIA for osteocalcin revealed that osteocalcin levels in 4-week postovariectomized (OVX) C57BL/6N mice (n = 10) were 80% higher than the sham-operated (n = 10) mice receiving vehicle. OVX mice receiving weekly injections of estradiol (400 microg/kg body weight) were 38% lower compared with the OVX group treated with vehicle. In conclusion, the peptide-based RIA has analytical and a discriminative power similar to that of the intact osteocalcin assay but has the advantage that the resources for this assay are much easier to accrue.

TWIST, a basic helix-loop-helix transcription factor, can regulate the human osteogenic lineage.

Basic helix-loop-helix (bHLH) transcription factors have been shown to play an important role in controlling cell type determination and differentiation. TWIST, a member of the bHLH transcription factor family, is involved in the development of mesodermally derived tissue, including the skeleton. We examined the role of human TWIST in osteoblast metabolism using stable expression of sense and antisense TWIST in human osteoblast HSaOS-2 cells. Changes in morphology and osteogenic phenotype characterized these stable clones. Cells that overexpressed TWIST exhibited a spindle shaped morphology, reduced levels of alkaline phosphatase, a reduced proliferation rate, and failed to respond to basic fibroblast growth factor (bFGF). In contrast, those that underexpressed TWIST demonstrated a cuboidal epithelial-like morphology characteristic of differentiated osteoblasts. TWIST antisense cells exhibited increased levels of alkaline phosphatase and type I collagen mRNA, initiated osteopontin mRNA expression, and had a reduced proliferation rate. These results indicate that TWIST overexpressing cells may de-differentiate and remain in an osteoprogenitor-like state, and antisense TWIST cells progress to a more differentiated mature osteoblast-like state. Therefore, the level of TWIST can influence osteogenic gene expression and may act as a master switch in initiating bone cell differentiation by regulating the osteogenic cell lineage.

Histomorphometric studies show that bone formation and bone mineral apposition rates are greater in C3H/HeJ (high-density) than C57BL/6J (low-density) mice during growth.

High-density C3H/HeJ (C3H) and low-density C57BL/6J (B6) mice, with femoral bone density differing by 50%, were chosen as a model to investigate the mechanisms controlling peak bone density and to map peak bone density genes. The present longitudinal study was undertaken to further establish the bone biologic phenotypes of these two inbred strains of mice. To evaluate phenotypic differences in bone formation parameters in C3H and B6 mice between the ages of 6 and 26 weeks, undecalcified ground sections from the diaphyses of the tibia and femur were prepared from mice receiving two injections of tetracycline. Histomorphometric analyses revealed that the cortical bone area was significantly greater (16%-56%, p < 0.001) in both the femur and tibia of the C3H mice than in the B6 mice at all timepoints. This difference in cortical bone area was due to significantly smaller medullary areas in the C3H mice than in the B6 mice. The bone formation rates (BFR) at the endosteum in both the femur and tibia were significantly greater (28%-117%,p < 0.001) in the young C3H mice (6-12 weeks old) than in B6 mice. The higher bone formation in C3H mice was associated with higher values of the bone mineral apposition rate (25%-94%, p < 0.001), and was not associated with higher values of the forming surface length as measured by tetracycline label length. Similar interstrain differences in mineral apposition and bone formation rates were observed in the periosteum of the femur and tibia. In conclusion, the greater bone area in the high-density C3H mice vs. the low-density B6 mice was, in part, due to the greater periosteal and endosteal bone formation rates during growth in the C3H mice. Because the C3H and B6 mice were maintained under identical environmental conditions (diet, lighting, etc.), the observed interstrain differences in bone parameters were the result of the action of genetic factors. Consequently, these two inbred strains of mice are suitable as a model to identify genetic factors responsible for high bone formation rates.

Osteoclast formation in bone marrow cultures from two inbred strains of mice with different bone densities.

For the purpose of identifying genes that affect bone volume, we previously identified two inbred mouse strains (C57BL/6J and C3H/HeJ) with large differences in femoral bone density and medullary cavity volume. The lower density and larger medullary cavity volume in C57BL/6J mice could result from either decreased formation or increased resorption or both. We recently reported evidence suggesting that bone formation was increased in vivo and that osteoblast progenitor cells are more numerous in the bone marrow of C3H/HeJ compared with C57BL/6J mice. In the present study, we determined whether osteoclast numbers in vivo and osteoclast formation from bone marrow cells in vitro might also differ between the two mouse strains. We have found that the number of osteoclasts on bone surfaces of distal humerus secondary spongiosa was 2-fold higher in 5.5-week-old C57BL/6J mice than in C3H/HeJ mice of the same age (p < 0.001). Bone marrow cells of C57BL/6J mice cocultured with Swiss/Webster mouse osteoblasts consistently produced more osteoclasts than did C3H/HeJ bone marrow cells at all ages tested from 3.5-14 weeks of age (p < 0.001). Osteoclast formation was also greater from spleen cells of 3.5-week-old C57BL/6J mice than C3H/HeJ mice. The distribution of nuclei per osteoclast and the 1, 25-dihydroxyvitamin D3 dose dependence of osteoclast production from bone marrow cells were similar. Osteoclasts that developed from both C57BL/6J and C3H/HeJ marrow cells formed pits in dentin slices. Cultures from C57BL/6J marrow cells formed 2.5-fold more pits than cultures from C3H/HeJ marrow cells (p < 0.02). We compared the abilities of C57BL/6J and C3H/HeJ osteoblasts to support osteoclast formation. When bone marrow cells from either C57BL/6J or C3H/HeJ mice were cocultured with osteoblasts from either C57BL/6J or C3H/HeJ newborn calvaria, the strain from which osteoblasts were derived did not affect the number of osteoclasts formed from marrow cells of either strain. Together, these observations suggest that genes affecting the bone marrow osteoclast precursor population may contribute to the relative differences in bone density that occur between C3H/HeJ and C57BL/6J mouse strains.

Insulin-like growth factor (IGF)-I, -II, IGF binding proteins (IGFBP)-3, -4, and -5 levels in the conditioned media of normal human bone cells are skeletal site-dependent.

The skeleton in its function of affording strength and support to the body is subject to differential mechanical loading which has been implicated to mediate some of its effects on bone formation via the insulin-like growth factors (IGFs), which are important regulators of bone metabolism. We, therefore, sought to conduct the present study with the hypothesis that the skeletal site-dependent differences in mechanical loading and other variables including stage of osteoblast differentiation would be associated with site-specific differences in the production of the IGF system components. To test this hypothesis, conditioned media (CM) from normal human bone cells (control and IGF-II-treated 48-h cultures) from five different skeletal sites were obtained and assayed for IGF-I, IGF-II (following separation of IGF binding proteins [IGFBPs]), IGFBP-3, IGFBP-4, and IGFBP-5 protein levels employing specific radioimmunoassays for each protein. IGF-I levels were lower than any other IGF system component but were significantly different between the various sites tested. IGF-II levels were greatest in the CM from mandibular cells, followed by calvarial and rib cells, and least in the marrow stromal cells. IGFBP-3 levels were highest in the CM of vertebral cells and lowest in the CM of rib and mandibular cells. The relative abundance of IGFBP-4 in decreasing order was observed in mandibular, calvarial, vertebral, rib, and stromal cells' CM. IGFBP-5 was produced maximally by the calvarial cells, followed by the mandibular, vertebral, stromal, and rib cells. IGFBP-4 appeared to be the IGF system component most abundantly produced by all the cell types from the skeletal sites tested. On a molar basis, the IGFBPs in general were estimated to be produced at a higher magnitude than the IGFs. These findings indicate that there are skeletal site-dependent differences in the production of IGF system components and suggest that the regulation of bone metabolism may vary at the various skeletal sites.

Mitogenic action of hydrochlorothiazide on human osteoblasts in vitro: requirement for platelet-derived growth factor.

Long-term use of hydrochlorothiazide (HCTZ), a common diuretic agent for hypertension, has been associated with increased bone density and reduced hip fracture rates in patients. In this study, we sought to examine whether HCTZ has an anabolic effect on the proliferation of human osteoblasts (derived from either vertebrate or rib bone samples) in vitro. Cell proliferation was determined by [3H]thymidine incorporation and cell number counting. In medium supplemented with 1% bovine calf serum, HCTZ significantly and reproducibly increased [3H]thymidine incorporation and cell number. The stimulatory effect was dose dependent in a biphasic manner, with the maximal stimulation (approximately 60% above control, P < 0.001) seen at 1 microM HCTZ. In fresh serum-free medium, HCTZ was ineffective as a bone cell mitogen, indicating that the bone cell mitogenic activity of HCTZ required a serum growth factor (GF). HCTZ at doses greater than 10 microM was inhibitory in the presence or the absence of serum, presumably because of the cytotoxic effects. The serum requirement for the bone cell mitogenic activity of HCTZ could be replaced with a conditioned medium (conditioned with normal human osteoblasts for 24 hours), or with a mitogenic dose (1 ng/ml) of PDGF. The GF requirement was specific for PDGF, because other bone cell-derived growth factors (i.e., TGFbeta, IGF-I, IGF-II, and bFGF) were unable to replace serum for the bone cell mitogenic activity of HCTZ. In summary, this study shows that (1) HCTZ stimulated the proliferation of normal, untransformed, human osteoblasts in vitro; (2) the bone cell mitogenic effect of HCTZ required the presence of a serum GF; (3) the serum requirement could be replaced with a bone cell GF in conditioned medium; (4) the GF requirement was specific for PDGF. In conclusion, we have demonstrated for the first time that HCTZ has a direct anabolic effect on human osteoblasts in vitro, and that the mitogenic activity is dependent on the presence of PDGF. Because increased bone cell proliferation is a key determinant of bone formation, these observations raise the interesting possibility that HCTZ could act directly on bone cells to stimulate bone formation in patients.

Truncation of the amino terminus of PTH alters its anabolic activity on bone in vivo.

In vitro studies of parathyroid hormone (PTH) structure and function have suggested that the anabolic effect of PTH on bone requires the presence of amino acid residues 28-34 (domains for protein kinase C activation and mitogenic activity), but not amino acid residues 1-7 (adenylate cyclase activation domain). We have tested this hypothesis with in vivo studies of human PTH (hPTH) analogs. Serum biomarkers and selected histomorphometric parameters of bone formation and resorption were assessed in adult, female, Sprague-Dawley rats following 19 daily injections of vehicle, 10 micrograms/kg body weight (bw) of hPTH(1-38), or a dose range of 10, 40, and 100 micrograms/100 g bw of hPTH(2-38) or hPTH(3-38). Treatment with hPTH(1-38) increased serum osteocalcin, the percentage of osteoblast surface, percentage of osteoid surface, percentage of bone volume, trabecular thickness, and bone formation rate, while it decreased the percentage of osteoclast surface. The hPTH(2-38) fragment exhibited 10%-25% of the in vivo anabolic activity of hPTH(1-38), while it had no effect on the percentage of osteoclast surface. The hPTH(3-38) fragment exhibited no biological activity on bone. In contrast, serum INS-PTH (intact-N-terminal specific PTH) levels were similarly and significantly increased above control in rats treated with hPTH(1-38), hPTH(2-38), or hPTH(3-38) at the same dose. This preliminary finding suggests that the differential activity of these peptides on bone is not due to differences in the circulating level of immunoreactive PTH (intact and amino-terminal fragments of PTH from endogenous and exogenous sources) several hours after PTH injection. However, we can draw no conclusion regarding the relative clearance rates of these peptides. Last, because hPTH(3-38) was without any detectable biological activity on rat bone in vivo, its mitogenic activity was confirmed in two osteoblast-like cell lines. In summary, the anabolic effect of hPTH(1-38) on bone in vivo was (1) diminished by removal of amino acid residue 1, and (2) abolished by the removal of amino acid residues 1 and 2. Although these findings suggest that the therapeutic benefits of exogenous PTH administration may depend upon activation of not only protein kinase C, but also adenylate cyclase, they do not rule out a differential PTH response due to other causes, e.g., metabolic inactivation.