Absence of bone sialoprotein (BSP) impairs cortical defect repair in mouse long bone

Matrix proteins of the SIBLING family interact with bone cells and with bone mineral and are thus in a key position to regulate bone development, remodeling and repair. Within this family, bone sialoprotein (BSP) is highly expressed by osteoblasts, hypertrophic chondrocytes and osteoclasts. We recently reported that mice lacking BSP (BSP−/−) have very low trabecular bone turnover. In the present study, we set up an experimental model of bone repair by drilling a 1 mm diameter hole in the cortical bone of femurs in both BSP−/− and +/+ mice. A non-invasive MRI imaging and bone quantification procedure was designed to follow bone regeneration, and these data were extended by μCT imaging and histomorphometry on undecalcified sections for analysis at cellular level. These combined approaches revealed that the repair process as reflected in defect-refilling in the cortical area was significantly delayed in BSP−/− mice compared to +/+ mice. Concomitantly, histomorphometry showed that formation, mineralization and remodeling of repair (primary) bone in the medulla were delayed in BSP−/− mice, with lower osteoid and osteoclast surfaces at day 15. In conclusion, the absence of BSP delays bone repair at least in part by impairing both new bone formation and osteoclast activity.     

Bone particles from osteopetrotic mice not cured by bone marrow transplants are resorbed in normal littermates

Osteosclerotic (oc/oc) and osteopetrotic (op/op) mice are not cured by bone marrow transplantation from normal littermates. The possibility that this is due to production of poorly resorbable bone was examined by comparing the fate of mutant and normal bone particles implanted subcutaneously in normal hosts. Bone, removed aseptically from calvarial and tibial sites of normal littermates and mutants, was cleaned of adherent soft tissue, ground and sieved to a particle size of 70–300 μm. Aliquots (17–20 mg) of bone from each phenotype of each stock were pelleted and implanted beneath the anterior thoracic skin of normal littermates for two weeks. Particle density in tissue sections was determined as percent of field by a point-counting method. Giant cell response was recorded as number per high-power field. Percent bone present initially was determined in pellets implanted for less than 24 hr. Bone particles were reduced in each pellet with time, about 25% of the original volume being removed in two weeks. No statistically significant differences were noted in the rates of disappearance of mutant and normal bone or in the percentage or number of giant cells in implants of mutant and normal bone in either stock. Furthermore, these values were not different from identical studies in microphthalmic mice, an osteopetrotic stock cured by bone marrow transplantation. These data suggest that the failure of osteopetrotic and osteosclerotic mice to be cured by bone marrow transplants from normal littermates is not due to the presence of unresorbable bone.     

Bone has a sexually dimorphic response to aromatase deficiency.

Aromatase synthesizes estrogen from androgen precursors. To better understand the role of estrogen in skeletal metabolism and growth, we have assessed long bone growth and histomorphometry in aromatase-deficient (ArKO) mice. The age range for the animals was 5-7 months. At this age mice have already achieved peak bone density but continue slow bone growth. Femur length, an index of long bone growth, showed decreased growth in ArKO males compared with wild-type (wt) littermates but no significant difference in females. Radiographically, compared with age- and sex- matched littermates both ArKO males and females showed osteopenia in the lumbar spine. Histologically, both ArKO males and females showed an osteoporotic-type picture, characterized by significant decreases in trabecular bone volume and trabecular thickness. However, compared with wt littermates female ArKO animals showed a bone remodeling picture consistent with increased bone turnover, much like early postmenopausal osteoporosis in humans. On the other hand, male ArKO animals showed decreases in both osteoblastic and osteoclastic surfaces compared with wt littermates, similar to age-related osteopenia. These findings suggest that osteoporosis seen in aromatase-deficient mice may arise from different bone remodeling activities between males and females. These results also show that the ArKO model exhibits the expected results of estrogen deficiency and may be a good model for investigating sex-specific responses to estrogen deficiency. Furthermore, they imply that estrogen is important for attaining peak bone mass in male as well as in female mice.     

Glucose-dependent insulinotropic polypeptide receptor knockout mice have altered bone turnover

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone, which is secreted from endocrine cells in the small intestine after meal ingestion. GIP has been shown to affect osteoblastic function in vitro; however, the in vivo effects of GIP on bone remodeling remain unclear. In the present study, we investigated the role of GIP in modulating bone turnover, by evaluating serum markers of bone turnover, bone density, bone morphology, and changes in biomechanical bone strength over time (one to five months) in GIP receptor knockout mice (GIPR−/− mice). The GIPR−/− mice showed a decreased bone size, lower bone mass, altered bone microarchitecture and biomechanical properties, and altered parameters for bone turnover, especially in bone formation. Moreover, the effects of GIP on bone mass were site-specific and compensatory mechanism developed over time and ameliorated the impact of the loss of GIP signaling on bone mass. Further, GIPR−/− mice had earlier age-related changes than wild-type mice in body composition, including bone mass, lean body mass, and fat percentage. In summary, our results indicate that GIP has an anabolic effect on bone mass and bone quality and suggests that GIP may be a hormonal link between nutrient ingestion and utilization.     

Impact of Pregnancy‐Associated Plasma Protein‐A Deletion on the Adult Murine Skeleton

Introduction : The metalloproteinase, pregnancy‐associated plasma protein‐A (PAPP‐A) functions to enhance local insulin‐like growth factor (IGF)‐I bioavailability through cleavage of inhibitory IGF binding proteins. Because IGF‐I is an important regulator of skeletal growth and remodeling and PAPP‐A is highly expressed by osteoblastic cells, we hypothesized that, in the absence of PAPP‐A, bone physiology would be compromised because of a blunting of local IGF‐I action even in the presence of normal circulating IGF‐I levels. Materials and Methods : pQCT, μCT, histomorphometry, and mechanical strength testing were performed on bones from PAPP‐A knockout (KO) mice and wildtype (WT) littermates at 2–12 mo of age. IGF‐I levels and bone formation and resorption markers were determined in sera from these animals. Results : Volumetric BMD in PAPP‐A KO mice measured by pQCT at the femoral midshaft, which is primarily cortical bone, was 10% less than WT at 2 mo. This difference was maintained at 4, 6, and 12 mo. Cortical thickness at this site was similarly decreased. On the other hand, trabecular bone at the distal femur (pQCT) and in the tibia (μCT) showed age‐progressive decreases in bone volume fraction in PAPP‐A KO compared with WT mice. Tibial μCT indicated a 46% relative decrease in trabecular bone volume/total volume (BV/TV) and a 28% relative decrease in trabecular thickness in PAPP‐A KO compared with WT mice at 6 mo. These trabecular deficiencies in PAPP‐A KO mice corresponded to a weakening of the bone. Serum markers and bone histomorphometry indicated that the primary impact of PAPP‐A is on skeletal remodeling resulting in a state of low‐turnover osteopenia in adult PAPP‐A KO mice. Circulating IGF‐I levels were not altered in PAPP‐A KO mice. Conclusions : PAPP‐A is a bone growth regulatory factor in vivo and, in its absence, mice show skeletal insufficiency in mass, density, architecture, and strength. The data suggest a primary role for PAPP‐A in modulating local IGF bioavailability for trabecular bone remodeling.     

Osteoblast-specific targeting of soluble colony-stimulating factor-1 increases cortical bone thickness in mice.

The soluble and membrane-bound forms of CSF-1 are synthesized by osteoblasts and stromal cells in the bone microenvironment. Transgenic mice, generated to selectively express sCSF-1 in bone, showed increased cortical thickness in the femoral diaphysis caused by new bone formation along the endosteal surface. The ability of sCSF-1 to enhance bone cell activity in vivo is potentially relevant for increasing cortical bone in a variety of disorders. INTRODUCTION: The soluble form of colony-stimulating factor-1 (sCSF-1) and the membrane-bound form of CSF-1 (mCSF-1) have been shown to support osteoclastogenesis in vitro; however, the effect of each peptide on bone remodeling in vivo is unclear. To determine the effect of sCSF-1, selectively expressed in bone, the skeletal phenotype of transgenic mice harboring the human sCSF-1 cDNA under the control of the osteocalcin promoter was assessed. METHODS: At 5 and 14 weeks, mice were analyzed for CSF-1 protein levels, weighed, and X-rayed, and femurs were removed for peripheral quantitative computed tomography, histology, and histomorphometry. RESULTS: High levels of human sCSF-1 were detected in bone extracts and, to a lesser extent, in plasma. Adult transgenic mice showed normal body weight and increased circulating monocytic cells. At 5 weeks, the femoral diaphysis was similar in CSF-1T and wt/wt littermates. However, by 14 weeks, the femoral diaphysis in CSF-1T mice showed increased cortical thickness and bone mineral density. In contrast to the diaphysis, the femoral metaphysis of CSF-1T mice showed normal cancellous bone comparable with wt/wt littermates at each time point. Histological sections demonstrated increased woven bone along the endosteal surface of the diaphysis and intracortical remodeling. Fluorochrome-labeling analysis confirmed endocortical bone formation in CSF-1T, with a 3.1-fold increase in the percentage of double-labeled surfaces and a 3.6-fold increase in the bone formation rate compared with wt/wt mice. Although remodeling resulted in a slightly porous cortex, sCSF-1 preferentially stimulated endocortical bone formation, leading to increased cortical thickness. CONCLUSIONS: These findings indicate that sCSF-1 is a key determinant of bone cell activity in the corticoendosteal envelope.     

Trabecular bone remodeling and bone balance in hyperthyroidism

In vivo tetracycline double-labeled iliac crest bone biopsies from 15 hyperthyroid patients were used for the reconstruction of curves describing the variation of resorption depth and formation thickness with time. The curves emerging were compared to curves reconstructed from 13 age- and sex-matched normal individuals (mean age 44 years). The median function period for resorptive cells in hyperthyroid patients (16 days) was about one-third the resorptive period in normals (51 days). No significant difference between the osteoclast-, mononuclear-, or preosteoblast-like cell resorption depths could be demonstrated between the two groups. Consequently, the median resorption rate in hyperthyroid patients (3.8 μm/day) was more than 3 times higher than the value in the control group (1.1 μm/day). Median Sigma_f, was shorter in the hyperthyroid group (109 days) than in the control group (151 days, P < 0.05), as was the median initial mineralization lag time (5 and 16 days, respectively, P < 0.01). No significant difference between the measured mean completed wall thickness (mcwT) values in the hyperthyroid groups and the control group could be demonstrated (58.1 and 60.5 μm respectively). Median initial mineralization rate in the hyperthyroid group (1.2 μm³/μm² per day) was not significantly higher than the value calculated in the control group (0.9 μm³/ μm² per day), but median initial matrix appositional rate in hyperthyroids (4.8 μm³/μm² per day) was 3 times higher than the value calculated for normals (1.6 μm³/μm² per day) (P < 0.01). Direct measurements of mean completed wall thickness in the hyperthyroid group gave results (58.1 μm) that were not in accordance with the mean completed wall thickness calculated from the growth curve (52.1 μm, P < 0.02). In normals no such discrepancy could be demonstrated. Using the mcwT value estimated from the growth curve, the bone formation period was calculated to 90 days for hyperthyroid patients. This maximal estimate for mcwT was also significantly lower than the mean resorption depth measured in the hyperthyroid group (61.7 μm, P < 0.05), which means that a net negative balance per remodeling cycle existed in the hyperthyroid group. Bone balance was preserved in the control group.     

Variations of microstructure, mineral density and tissue elasticity in B6/C3H mice

200-MHz scanning acoustic microscopy (SAM) and synchrotron radiation μCT (SR-μCT) were used to assess microstructural parameters, acoustic impedance Z and tissue degree of mineralization of bone (DMB) in site-matched regions of interest in femoral bone of two inbred strains. Transverse femoral sections taken from 5 C57BL/6J@Ico (B6) and 5 C3H/HeJ@Ico (C3H) mice (5.5 months old) were explored. Mass density ρ, elastic coefficient c₁₁ and Young's modulus E₁ were locally derived in the distal epiphysis, distal metaphysis for trabecular bone and mid-diaphysis for cortical bone using a rule-of-mixture model. Structural parameter estimations obtained from X-ray tomographic and acoustic images were almost identical. Both strains had the same bone diameter, but the C3H mice had greater cortical thickness and smaller cancellous diameter than did B6 mice. The average DMB and impedance values were in the range between 1.13 and 1.33 g cm^− 3 and 5.8 and 7.8 Mrayl, respectively. All tissue parameters were lower in B6 mice than in C3H mice. However, interstrain differences of DMB were much less (up to 3.8%) than differences of Z (up to 13.2%). SAM and SR-μCT fulfill the requirement for a simultaneous evaluation of cortical bone microstructure and material properties at the tissue level. However, SAM provides a quantitative estimate of elastic properties at the tissue level that cannot be captured by SR-μCT. The strong differences in the measured acoustic impedances among the two inbred strains indicate that the impedance is a good parameter to detect genetic variations of the skeletal phenotype in small animal models.     

The skeletal structure of insulin-like growth factor I-deficient mice.

The importance of insulin-like growth factor I (IGF-I) for growth is well established. However, the lack of IGF-I on the skeleton has not been examined thoroughly. Therefore, we analyzed the structural properties of bone from mice rendered IGF-I deficient by homologous recombination (knockout [k/o]) using histomorphometry, peripheral quantitative computerized tomography (pQCT), and microcomputerized tomography (muCT). The k/o mice were 24% the size of their wild-type littermates at the time of study (4 months). The k/o tibias were 28% and L1 vertebrae were 26% the size of wild-type bones. Bone formation rates (BFR) of k/o tibias were 27% that of the wild-type littermates. The k/o bones responded normally to growth hormone (GH; 1.7-fold increase) and supranormally to IGF-I (5.2-fold increase) with respect to BFR. Cortical thickness of the proximal tibia was reduced 17% in the k/o mouse. However, trabecular bone volume (bone volume/total volume [BV/TV]) was increased 23% (male mice) and 88% (female mice) in the k/o mice compared with wild-type controls as a result of increased connectivity, increased number, and decreased spacing of the trabeculae. These changes were either less or not found in L1. Thus, lack of IGF-I leads to the development of a bone structure, which, although smaller, appears more compact.     

Effect of slow-release sodium fluoride on cancellous bone histology and connectivity in osteoporosis

We have previously demonstrated that a treatment regimen of slow-release sodium fluoride (SRNaF) and continuous calcium citrate increases lumbar bone mass, improves cancellous bone material quality, and significantly reduces vertebral fracture rate in osteoporotic patients. In order to assess whether such treatment also improves trabecular structure, we quantitated cancellous bone connectivity before and following 2 years of therapy with SRNaF in 23 patients with osteoporosis and vertebral fractures. In addition, we performed bone histomorphometry on the same sections used for connectivity measurements. There was a significant increase in L2-L4 bone mineral density during therapy (0.827 ± 0.176 g/cm² SD to 0.872 ± 0.166, p = 0.0004). Significant histomorphometric changes were represented by increases in mineral apposition rate (0.6 ± 0.4μm/d to 1.1 ± 0.7, p = 0.0078) and adjusted apposition rate (0.4 ± 0.3 μm/d to 0.6 ± 0.4, p = 0.016). On the other hand, trabecular spacing significantly declined (from 1375 ± 878 μm to 1052 ± 541, p = 0.05). Two-dimensional quantitation of trabecular struts on iliac crest histological sections disclosed significant increases in mean node number per mm² of cancellous tissue area (0.22 ± 0.12 vs. 0.39 ± 0.27, p = 0.0077), the mean node to free-end ratio (0.23 ± 0.21 vs. 0.41 ± 0.46, p < 0.05), and in the mean node to node strut length per mm² of cancellous area (0.098 ± 0.101 vs. 0.212 ± 0.183, p < 0.01). There were no significant changes in any of the measurements associated with free-end number or free-end to free-end strut length. When patients were divided into those with severe and mild-modest spinal bone loss (based upon initial lumbar bone density) the significant changes in connectivity occurred in patients with mild-moderate bone loss, but not in those with severe bone loss, suggesting that fluoride's effect is in part dependent on the presence of a certain critical amount of bone. This finding in combination with the previously reported increases in bone mass and bone material quality may explain the significant reduction in vertebral fracture rate observed with this particular fluoride regimen.     

The protein tyrosine phosphatase Rptpzeta is expressed in differentiated osteoblasts and affects bone formation in mice

Tyrosine phosphorylation of intracellular substrates is one mechanism to regulate cellular proliferation and differentiation. Protein tyrosine phosphatases (PTPs) act by dephosphorylation of substrates and thereby counteract the activity of tyrosine kinases. Few PTPs have been suggested to play a role in bone remodeling, one of them being Rptpzeta, since it has been shown to be suppressed by pleiotrophin, a heparin-binding molecule affecting bone formation, when over-expressed in transgenic mice. In a genome-wide expression analysis approach we found that Ptprz1, the gene encoding Rptpzeta, is strongly induced upon terminal differentiation of murine primary calvarial osteoblasts. Using RT-PCR and Western Blotting we further demonstrated that differentiated osteoblasts, in contrast to neuronal cells, specifically express the short transmembrane isoform of Rptpzeta. To uncover a potential role of Rptpzeta in bone remodeling we next analyzed the skeletal phenotype of a Rptpzeta-deficient mouse model using non-decalcified histology and histomorphometry. Compared to wildtype littermates, the Rptpzeta-deficient mice display a decreased trabecular bone volume at the age of 50 weeks, caused by a reduced bone formation rate. Likewise, Rptpzeta-deficient calvarial osteoblasts analyzed ex vivo display decreased expression of osteoblast markers, indicating a cell-autonomous defect. This was confirmed by the finding that Rptpzeta-deficient osteoblasts had a diminished potential to form osteocyte-like cellular extensions on Matrigel-coated surfaces. Taken together, these data provide the first evidence for a physiological role of Rptpzeta in bone remodeling, and thus identify Rptpzeta as the first PTP regulating bone formation in vivo.     

Increased bone formation in mice lacking apolipoprotein E.

ApoE is a plasma protein that plays a major role in lipoprotein metabolism. Here we describe that ApoE expression is strongly induced on mineralization of primary osteoblast cultures. ApoE-deficient mice display an increased bone formation rate compared with wildtype controls, thereby showing that ApoE has a physiologic function in bone remodeling. INTRODUCTION: Apolipoprotein E (ApoE) is a protein component of lipoproteins and facilitates their clearance from the circulation. This is confirmed by the phenotype of ApoE-deficient mice that have high plasma cholesterol levels and spontaneously develop atherosclerotic lesions. The bone phenotype of these mice has not been analyzed to date, although an association between certain ApoE alleles and BMD has been reported. MATERIALS AND METHODS: Primary osteoblasts were isolated from newborn mouse calvariae and mineralized ex vivo. A genome-wide expression analysis was performed during the course of differentiation using the Affymetrix gene chip system. Bones from ApoE-deficient mice and wildtype controls were analyzed using radiography, micro CT imaging, and undecalcified histology. Cellular activities were assessed using dynamic histomorphometry and by measuring urinary collagen degradation products. Lipoprotein uptake assays were performed with (125)I-labeled triglyceride-rich lipoprotein-remnants (TRL-R) using primary osteoblasts from wildtype and ApoE-deficient mice. Serum concentrations of osteocalcin were determined by radioimmunoassay after hydroxyapatite chromatography. RESULTS: ApoE expression is strongly induced on mineralization of primary osteoblast cultures ex vivo. Mice lacking ApoE display a high bone mass phenotype that is caused by an increased bone formation rate, whereas bone resorption is not affected. This phenotype may be explained by a decreased uptake of triglyceride-rich lipoproteins by osteoblasts, resulting in elevated levels of undercarboxylated osteocalcin in the serum of ApoE-deficient mice. CONCLUSION: The specific induction of ApoE gene expression during osteoblast differentiation along with the increased bone formation rate observed in ApoE-deficient mice shows that ApoE has a physiologic role as a regulator of osteoblast function.     

Estimation of distal radius failure load with micro-finite element analysis models based on three-dimensional peripheral quantitative computed tomography images

There is increasing evidence that, in addition to bone mass, bone microarchitecture and its mechanical load distribution are important factors for the determination of bone strength. Recently, it has been shown that new high-resolution imaging techniques in combination with new modeling algorithms based on the finite element (FE) method can account for these additional factors. Such models thus could provide more relevant information for the estimation of bone failure load. The purpose of the present study was to determine whether results of whole-bone micro-FE (μFE) analyses with models based on three-dimensional peripheral quantitative computer tomography (3D-pQCT) images (isotropic voxel resolution of 165 μm) could predict the failure load of the human radius more accurately than results with dual-energy X-ray absorptiometry (DXA) or bone morphology measurements. For this purpose, μFE models were created using 54 embalmed cadaver arms. It was assumed that bone failure would be initiated if a certain percentage of the bone tissue (varied from 1% to 7%) would be strained beyond the tissue yield strain. The external force that produced this tissue strain was calculated from the FE analyses. These predictions were correlated with results of real compression testing on the same cadaver arms. The results of these compression tests were also correlated with results of DXA and structural measurements of these arms. The compression tests produced Colles-type fractures in the distal 4 cm of the radius. The predicted failure loads calculated from the FE analysis agreed well with those measured in the experiments (R² = 0.75 p < 0.001). Lower correlations were found with bone mass (R² = 0.48, p < 0.001) and bone structural parameters (R² = 0.57 p < 0.001). We conclude that application of the techniques investigated here can lead to a better prediction of the bone failure load for bone in vivo than is possible from DXA measurements, structural parameters, or a combination thereof.     

Human parathyroid hormone (1-34) accelerates natural fracture healing process in the femoral osteotomy model of cynomolgus monkeys

Several studies in rats have demonstrated that parathyroid hormone accelerates fracture healing by increasing callus formation or stimulating callus remodeling. However the effect of PTH on fracture healing has not been tested using large animals with Haversian remodeling system. Using cynomolgus monkey that has intracortical remodeling similar to humans, we examined whether intermittent treatment with human parathyroid hormone [hPTH(1–34)] accelerates the fracture healing process, especially callus remodeling, and restores geometrical shapes and mechanical properties of osteotomized bone.

Seventeen female cynomolgus monkeys aged 18–19 years were allocated into three groups: control (CNT, n = 6), low-dose PTH (0.75 μg/kg; PTH-L, n = 6), and high-dose PTH (7.5 μg/kg; PTH-H, n = 5) groups. In all animals, twice a week subcutaneous injection was given for 3 weeks. Then fracture was produced surgically by transversely cutting the midshaft of the right femur and fixing with stainless plate. After fracture, intermittent PTH treatment was continued until sacrifice at 26 weeks after surgery. The femora were assessed by soft X-ray, three-point bending mechanical test, histomorphometry, and degree of mineralization in bone (DMB) measurement. Soft X-ray showed that complete bone union occurred in all groups, regardless of treatment. Ultimate stress and elastic modulus in fractured femur were significantly higher in PTH-H than in CNT. Total area and percent bone area of the femur were significantly lower in both PTH-L and PTH-H than in CNT. Callus porosity decreased dose-dependently following PTH treatment. Mean DMB of callus was significantly higher in PTH-H than in CNT or PTH-L. These results suggested that PTH decreased callus size and accelerated callus maturation in the fractured femora.

PTH accelerates the natural fracture healing process by shrinking callus size and increasing degree of mineralization of the fracture callus, thereby restoring intrinsic material properties of osteotomized femur shaft in cynomolgus monkeys although there were no significant differences among the groups for structural parameters.     

Calcitonin deficiency in mice progressively results in high bone turnover.

Although the pharmacological action of calcitonin (CT) as an inhibitor of bone resorption is well established, there is still some controversy regarding its physiological function. Unexpectedly, Calca-deficient mice lacking CT and alpha-calcitonin gene-related peptide (alphaCGRP) were described to have a high bone mass phenotype caused by increased bone formation with normal bone resorption. Here we show that these mice develop a phenotype of high bone turnover with age, suggesting that CT is a physiological inhibitor of bone remodeling. INTRODUCTION: The absence of significant changes in bone mineral density caused by decline or overproduction of CT in humans has raised the question, whether the pharmacological action of CT as an inhibitor of bone resorption is also of physiological relevance. To study the physiological role of mammalian CT, we have analyzed the age-dependent bone phenotype of two mouse models, one lacking CT and alphaCGRP (Calca-/-), the other one lacking only alphaCGRP (alphaCGRP-/-). MATERIALS AND METHODS: Bones from wildtype, Calca-/- -mice and alphaCGRP-/- -mice were analyzed at the ages of 6, 12 and 18 months using undecalcified histology. Differences of bone remodeling were quantified by static and dynamic histomorphometry as well as by measuring the urinary collagen degradation products. To rule out secondary mechanisms underlying the observed phenotype, we determined serum concentrations of relevant hormones using commercially available antibody-based detection kits. RESULTS: Whereas alphaCGRP-/- -mice display an osteopenia at all ages analyzed, the Calca-/- -mice develop a phenotype of high bone turnover with age. Histomorphometric analysis performed at the age of 12 months revealed significant increases of bone formation and bone resorption specifically in the Calca-/- -mice. This severe phenotype that can result in hyperostotic lesions, can not be explained by obvious endocrine abnormalities other than the absence of CT. CONCLUSIONS: In addition to the previously described increase of bone formation in the Calca-deficient mice, we have observed that there is also an increase of bone resorption with age. This suggests that CT has a dual action as an inhibitor of bone remodeling, which may explain why alterations of CT serum levels in humans do not result in major changes of bone mineral density.     

In vivo inhibition of osteoblastic metalloproteinases leads to increased trabecular bone mass.

Mice specifically overexpressing TIMP-1 in osteoblasts have been generated to investigate the role of MMPs in bone in vivo. These mice displayed increased trabecular bone volume and decreased bone turnover. This model provides evidence of the role played by the MMPs in bone remodeling and balance. INTRODUCTION: Although it has been suggested that the matrix metalloproteinases (MMPs) may play a role in initiating the bone resorption process in vitro, there is no evidence that they play any role in in vivo bone maintenance. MATERIALS AND METHODS: We used an artificial promoter specifically driving cells of the osteoblastic lineage to overexpress the tissue inhibitor of MMPs (TIMP-1) cDNA in mice. Densitometric analysis, using DXA and pQCT, and static and dynamic histomorphometry were used to evaluate the bone phenotype both in male and female transgenic mice. We evaluated osteoblastic differentiation using a primary osteoblast culture and osteoclast activity using an ex vivo organ culture. RESULTS AND CONCLUSION: We showed that at 1 and 2.5 months of age, only the female mice exhibited a bone phenotype. These mice displayed specific increases in the BMD and bone volume of trabecular bone. This increase was accompanied by decreased trabecular separation, suggesting a decrease in bone resorption. Using an ex vivo resorption assay, we demonstrated that parathyroid hormone (PTH)-stimulated bone resorption was reduced in these mice. Evaluation of the bone histomorphometric dynamic parameters showed that the mineralizing surfaces and bone formation rate were both reduced. There was no change in the mineralization lag time or number of osteocyte lacunae. Using primary osteoblast culture and molecular analysis, we showed that the differentiation and function of osteoblasts from transgenic mice were normal, but that the ex vivo formation of mineralized nodules was delayed. This model is the first to show that in vivo MMPs play a role in bone remodeling and bone balance. Moreover, our data suggest that MMP activity could be involved in the hormonal regulation of bone resorption by osteoblasts.     

Mechanical Properties of Ewe Vertebral Cancellous Bone Compared With Histomorphometry and High-Resolution Computed Tomography Parameters

The goal of the present study was to determine if a high-resolution computed tomography (HRCT) system with 150 μm resolution was sufficient to predict mechanical properties in ewe lumbar vertebrae. To answer this question, we used a triangular comparison between: HRCT; biomechanics (compression and shear tests); and histomorphometry, which was the reference method for the measurements of morphometric parameters. Two dissected lumbar vertebrae (L-4 and L-5) from 32 ewes were used. Both compressive and shear properties correlated significantly with amount of bone and structural parameters evaluated by histomorphometry (bone volume/tissue volume, trabecular thickness, trabecular separation), but no significant correlation was found with the trabecular number. With our shear test involving the trabecular architecture itself more significant correlations were found with the node-strut analysis parameters than from the compressive test. Significant correlations were also found between HRCT and histological parameters (bone volume/tissue volume, bone surface/bone volume, trabecular separation, trabecular number, total strut length, number of nodes, and number of termini). Correlations between HRCT structural parameters and mechanical properties on L-4 were of the same magnitude as the correlations between the histomorphometric structural parameters and mechanical results on L-5 but with the remarkable advantage that HRCT is a noninvasive method. In spite of the resolution (150 μm) of our HRCT system, which entailed mainly an enlargement of the thinnest trabeculae or their loss during the segmentation process, we obtained coherent relationships between mechanical and tomographic parameters. The thinnest trabeculae probably had little effect on the mechanical strength. Also, this type of resolution allows us to consider the possibility of perfecting an in vivo HRCT system. However, physical density and bone mineral density correlated much better with strength than either classical histomorphometric or tomographic parameters. The current conclusion is fairly negative with respect to the ability of HRCT to assess mechanical properties nondestructively as compared with dual-energy X-ray absorptiometry. But, the noninvasive nature of the imaging modality and the capacity for three-dimensional imaging at arbitrary orientation make HRCT a promising tool in the quantitative assessment of cancellous architecture.     

Delayed fracture healing in tetranectin-deficient mice

Tetranectin is a plasminogen-binding protein that enhances plasminogen activation, which has been suggested to play a role in tissue remodeling. Recently, we showed that tetranectin has a role in the wound-healing process. In this study, we investigated whether tetranectin plays a role in fracture healing. The fracture-healing process was studied using a femoral osteotomy model in tetranectin-null mice, previously generated by the authors. Radiographic imaging, micro-computed tomography (μCT), and histological analysis were used to evaluate osteotomy healing. In wild-type mice, a callus was apparent from 7 days, and most samples showed marked callus formation and rebridging of the cortices at the osteotomy site at 21 days. In contrast, in the tetranectin-null mice there was no callus formation at 7 days and much less callus formation and no bridging of cortices were observed at 21 days. At 35 days, all osteotomy sites showed clear rebridging, and secondary bone formation was achieved in wild-type mice by 42 days. In contrast, no clear rebridging or secondary bone formation was observed at 42 days in the tetranectin-null mice. Analysis using μCT at 21 days after osteotomy revealed that the callus area in tetranectin-null mice was smaller than that in wild-type mice. Histological analysis also showed that soft tissue and callus formation were smaller in the tetranectin-null mice at the early stage of the healing process after drill-hole injury. These results suggested that tetranectin could have a role in the positive regulation at the early stage of the fracture-healing process, which was reflected in the delayed fracture healing in tetranectin-deficient mice.     

Mechanical force-induced midpalatal suture remodeling in mice

Mechanical stress is an important epigenetic factor for regulating skeletal remodeling, and application of force can lead to remodeling of both bone and cartilage. Chondrocytes, osteoblasts and osteoclasts all participate and interact with each other in this remodeling process. To study cellular responses to mechanical stimuli in a system that can be genetically manipulated, we used mouse midpalatal suture expansion in vivo. Six-week-old male C57BL/6 mice were subjected to palatal suture expansion by opening loops with an initial force of 0.56 N for the periods of 1, 3, 5, 7, 14 or 28 days. Periosteal cells in expanding sutures showed increased proliferation, with Ki67-positive cells representing 1.8 ± 0.1% to 4.5 ± 0.4% of total suture cells in control groups and 12.0 ± 2.6% to 19.9 ± 1.2% in experimental/expansion groups (p < 0.05). Starting at day 1, cells expressing alkaline phosphatase and type I collagen were seen. New cartilage and bone formation was observed at the oral edges of the palatal bones at day 7; at the nasal edges only bone formation without cartilage appeared to occur. An increase in osteoclast numbers suggested increased bone remodeling, ranging from 60 to 160% throughout the experimental period. Decreased Saffranin O staining after day 3 suggested decreased proteoglycan content in the secondary cartilage. Micro-CT showed a significant increase in maxillary width at days 14 and 28 (from 2334 ± 4 μm to 2485 ± 3 μm at day 14 and from 2383 ± 5 μm to 2574 ± 7 μm at day 28, p < 0.001). The suture width was increased at days 14 and 28, except in the oral third region at day 28 (from 48 ± 5 μm to 36 ± 4 μm, p < 0.05). Bone volume/total volume was significantly reduced at days 14 and 28 (50.2 ± 0.7% vs. 68.0 ± 3.7% and 56.5 ± 1.0% vs. 60.9 ± 1.3%, respectively, p < 0.05), indicative of increased bone marrow space. These findings demonstrate that expansion forces across the midpalatal suture promote bone resorption through activation of osteoclasts and bone and cartilage formation via increased proliferation and differentiation of periosteal cells. Mouse midpalatal suture expansion would be useful in further studies of the ability of mineralized tissues to respond to mechanical stimulation.     

Thrombin receptor deficiency leads to a high bone mass phenotype by decreasing the RANKL/OPG ratio

Thrombin and its receptor (TR) are, respectively, expressed in osteoclasts and osteoblasts. However, their physiological roles on bone metabolism have not been fully elucidated. Here we investigated the bone microarchitecture by micro-computed tomography (μCT) and demonstrated increased trabecular and cortical bone mass in femurs of TR KO mice compared to WT littermates. Trabecular thickness and connectivity were significantly enhanced. The physiological role of TR on both inorganic and organic phases of bone is illustrated by a significant increase in BMD and a decrease in urinary deoxypyridinoline (DPD) crosslink concentration in TR KO mice. Moreover, TR KO cortical bone expanded and had a higher polar moment of inertia (J), implying stronger bone. Bone histomorphometry illustrated unaltered osteoblast and osteoclast number and surface in femoral metaphyses, indicating that thrombin/TR regulates osteoblasts and osteoclasts at functional levels. Serum analysis showed a decrease in RANKL and an increase in osteoprotegerin (OPG) levels and reflected a reduced RANKL/OPG ratio in the TR KO group. In vitro experiments using MC3T3 pre-osteoblasts demonstrated a TR-dependent stimulatory effect of thrombin on the RANKL/OPG ratio. This effect was blocked by TR antagonist and p42/p44-ERK inhibitor. In addition, thrombin also intensified p42/p44-ERK expression and phosphorylation. In conclusion, the thrombin/TR system maintains normal bone remodeling by activating RANKL and limiting OPG synthesis by osteoblasts through the p42/44-ERK signaling pathway. Consequently, TR deficiency inhibits osteoclastogenesis, resulting in a high bone mass phenotype.