Peritoneal Delivery of Sodium Pyrophosphate Blocks the Progression of Pre-existing Vascular Calcification in Uremic Apolipoprotein-E Knockout Mice

Chronic kidney disease (CKD) is generally associated with disturbances of mineral and bone metabolism. They contribute to the development of vascular calcification (VC), a strong, independent predictor of cardiovascular risk. Pyrophosphate (PPi), an endogenous inhibitor of hydroxyapatite formation, has been shown to slow the progression of VC in uremic animals. Since in patients with CKD treatment is usually initiated for already existing calcifications, we aimed to compare the efficacy of PPi therapy with that of the phosphate binder sevelamer, using a uremic apolipoprotein-E knockout mouse model with advanced VCs. After CKD creation or sham surgery, 12-week-old female mice were randomized to one sham group and four CKD groups (n = 18–19/group). Treatment was initiated 8 weeks after left nephrectomy allowing prior VC development. Uremic groups received either intraperitoneal PPi (high dose, 1.65 mg/kg or low dose, 0.33 mg/kg per day), oral sevelamer (3 % in diet), or placebo treatment for 8 weeks. Both intima and media calcifications worsened with time in placebo-treated CKD mice, based on both quantitative image analysis and biochemical measurements. Progression of calcification between 8 and 16 weeks was entirely halted by PPi treatment, as it was by sevelamer treatment. PPi did not induce consistent bone histomorphometry changes. Finally, the beneficial vascular action of PPi probably involved mechanisms different from that of sevelamer. Further studies are needed to gain more precise insight into underlying mechanisms and to see whether PPi administration may also be useful in patients with CKD and VC.     

Apolipoprotein E-dependent inverse regulation of vertebral bone and adipose tissue mass in C57Bl/6 mice: Modulation by diet-induced obesity

The long prevailing view that obesity is generally associated with beneficial effects on the skeleton has recently been challenged. Apolipoprotein E (apoE) is known to influence both adipose tissue and bone. The goal of the current study was to examine the impact of apoE on the development of fat mass and bone mass in mice under conditions of diet-induced obesity (DIO).Four week-old male C57BL/6 (WT) and apoE-deficient (apoE^?/?) mice received a control or a diabetogenic high-fat diet (HFD) for 16 weeks. The control-fed apoE^?/? animals displayed less total fat mass and higher lumbar trabecular bone volume (BV/TV) than WT controls. When stressed with HFD to induce obesity, apoE^?/? mice had a lower body weight, lower serum glucose, insulin and leptin levels and accumulated less white adipose tissue mass at all sites including bone marrow. While WT animals showed no significant change in BV/TV and bone formation rate (BFR), apoE deficiency led to a decrease of BV/TV and BFR when stressed with HFD. Bone resorption parameters were not affected by HFD in either genotype.Taken together, under normal dietary conditions, apoE-deficient mice acquire less fat mass and more bone mass than WT littermates. When stressed with HFD to develop DIO, the difference of total body fat mass becomes larger and the difference of bone mass smaller between the genotypes. We conclude that apoE is involved in an inverse regulation of bone mass and fat mass in growing mice and that this effect is modulated by diet-induced obesity.     

Chronic Kidney Disease–Induced Vascular Calcification Impairs Bone Metabolism

An association between lower bone mineral density (BMD) and presence of vascular calcification (VC) has been reported in several studies. Chronic kidney disease (CKD) causes detrimental disturbances in the mineral balance, bone turnover, and development of severe VC. Our group has previously demonstrated expression of Wnt inhibitors in calcified arteries of CKD rats. Therefore, we hypothesized that the CKD-induced VC via this pathway signals to bone and induces bone loss. To address this novel hypothesis, we developed a new animal model using isogenic aorta transplantation (ATx). Severely calcified aortas from uremic rats were transplanted into healthy rats (uremic ATx). Transplantation of normal aortas into healthy rats (normal ATx) and age-matched rats (control) served as control groups. Trabecular tissue mineral density, as measured by μCT, was significantly lower in uremic ATx rats compared with both control groups. Uremic ATx rats showed a significant upregulation of the mineralization inhibitors osteopontin and progressive ankylosis protein homolog in bone. In addition, we found significant changes in bone mRNA levels of several genes related to extracellular matrix, bone turnover, and Wnt signaling in uremic ATx rats, with no difference between normal ATx and control. The bone histomorphometry analysis showed significant lower osteoid area in uremic ATx compared with normal ATx along with a trend toward fewer osteoblasts as well as more osteoclasts in the erosion lacunae. Uremic ATx and normal ATx had similar trabecular number and thickness. The bone formation rate did not differ between the three groups. Plasma biochemistry, including sclerostin, kidney, and mineral parameters, were similar between all three groups. ex vivo cultures of aorta from uremic rats showed high secretion of the Wnt inhibitor sclerostin. In conclusion, the presence of VC lowers BMD, impairs bone metabolism, and affects several pathways in bone. The present results prove the existence of a vasculature to bone tissue cross-talk. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

Low turnover osteodystrophy and vascular calcification are amenable to skeletal anabolism in an animal model of chronic kidney disease and the metabolic syndrome

LDL receptor (LDLR)-null mice fed high-fat/cholesterol diets, a model of the metabolic syndrome, have vascular calcification (VC) worsened by chronic kidney disease (CKD) and ameliorated by bone morphogenetic protein-7 (BMP-7), an efficacious agent in treating animal models of renal osteodystrophy. Here, LDLR-/- high-fat-fed mice without CKD were shown to have significant reductions in bone formation rates, associated with increased VC and hyperphosphatemia. Superimposing CKD resulted in a low turnover osteodystrophy, whereas VC worsened and hyperphosphatemia persisted. BMP-7 treatment corrected the hyperphosphatemia, corrected the osteodystrophy, and prevented VC, compatible with skeletal phosphate deposition leading to reduced plasma phosphate and removal of a major stimulus to VC. A pathologic link between abnormal bone mineralization and VC through the serum phosphorus was supported by the partial effectiveness of directly reducing the serum phosphate by a phosphate binder that had no skeletal action. Thus, in this model of the metabolic syndrome with CKD, a reduction in bone-forming potential of osteogenic cells leads to low bone turnover rates, producing hyperphosphatemia and VC, processes ameliorated by the skeletal anabolic agent BMP-7, in part through deposition of phosphate and increased bone formation.     

Successful treatment of an adynamic bone disorder with bone morphogenetic protein-7 in a renal ablation model

An adynamic bone disorder (ABD) is an important complication of chronic kidney disease (CKD) of unknown etiology for which there is no adequate treatment. Reported is an animal model of ablative CKD complicated by an ABD characterized by the absence of secondary hyperparathyroidism and its successful treatment with a skeletal anabolic factor, bone morphogenetic protein-7 (BMP-7). Adult mice were subjected to electrocautery of the right kidney followed by left nephrectomy. Animals were randomized into groups fed normal chow or fed low-phosphate chow supplemented with calcitriol to maintain normophosphatemia in CKD. All groups were maintained on the regimens for 12 wk. Hyperphosphatemia, secondary hyperparathyroidism, and a mild osteodystrophy developed in the CKD/chow-fed group, as expected. When dietary phosphorus was restricted and calcitriol was administered in the CKD low-phosphate/calcitriol group (ABD), Ca, PO(4), and parathyroid hormone levels were maintained normal. A significant ABD developed in the ABD group characterized by significant depressions in osteoblast number, perimeters, bone formation rates, and mineral apposition rates when compared with the sham-operated, chow-fed group. The abnormal skeletal histomorphometry was reversed by BMP-7 therapy to normal values and significantly improved from the ABD group (P < 0.05). The sham-operated low-phosphate/calcitriol-fed control group and the CKD low-phosphate/calcitriol/BMP-7 groups had reduced phosphate levels compared with the other groups (P < 0.05). ABD produced in mice with CKD in the absence of hyperparathyroidism was successfully reversed with a bone anabolic, BMP-7, associated with a reduction in plasma phosphorus.     

Reversal of the adynamic bone disorder and decreased vascular calcification in chronic kidney disease by sevelamer carbonate therapy

A model of chronic kidney disease (CKD)-induced vascular calcification (VC) that complicates the metabolic syndrome was produced. In this model, the metabolic syndrome is characterized by severe atherosclerotic plaque formation, hypertension, type 2 diabetes, obesity, and hypercholesterolemia, and CKD stimulates calcification of the neointima and tunica media of the aorta. The CKD in this model is associated the adynamic bone disorder form of renal osteodystrophy. The VC of the model is associated with hyperphosphatemia, and control of the serum phosphorus both in this animal model and in humans has been preventive in the development of VC. This article reports studies that demonstrate reduction of established VC by the addition of sevelamer carbonate to the diets of this murine metabolic syndrome model with CKD. Sevelamer, besides normalizing the serum phosphorus, surprisingly, reversed the CKD-induced trabecular osteopenia. Sevelamer therapy increased osteoblast surfaces in the metaphyseal trabeculae of the tibia and femur. It also increased osteoid surfaces and, importantly, bone formation rates. In addition, sevelamer was found to be effective in decreasing serum cholesterol levels. These results suggest that sevelamer may have important actions in decreasing diabetic and uremic vasculopathy and that sevelamer carbonate may be capable of increasing bone formation rates that are suppressed by diabetic nephropathy.     

Interferon‐γ plays a role in bone formation in vivo and rescues osteoporosis in ovariectomized mice

Interferon γ (IFN‐γ) is a cytokine produced locally in the bone microenvironment by cells of immune origin as well as mesenchymal stem cells. However, its role in normal bone remodeling is still poorly understood. In this study we first examined the consequences of IFN‐γ ablation in vivo in C57BL/6 mice expressing the IFN‐γ receptor knockout phenotype (IFNγR1^?/?). Compared with their wild‐type littermates (IFNγR1^+/+), IFNγR1^?/? mice exhibit a reduction in bone volume associated with significant changes in cortical and trabecular structural parameters characteristic of an osteoporotic phenotype. Bone histomorphometry of IFNγR1^?/? mice showed a low‐bone‐turnover pattern with a decrease in bone formation, a significant reduction in osteoblast and osteoclast numbers, and a reduction in circulating levels of bone‐formation and bone‐resorption markers. Furthermore, administration of IFN‐γ (2000 and 10,000 units) to wild‐type C57BL/6 sham‐operated (SHAM) and ovariectomized (OVX) female mice significantly improved bone mass and microarchitecture, mechanical properties of bone, and the ratio between bone formation and bone resorption in SHAM mice and rescued osteoporosis in OVX mice. These data therefore support an important physiologic role for IFN‐γ signaling as a potential new anabolic therapeutic target for osteoporosis. © 2011 American Society for Bone and Mineral Research.     

Telomerase‐deficient mice exhibit bone loss owing to defects in osteoblasts and increased osteoclastogenesis by inflammatory microenvironment

Telomere shortening owing to telomerase deficiency leads to accelerated senescence of human skeletal (mesenchymal) stem cells (MSCs) in vitro, whereas overexpression leads to telomere elongation, extended life span, and enhanced bone formation. To study the role of telomere shortening in vivo, we studied the phenotype of telomerase‐deficient mice (Terc^?/?). Terc^?/? mice exhibited accelerated age‐related bone loss starting at 3 months of age and during 12 months of follow‐up revealed by dual‐energy X‐ray absorptiometric (DXA) scanning and by micro–computed tomography (µCT). Bone histomorphometry revealed decreased mineralized surface and bone‐formation rate as well as increased osteoclast number and size in Terc^?/? mice. Also, serum total deoxypyridinoline (tDPD) was increased in Terc^?/? mice. MSCs and osteoprogenitors isolated from Terc^?/? mice exhibited intrinsic defects with reduced proliferating cell number and impaired osteogenic differentiation capacity. In addition, the Terc^?/?‐MSC cultures accumulated a larger proportion of senescent β‐galactosidase⁺ cells and cells exhibiting DNA damage. Microarray analysis of Terc^?/? bone revealed significant overexpression of a large number of proinflammatory genes involved in osteoclast (OC) differentiation. Consistently, serum obtained from Terc^?/? mice enhanced OC formation of wild‐type bone marrow cultures. Our data demonstrate two mechanisms for age‐related bone loss caused by telomerase deficiency: intrinsic osteoblastic defects and creation of a proinflammatory osteoclast‐activating microenvironment. Thus telomerization of MSCs may provide a novel approach for abolishing age‐related bone loss. © 2011 American Society for Bone and Mineral Research.     

Genetic Disruption of All NO Synthase Isoforms Enhances BMD and Bone Turnover in Mice In Vivo: Involvement of the Renin‐Angiotensin System

Introduction : NO is synthesized by three different NO synthase (NOS) isoforms, including neuronal (nNOS), inducible (iNOS) and endothelial NOS (eNOS). The roles of NO in bone metabolism have been extensively investigated in pharmacological studies and in studies with NOS isoform–deficient mice. However, because of the nonspecificity of agents and compensation among the NOS isoforms, the ultimate roles of endogenous NO are still poorly understood. To address this point, we successfully generated mice in which all three NOS genes are completely disrupted. In this study, we examined whether bone metabolism is abnormal in those mice. Materials and Methods : Experiments were performed in 12‐wk‐old male wildtype, singly nNOS^?/?, iNOS^?/?, and eNOS^?/? and triply n/i/eNOS^?/? mice. BMD was assessed by DXA. The kinetics of osteoblastic bone formation and those of osteoclastic bone resorption were evaluated by measurements of morphological and biochemical markers. Results : BMD was significantly higher only in the triply NOS^?/? mice but not in any singly NOS^?/? mice compared with the wildtype mice. Markers of osteoblastic bone formation, including bone formation rate, mineral apposition rate, and serum alkaline phosphatase concentration, were also significantly larger only in the triply NOS^?/? mice compared with wildtype mice. Furthermore, markers of osteoclastic bone resorption, including osteoclast number, osteoclast surface, and urinary deoxypyridinoline excretion, were again significantly greater only in the triply NOS^?/? mice. Importantly, the renin‐angiotensin system in bone was significantly activated in the triply NOS^?/? mice, and long‐term oral treatment with an angiotensin II type 1 (AT₁) receptor blocker normalized this pathological bone remodeling in those mice. Conclusions : These results provide the first direct evidence that genetic disruption of the whole NOS system enhances BMD and bone turnover in mice in vivo through the AT₁ receptor pathway, showing the critical role of the endogenous NO/NOS system in maintaining bone homeostasis.     

Endogenous parathyroid hormone–related protein compensates for the absence of parathyroid hormone in promoting bone accrual in vivo in a model of bone marrow ablation

To assess the effect of hypoparathyroidism on osteogenesis and bone turnover in vivo, bone marrow ablation (BMXs) were performed in tibias of 8‐week‐old wild‐type and parathyroid hormone–null (PTH^?/?) mice and newly formed bone tissue was analyzed from 5 days to 3 weeks after BMX. At 1 week after BMX, trabecular bone volume, osteoblast numbers, alkaline phosphatase‐positive areas, type I collagen‐positive areas, PTH receptor–positive areas, calcium sensing receptor–positive areas, and expression of bone formation–related genes were all decreased significantly in the diaphyseal regions of bones of PTH^?/? mice compared to wild‐type mice. In contrast, by 2 weeks after BMX, all parameters related to osteoblastic bone accrual were increased significantly in PTH^?/? mice. At 5 days after BMX, active tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts had appeared in wild‐type mice but were undetectable in PTH^?/? mice, Both the ratio of mRNA levels of receptor activator of NF‐κB ligand (RANKL)/osteoprotegerin (OPG) and TRAP‐positive osteoclast surface were still reduced in PTH^?/? mice at 1 week but were increased by 2 weeks after BMX. The expression levels of parathyroid hormone–related protein (PTHrP) at both mRNA and protein levels were upregulated significantly at 1 week and more dramatically at 2 weeks after BMX in PTH^?/? mice. To determine whether the increased newly formed bones in PTH^?/? mice at 2 weeks after BMX resulted from the compensatory action of PTHrP, PTH^?/?PTHrP^+/? mice were generated and newly formed bone tissue was compared in these mice with PTH^?/? and wild‐type mice at 2 weeks after BMX. All parameters related to osteoblastic bone formation and osteoclastic bone resorption were reduced significantly in PTH^?/?PTHrP^+/? mice compared to PTH^?/? mice. These results demonstrate that PTH deficiency itself impairs osteogenesis, osteoclastogenesis, and osteoclastic bone resorption, whereas subsequent upregulation of PTHrP in osteogenic cells compensates by increasing bone accrual. © 2013 American Society for Bone and Mineral Research     

Connections between vascular calcification and progression of chronic kidney disease: therapeutic alternatives

We have shown that renal injury and chronic kidney disease (CKD) directly inhibit skeletal anabolism, and that stimulation of bone formation decreases the serum phosphate. Most recently, these observations were rediscovered in low-density lipoprotein receptor null mice fed high-fat/cholesterol diets, a model of the metabolic syndrome (hypertension, obesity, dyslipidemia, and insulin resistance). We had demonstrated that these mice have vascular calcification (VC) of both the intimal atherosclerotic type and medial type. We have shown that VC is worsened by CKD and ameliorated by bone morphogenetic protein -7 (BMP-7). The finding that high-fat-fed low-density lipoprotein receptor null animals without CKD have hyperphosphatemia led us to examine the skeletons of these mice. We found significant reductions in bone formation rates, associated with increased VC and superimposing CKD results in the adynamic bone disorder (ABD), while VC was worsened and hyperphosphatemia persisted. A pathological link between abnormal bone mineralization and VC through the serum phosphorus was demonstrated by the partial effectiveness of directly reducing the serum phosphate by a phosphate binder that had no skeletal action. BMP-7 treatment corrected the ABD and corrected hyperphosphatemia, compatible with BMP-7-driven stimulation of skeletal phosphate deposition reducing plasma phosphate and thereby removing a major stimulus to VC. Thus, in the metabolic syndrome with CKD, a reduction in bone-forming potential of osteogenic cells leads to ABD producing hyperphosphatemia and VC, processes ameliorated by the skeletal anabolic agent BMP-7, in part through increased bone formation and skeletal deposition of phosphate, and in part through direct actions on vascular smooth muscle cells. We have demonstrated that the processes leading to vascular calcification begin with even mild levels of renal injury before demonstrable hyperphosphatemia, and they are preventable and treatable. Therefore, early intervention in CKD is warranted and may affect mortality of the disease.     

Effects of intravenous zoledronic acid once yearly on bone remodeling and bone structure.

In a substudy of the HORIZON pivotal fracture trial, in which yearly intravenous zoledronic acid 5 mg was found to significantly reduce risk of various fracture types in patients with postmenopausal osteoporosis, 152 patients underwent bone biopsy. Zoledronic acid reduced bone turnover by 63% and preserved bone structure and volume, with evidence of ongoing bone remodeling in 99% of biopsies obtained. INTRODUCTION: In the HORIZON pivotal fracture trial (PFT), enrolling 7,736 women with postmenopausal osteoporosis, three annual intravenous infusions of the bisphosphonate zoledronic acid (5 mg) significantly reduced morphometric vertebral, clinical vertebral, hip, and nonvertebral fractures by 70%, 77%, 41%, and 25%, respectively. Whereas 79% of patients received zoledronic acid/placebo only (stratum I, n = 6,113), 21% received concomitant treatment with other antiresorptive drugs, excluding other bisphosphonates, PTH, and strontium (stratum II, n = 1,652). MATERIALS AND METHODS: To determine effects on bone remodeling and bone architecture, iliac crest bone biopsies were obtained in 152 patients on active treatment or placebo at 3 yr after double tetracycline labeling. In five patients, only qualitative histology was performed, leaving 147 biopsy cores (79 on active treatment and 68 on placebo) for microCT analysis and histomorphometry. RESULTS: Analysis of bone structure by microCT revealed higher trabecular bone volume (BV/TV) in the zoledronic acid group (median, 16.6% versus 12.8%; p = 0.020). In addition, patients treated with zoledronic acid exhibited higher trabecular numbers (p = 0.008), decreased trabecular separation (p = 0.011), and a trend toward improvement in connectivity density (p = 0.062), all indicating better preservation of trabecular structure after treatment with zoledronic acid. Qualitative analysis revealed presence of tetracycline label in 81 of 82 biopsies from patients on zoledronic acid and all 70 biopsies from placebo patients, indicative of continued bone remodeling. No bone pathology was observed. Zoledronic acid induced a 63% median (71% mean) reduction of the activation frequency (Ac.f; p < 0.0001) and reduced mineralizing surface (MS/BS; p < 0.0001) and volume referent bone formation rate (BFR/BV) versus placebo, indicating reduced bone turnover. Mineral appositional rate was higher in the zoledronic acid group (p = 0.0002), suggesting improved osteoblast function compared with placebo. Mineralization lag time was similar in the two groups, whereas osteoid volume (OV/BV; p < 0.0001) and osteoid thickness (O.Th; p = 0.0094) were lower in zoledronic acid-treated patients, indicating normal osteoid formation and mineralization of newly formed bone. Concomitant administration of other antiresorptive osteoporosis therapies (e.g., raloxifene, tamoxifen, tibolone, ipriflavone) did not significantly alter the tissue level response to zoledronic acid. CONCLUSIONS: Annual dosing for 3 yr with zoledronic acid 5 mg intravenously resulted in a median 63% (mean, 71%) reduction of bone turnover and preservation of bone structure and mass without any signs of adynamic bone. Concomitant treatment with other osteoporosis therapies did not significantly affect the bone response to zoledronic acid.     

Genetic Background Modifies the Effects of Type 2 Cannabinoid Receptor Deficiency on Bone Mass and Bone Turnover

Cannabinoid receptors and their ligands play significant roles in regulating bone metabolism. Previous studies of type 1 cannabinoid receptor-deficient mice have shown that genetic background influences the skeletal phenotype. Here, we investigated the effects of genetic background on the skeletal phenotype of mice with type 2 cannabinoid receptor deficiency (Cnr2 ^?/?). We studied Cnr2 ^?/? mice on a CD1 background and compared the findings with those previously reported in Cnr2 ^?/? C57BL/6 mice. Young female Cnr2 ^?/? CD1 mice had low bone turnover and high trabecular bone mass compared with wild-type (WT), contrasting with the situation in Cnr2 ^?/? C57BL/6 mice where trabecular bone mass has been reported to be similar to WT. The Cnr2 ^?/? CD1 mice lost more trabecular bone at the tibia with age than WT due to reduced bone formation, and at 12 months there was no difference in trabecular bone volume between genotypes. This differs from the phenotype previously reported in C57BL/6 Cnr2 ^?/? mice, where bone turnover is increased and bone mass reduced with age. There were no substantial differences in skeletal phenotype between Cnr2 ^?/? and WT in male mice. Cortical bone phenotype was similar in Cnr2 ^?/? and WT mice of both genders. Deficiency of Cnr2 has site- and gender-specific effects on the skeleton, mainly affecting trabecular bone, which are influenced by genetic differences between mouse strains. Further evaluation of the pathways responsible might yield new insights into the mechanisms by which cannabinoid receptors regulate bone metabolism.     

SDF‐1/CXCR4 Axis in Tie2‐Lineage Cells Including Endothelial Progenitor Cells Contributes to Bone Fracture Healing

CXC chemokine receptor 4 (CXCR4) is a specific receptor for stromal‐derived‐factor 1 (SDF‐1). SDF‐1/CXCR4 interaction is reported to play an important role in vascular development. On the other hand, the therapeutic potential of endothelial progenitor cells (EPCs) in fracture healing has been demonstrated with mechanistic insight of vasculogenesis/angiogenesis and osteogenesis enhancement at sites of fracture. The purpose of this study was to investigate the influence of the SDF‐1/CXCR4 pathway in Tie2‐lineage cells (including EPCs) in bone formation. We created CXCR4 gene conditional knockout mice using the Cre/loxP system and set two groups of mice: Tie2‐Cre^ER CXCR4 knockout mice (CXCR4^?/?) and wild‐type mice (WT). We report here that in vitro, EPCs derived from of CXCR4^?/? mouse bone marrow demonstrated severe reduction of migration activity and EPC colony‐forming activity when compared with those derived from WT mouse bone marrow. In vivo, radiological and morphological examinations showed fracture healing delayed in the CXCR4^?/? group and the relative callus area at weeks 2 and 3 was significantly smaller in CXCR4^?/? group mice. Quantitative analysis of capillary density at perifracture sites also showed a significant decrease in the CXCR4^?/? group. Especially, CXCR4^?/?group mice demonstrated significant early reduction of blood flow recovery at fracture sites compared with the WT group in laser Doppler perfusion imaging analysis. Real‐time RT‐PCR analysis showed that the gene expressions of angiogenic markers (CD31, VE‐cadherin, vascular endothelial growth factor [VEGF]) and osteogenic markers (osteocalcin, collagen 1A1, bone morphogenetic protein 2 [BMP2]) were lower in the CXCR4^?/? group. In the gain‐of‐function study, the fracture in the SDF‐1 intraperitoneally injected WT group healed significantly faster with enough callus formation compared with the SDF‐1 injected CXCR4^?/? group. We demonstrated that an EPC SDF‐1/CXCR4 axis plays an important role in bone fracture healing using Tie2‐Cre^ER CXCR4 conditional knockout mice. © 2014 American Society for Bone and Mineral Research.     

Role of Bcl2 in Osteoclastogenesis and PTH Anabolic Actions in Bone

Introduction : B‐cell leukemia/lymphoma 2 (Bcl2) is a proto‐oncogene best known for its ability to suppress cell death. However, the role of Bcl2 in the skeletal system is unknown. Bcl2 has been hypothesized to play an important anti‐apoptotic role in osteoblasts during anabolic actions of PTH. Although rational, this has not been validated in vivo; hence, the impact of Bcl2 in bone remains unknown. Materials and Methods : The bone phenotype of Bcl2 homozygous mutant (Bcl2^?/?) mice was analyzed with histomorphometry and μCT. Calvarial osteoblasts were isolated and evaluated for their cellular activity. Osteoclastogenesis was induced from bone marrow cells using RANKL and macrophage‐colony stimulating factor (M‐CSF), and their differentiation was analyzed. PTH(1–3;34) (50 μg/kg) or vehicle was administered daily to Bcl2^+/+ and Bcl2^?/? mice (4 days old) for 9 days to clarify the influence of Bcl2 ablation on PTH anabolic actions. Western blotting and real‐time PCR were performed to detect Bcl2 expression in calvarial osteoblasts in response to PTH ex vivo. Results : There were reduced numbers of osteoclasts in Bcl2^?/? mice, with a resultant increase in bone mass. Bcl2^?/? bone marrow–derived osteoclasts ex vivo were significantly larger in size and short‐lived compared with wildtype, suggesting a pro‐apoptotic nature of Bcl2^?/? osteoclasts. In contrast, osteoblasts were entirely normal in their proliferation, differentiation, and mineralization. Intermittent administration of PTH increased bone mass similarly in Bcl2^+/+ and Bcl2^?/? mice. Finally, Western blotting and real‐time PCR showed that Bcl2 levels were not induced in response to PTH in calvarial osteoblasts. Conclusions : Bcl2 is critical in osteoclasts but not osteoblasts. Osteoclast suppression is at least in part responsible for increased bone mass of Bcl2^?/? mice, and Bcl2 is dispensable in PTH anabolic actions during bone growth.     

LIM kinase 1 deficient mice have reduced bone mass

The cytoskeleton determines cell shape and is involved in cell motility. It also plays a role in differentiation and in modulating specialized cellular functions. LIM kinase 1 (LIMK1) participates in cytoskeletal remodeling by phosphorylating and inactivating the actin-severing protein, cofilin. Severing F-actin to release G-actin monomers is required for actin cytoskeletal remodeling. Although less well established, LIMK1 may also influence the cell cycle and modulate metalloproteinase activity. Since the role of LIMK1 in bone cell biology has not been reported, the skeletal phenotype of LIMK1^?/? mice was examined. LIMK1^?/? mice had significantly reduced trabecular bone mass when analyzed by microCT (p < 0.01). Histomorphometric analyses demonstrated a 31% reduction in the number of osteoblasts (p = 0.0003) and a 23% reduction in osteoid surface (p = 0.0005). The number of osteoclasts was no different in control and knock out animals. Consistent with the in vivo findings in osteoblasts, the number of osteoblast colony forming units in LIMK1^?/? bone marrow was reduced by nearly 50%. Further, osteoblasts isolated from LIMK1^?/? mice showed significantly reduced rates of mineralization in vitro. Osteoclasts from LIMK1^?/? mice evidenced more rapid cytoskeletal remodeling in response to treatment with CSF1. In keeping with this latter finding, basal levels of phospho-cofilin were reduced in LIMK1^?/? osteoclasts. LIMK1^?/? osteoclasts also resorbed dentine slices to a greater extent in vitro and were more active in a pit assay. These data support the hypothesis that LIMK1 is required for normal osteoblast differentiation. In addition, its absence leads to increased cytoskeletal remodeling and bone resorption in osteoclasts.     

18F-fluoride Positron Emission Tomography Measurements of Regional Bone Formation in Hemodialysis Patients with Suspected Adynamic Bone Disease

¹⁸F-fluoride positron emission tomography (¹⁸F-PET) allows the assessment of regional bone formation and could have a role in the diagnosis of adynamic bone disease (ABD) in patients with chronic kidney disease (CKD). The purpose of this study was to examine bone formation at multiple sites of the skeleton in hemodialysis patients (CKD5D) and assess the correlation with bone biopsy. Seven CKD5D patients with suspected ABD and 12 osteoporotic postmenopausal women underwent an ¹⁸F-PET scan, and bone plasma clearance, K _i, was measured at ten skeletal regions of interest (ROI). Fifteen subjects had a transiliac bone biopsy following double tetracycline labeling. Two CKD5D patients had ABD confirmed by biopsy. There was significant heterogeneity in K _i between skeletal sites, ranging from 0.008 at the forearm to 0.028 mL/min/mL at the spine in the CKD5D group. There were no significant differences in K _i between the two study groups or between the two subjects with ABD and the other CKD5D subjects at any skeletal ROI. Five biopsies from the CKD5D patients had single tetracycline labels only, including the two with ABD. Using an imputed value of 0.3 μm/day for mineral apposition rate (MAR) for biopsies with single labels, no significant correlations were observed between lumbar spine K _i corrected for BMAD (K _i/BMAD) and bone formation rate (BFR/BS), or MAR. When biopsies with single labels were excluded, a significant correlation was observed between K _i/BMAD and MAR (r = 0.81, p = 0.008) but not BFR/BS. Further studies are required to establish the sensitivity of ¹⁸F-PET as a diagnostic tool for identifying CKD patients with ABD.     

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.     

The P2Y13 receptor regulates extracellular ATP metabolism and the osteogenic response to mechanical loading

ATP release and subsequent activation of purinergic receptors has been suggested to be one of the key transduction pathways activated by mechanical stimulation of bone. The P2Y₁₃ receptor, recently found to be expressed by osteoblasts, has been suggested to provide a negative feedback pathway for ATP release in different cell types. Therefore, we hypothesized that the P2Y₁₃ receptor may contribute to the mediation of osteogenic responses to mechanical stimulation by regulating ATP metabolism by osteoblasts. To test this hypothesis, wild‐type (WT) and P2Y₁₃ receptor knockout (P2Y₁₃R^?/?) mice were subject to non‐invasive axial mechanical loading of the left tibiae to induce an osteogenic response. Micro‐computed tomography analysis showed mechanical loading induced an osteogenic response in both strains of mice in terms of increased total bone volume and cortical bone volume, with the P2Y₁₃R^?/? mice having a significantly greater response. The extent of the increased osteogenic response was defined by dynamic histomorphometry data showing dramatically increased bone formation and mineral apposition rates in P2Y₁₃R^?/? mice compared with controls. In vitro, primary P2Y₁₃R^?/? osteoblasts had an accumulation of mechanically induced extracellular ATP and reduced levels of hydrolysis. In addition, P2Y₁₃R^?/? osteoblasts also had a reduction in their maximal alkaline phosphatase (ALP) activity, one of the main ecto‐enzymes expressed by osteoblasts, which hydrolyzes extracellular ATP. In conclusion, deletion of the P2Y₁₃ receptor leads to an enhanced osteogenic response to mechanical loading in vivo, possibly because of the reduced extracellular ATP degradation by ALP. The augmented osteogenic response to mechanical stimulation, combined with suppressed bone remodeling activities and protection from OVX‐induced bone loss after P2Y₁₃ receptor depletion as previously described, suggests a potential role for P2Y₁₃ receptor antagonist‐based therapy, possibly in combination with mechanical loading, for the treatment of osteoporosis.