Increase In Number Of Osteoblasts Research Articles

Nephrectomy Does not Exacerbate Cancellous Bone loss in Thalassemic Mice

Patients with β-thalassemia have an increased risk of developing chronic kidney disease which is associated with osteoporosis and periodontitis. The purpose of this study was to evaluate mandibular and femoral bone change in heterozygous β-globin knockout (BKO) mice following 5/6 nephrectomy (Nx). Female and male BKO mouse blood smears demonstrated microcytic hypochromic anemia. Serum urea nitrogen, creatinine, calcium, and phosphorus levels were not changed in BKO mice. Nx increased the serum levels of urea nitrogen in both wild type (WT) and BKO mice and the level was much higher in BKO males. Serum level of creatinine was increased in Nx WT but not BKO mice. However, serum calcium and phosphorus levels were not altered. Nx induced comparable renal fibrosis in BKO mice and WT controls. Bone loss was observed in mandibular cancellous bone but not cortical bone of both male and female BKO mice. Nx decreased cancellous bone volume and cortical thickness in WT. Interestingly, BKO mice were resistant to Nx-induced cancellous bone loss. However, cortical thickness and cortical bone mineral density were reduced in Nx male BKO mice. Nx increased mRNA levels of type I collagen, Osx and Trap in WT but not BKO mice. Similarly, Nx reduced cancellous bone volume in femurs and increased osteoblast number and osteoclast number in WT not BKO mice. Serum FGF23 and erythropoietin levels were markedly increased in BKO mice. Nx decreased serum erythropoietin but not FGF23 levels. Since WT treated with erythropoietin exhibited a significant reduction in cancellous bone volume, it was possible that lower level of erythropoietin in Nx BKO mice prevented the Nx-induced cancellous bone loss.

OZONE IMPROVES AUTOGENOUS GRAFT HEALING IN EXPERIMENTAL DIABETES MELLITUS: A MORPHOMETRIC AND IMMUNOHISTOCHEMICAL STUDY

Backround: Diabetes mellitus (DM) is a metabolic disorder which plays crucial role in the pathogenesis of periodontitis. Ozone have several actions such as antimicrobial ,immunostimulating, antihypoxic effects, and activates antioxidant enzymes and angiogenesis Aim : The aim of this study is to investigate the effect of gaseous ozone on bone healing in diabetic rat calvarial defects treated with autogenous bone graft, morphometrically and immunohistochemically. Material and Methods: Diabetes was induced and critical size defects were created on rats. Study groups: 1-Empty defect (Control, n=14) group, 2-Autograft (AG, n=14) group, 3-Empty defect+ozone therapy (Control+Ozone, n=14) group, 4-Autograft+ozone application (AG+Ozone, n=14) group. Gaseous ozone was applied on the operation day and the following 2 weeks daily ( 140ppm @ 2L/d, 2.24 mg ). Total bone area was measured. Osteocalcin and Bone morphogenic protein-2 protein expressions were evaluated . Results : Control and Control+Ozone groups had no osteoclast and residuel lacunae during the study . Osteoblasts in AG+Ozone group were higher than AG group at 4th week (p>0.05). AG+Ozone group had more total bone area than AG group at 4th week. AG+Ozone group revealed more BMP-2 immune positivity compared to the other groups. Osteocalcin immune positivity in AG groups was higher than those of the Control groups. Conclusion : Within the limitations of this study, gaseous ozone application decreased osteoclast number and increased osteoblast number and bone regeneration, especially, in early stages of bone regeneration in diabetic rats. KEYWORDS: autograft, bone regeneration , calvarial defect , experimental diabetes mellitus , ozone

Postnatal Conditional Deletion of Bmal1 in Osteoblasts Enhances Trabecular Bone Formation Via Increased BMP2 Signals.

A large number of studies in recent years indicated the involvement of peripheral circadian clock in varied pathologies. However, evidence regarding how peripheral clocks regulate bone metabolism is still very limited. The present study aimed to investigate the direct role of Bmal1 (the key activator of peripheral circadian clock system) in vivo during bone developmental and remodeling stages using inducible osteoblast-specific Bmal1 knockout mice. Unexpectedly, the removal of Bmal1 in osteoblasts caused multiple abnormalities of bone metabolism, including a progressive increase in trabecular bone mass in as early as 8 weeks, manifested by an 82.3% increase in bone mineral density and 2.8-fold increase in bone volume per tissue volume. As mice age, an increase in trabecular bone mass persists while cortical bone mass decreases by about 33.7%, concomitant with kyphoscoliosis and malformed intervertebral disk. The increased trabecular bone mass is attributed to increased osteoblast number and osteoblast activity coupled with decreased osteoclastogenesis. Remarkably, the ablation of Bmal1 in osteoblasts promoted the expression level of Bmp2 and phosphorylation of SMAD1, whereas the attenuation of BMP2/SMAD1 signaling partially alleviated the effects of Bmal1 deficiency on osteoblast differentiation and activity. The results revealed that Bmal1 was a transcriptional silencer of Bmp2 by targeting the Bmp2 promoter. The peripheral clock gene Bmal1 in osteoblasts was crucial to coordinate differential effects on trabecular and cortical bones through regulating BMP2/SMAD1 during bone development, thus providing novel insights into a key role of osteoblast Bmal1 in homeostasis and integrity of adult bones. © 2020 American Society for Bone and Mineral Research.

A chitosan film containing quercetin-loaded transfersomes for treatment of secondary osteoporosis.

Osteoporosis, due to its prevalence worldwide, is a serious health problem. Topical administration of quercetin, a phytoestrogen, in the form of deformable transfersomes, could be used to treat osteoporosis to overcome its low oral solubility and bioavailability. Formulation process of transfersomes was screened by fractional factorial design and further optimized using full factorial design. Transfersomes showed good characteristics such as entrapment efficiency, particle size, zeta potential, and polydispersity index (83.0 ± 2.2%, 75.95 ± 2nm, - 13.6 ± 6mv and 0.333, respectively). Transfersomes were further loaded into chitosan film and showed good permeation through rat skin. Further, glucocorticoid-induced osteoporosis rat model showed induction of osteoporosis after day 30. On day 45, treatment with chitosan film containing quercetin-loaded transfersomes showed remarkable rise in femur thickness, length, density as well as in serum biochemical parameters such as calcium, phosphorous, alkaline phosphatase, and tartrate-resistant alkaline phosphatase compared to positive control group. Tensile strength of osteoporotic femur bone was also found to be increased and was comparable with normal group. Histomicrographic analysis of femur bone exhibited less disruptive and lytic changes. Thus, all the above findings indicated the beneficial effects of quercetin-loaded transfersome chitosan film, due to decline in osteoclastogenesis and osteoblast apoptosis, which further favored increase in osteoblast numbers and mineralization of bones. Thus, chitosan film containing quercetin-loaded transfersomes was found to be good alternative to oral administration of quercetin to treat osteoporosis, while easy applicability of film in the form of wrist band anytime, anywhere, and even at work achieve patient compliance. Graphical abstract.

Evaluation of Neo-Osteogenesis in Eosinophilic Chronic Rhinosinusitis Using a Nasal Polyp Murine Model.

PurposeOsteitis refers to the development of new bone formation and remodeling of bone in chronic rhinosinusitis (CRS) patients; it is typically associated with eosinophilia, nasal polyps (NPs), and recalcitrant CRS. However, the roles of ossification in CRS with or without NPs remain unclear due to the lack of appropriate animal models. Thus, it is necessary to have a suitable animal model for greater advances in the understanding of CRS pathogenesis.MethodsBALB/c mice were administered ovalbumin (OVA) and staphylococcal enterotoxin B (SEB). The numbers of osteoclasts and osteoblasts and bony changes were assessed. Micro computed tomography (micro-CT) scans were conducted to measure bone thickness. Immunofluorescence, immunohistochemistry, and quantitative polymerase chain reaction were performed to evaluate runt-related transcription factor 2 (RUNX2), osteonectin, interleukin (IL)-13, and RUNX2 downstream gene expression. Gene set enrichment analysis was performed in mucosal tissues from control and CRS patients. The effect of resveratrol was evaluated in terms of osteogenesis in a murine eosinophilic CRS NP model.ResultsThe histopathologic changes showed markedly thickened bones with significant increase in osteoblast numbers in OVA/SEB-treated mice compared to the phosphate-buffered saline-treated mice. The structural changes in bone on micro-CT were consistent with the histopathological features. The expression of RUNX2 and IL-13 was increased by the administration of OVA/SEB and showed a positive correlation. RUNX2 expression mainly co-localized with osteoblasts. Bioinformatic analysis using human CRS transcriptome revealed that IL-13-induced bony changes via RUNX2. Treatment with resveratrol, a candidate drug against osteitis, diminished the expression of IL-13 and RUNX2, and the number of osteoblasts in OVA/SEB-treated mice.ConclusionsIn the present study, we found the histopathological and radiographic evidence of osteogenesis using a previously established murine eosinophilic CRS NP model. This animal model could provide new insights into the pathophysiology of neo-osteogenesis and provide a basis for developing new therapeutics.

Mature Osteoblasts Promote Multiple Myeloma Survival through Cell-Cell Contact and Immune Modulation Mechanisms

The bone marrow (BM) microenvironment (TME) is a complex mix of cellular and non-cellular components that has a profound effect on hematopoiesis and hematological malignancies. Multiple myeloma (MM), a tumor of plasma cells that originates in the BM is highly influenced by TME components including stromal cells, osteoclasts, and immune cells. Among these cells, osteoblasts are the most versatile regulators of hematopoiesis and also play an integral role in malignant transformations of leukemia and lung cancer. MM is characterized by osteolytic bone lesions partly due to decreased numbers of osteoblasts. Therapies such as denosumab and zoledronic acid aim to restore bone health by inhibiting osteoclastogenesis. However, the contribution of osteoblasts to the initiation and progression of MM is not well understood. We previously demonstrated that increased osteoblast numbers using Activin A inhibition in an in vivo humanized myeloma model resulted in inhibition of MM growth (Vallet et al., 2010). Given that osteoblast numbers are on the decline in a typical MM patient, we hypothesized that the loss of osteoblasts contributes to the initiation and progression of MM. To assess the effects of osteoblasts on MM cells, MC3T3 murine pre-osteoblasts were differentiated into mature osteoblasts by supplementing media with ascorbic acid and β-glycerophosphate. Co-culture of 5TGM1 murine MM cells with the osteoblasts showed a significant decrease in the proliferation of MM cells by 40% (N=4). Osteoblasts go through distinct stages of maturation i.e., pre-osteoblasts, committed osteoblasts, and mature osteoblasts. These individual osteoblast populations were FACS sorted from mice to isolate pre-osteoblasts from the long bones of Osterix-GFP+ (Osx+) mice, committed osteoblasts from Collagen 2.3-GFP+ (Col2.3+) mice, and mature osteoblasts from Osteocalcin-YFP+ (OCN+) mice. Each of the osteoblast populations was co-cultured with 5TGM1 MM cells. Although all the osteoblast populationssignificantly suppressed MM proliferation, the OCN+ mature osteoblasts suppressed MM proliferation the most (N=3). Therefore, we focused our studies on the role of mature osteoblasts in MM progression. We generated mice in which mature osteoblasts could be postnatally deleted in an inducible and reversible manner. To achieve this, mice carrying floxed diphtheria toxin receptor (DTR) alleles were mated with mice expressing Cre-recombinase driven by the osteocalcin promoter (OC-Cre) to generate OC-Cre/iDTR mice. The control mice were littermates lacking the OC-Cre allele. The OC-Cre/iDTR mice were indistinguishable from the controls until treated with diphtheria toxin (DT). To induce postnatal deletion of mature osteoblasts, the OC-Cre/iDTR and control mice both were treatedwith 50 µg/Kg DT once a week beginning at 8-weeks of age. To study MM engraftment and progression, 3x1065TGM1-Luciferase MM cells were injected into the tibia of OC-Cre/iDTR and control mice followed by weeklyinjection of DT for 8-weeks. Bioluminescence imaging (BLI) showed that 4-weeks onwards the OC-iDTR mice, but not the control mice, continued to express and increase the BLI signal (N=6). This data suggeststhatunder physiological conditions, mature osteoblasts activelysuppress MM engraftment and progression. We hypothesized that osteoblasts provide niche support to MM cells via direct cell-to-cell contact. To begin to identify the molecular mechanisms, we compared gene expression changes in primary murine osteoblasts between the undifferentiated and 30-day osteogenic differentiation time points. We also examined the BM TME by quantitative protein antibody arrays at 2-weeks following 5TGM1MM intratibial injection into OC-Cre/iDTR and control mice. The mature osteoblasts showed a significant increase in the expression of integrins, including integrin α4, and several immunomodulatory markers. The cytokine array analysis showed altered expressions of cell-cell communication proteins MAdCAM1, BAFF-R, TACI, and immunomodulatory factors IL-33, IL-17F, and IL-13. Taken together, using in vivo and in vitro models, we show that mature osteoblasts may have a negative regulatory impact on MM cells through cell-cell communication or immunomodulatory mechanisms. Expanding the osteoblast niche may provide novel therapeutic avenues to reduce disease burden and create an environment for long term tumor control. Disclosures Raje: Merck: Consultancy; Amgen Inc.: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene Corporation: Consultancy; Takeda: Consultancy; Janssen: Consultancy.

\u03b1SMA Osteoprogenitor Cells Contribute to the Increase in Osteoblast Numbers in Response to Mechanical Loading

Bone is a dynamic tissue that site-specifically adapts to the load that it experiences. In response to increasing load, the cortical bone area is increased, mainly through enhanced periosteal bone formation. This increase in area is associated with an increase in the number of bone-forming osteoblasts; however, the origin of the cells involved remains unclear. Alpha-smooth muscle actin (αSMA) is a marker of early osteoprogenitor cells in the periosteum, and we hypothesized that the new osteoblasts that are activated by loading could originate from αSMA-expressing cells. Therefore, we used an in vivo fate-mapping approach in an established axial loading model to investigate the role of αSMA-expressing cells in the load-induced increase in osteoblasts. Histomorphometric analysis was applied to measure the number of cells of different origin on the periosteal surface in the most load-responsive region of the mouse tibia. A single loading session failed to increase the number of periosteal αSMA-expressing cells and osteoblasts. However, in response to multiple episodes of loading, the caudal, but not the cranial, periosteal surface was lined with an increased number of osteoblasts originating from αSMA-expressing cells 5 days after the initial loading session. The proportion of osteoblasts derived from αSMA-labeled progenitors increased by 70% (p < 0.05), and the proportion of αSMA-labeled cells that had differentiated into osteoblasts was doubled. We conclude that αSMA-expressing osteoprogenitors can differentiate and contribute to the increase in periosteal osteoblasts induced by mechanical loading in a site-specific manner.

Deficiency of stress-associated gene Nupr1 increases bone volume by attenuating differentiation of osteoclasts and enhancing differentiation of osteoblasts.

Nuclear protein 1 (NUPR1) is a multifunctional stress-induced protein involved in regulating tumorigenesis, apoptosis, and autophagy. Bone homeostasis is maintained by bone-resorbing osteoclasts and bone-forming osteoblasts and osteocytes. We aimed to determine the role of NUPR1 in bone metabolism. Using microcomputed tomography, we found that mice lacking Nupr1 exhibited increased bone volume. Histologic analysis showed that Nupr1 deficiency decreased osteoclast numbers but increased osteoblast numbers and osteoid formation. In vitro culture of bone marrow macrophages showed that receptor activator of NF-κB ligand-induced osteoclastogenesis was down-regulated in Nupr1-deficient mice. In contrast, primary osteoblasts from Nupr1-deficient mice revealed that proliferation of osteoblasts and expression of bone matrix proteins were markedly enhanced. In addition, expression of autophagy-related genes, formation of autophagosomes, and cell survival were up-regulated in Nupr1-deficient osteoblasts. In contract, deletion of Nupr1 reduced the formation of osteocyte cellular projection, which is an indicator of mature osteocytes. Importantly, we found that the expression of sclerostin (Sost), an inhibitor of bone formation, was down-regulated in the osteoblasts and osteocytes of Nupr1-deficient mice. Conversely, Nupr1 overexpression enhanced Sost expression in primary osteoblasts. Collectively, these results indicate that Nupr1 deficiency increases bone volume by attenuating production of Sost and osteoclastogenesis and enhancing differentiation of osteoblasts.-Shiraki, M., Xu, X., Iovanna, J. L., Kukita, T., Hirata, H., Kamohara, A., Kubota, Y., Miyamoto, H., Mawatari, M., Kukita, A. Deficiency of stress-associated gene Nupr1 increases bone volume by attenuating differentiation of osteoclasts and enhancing differentiation of osteoblasts.

Agonist-induced activation of the S1P receptor 2 constitutes a novel osteoanabolic therapy for the treatment of osteoporosis in mice.

Osteoporosis is a worldwide epidemic but pharmacological agents to stimulate new bone formation are scarce. We have shown that increasing tissue levels of sphingosine-1-phosphate (S1P) by blocking its degradation by the S1P lyase has pronounced osteoanabolic effect in mouse osteoporosis models by stimulating osteoblast differentiation through the S1P receptor 2 (S1P2). However, S1P lyase inhibitors have side effects complicating potential clinical use. Here, we tested whether direct S1P2 engagement by the S1P2 agonist CYM5520 exerted osteoanabolic potential in estrogen deficiency-induced osteopenia in mice. We compared its efficacy to LX2931, a novel S1P lyase inhibitor currently tested in rheumatoid arthritis. CYM5520, LX2931 or vehicle were administered to ovariectomized mice for 6 weeks beginning 5 weeks after ovariectomy, Bone mass, cellular composition and mechanical strength were assessed by microCT, histomorphometry and three point bending tests. Plasma markers of bone metabolism were analyzed by ELISA. Therapeutic treatment with CYM5520 and LX2931 clearly increased long bone and vertebral bone mass to impressive 3-5 fold over vehicle in osteopenic ovariectomized mice. As expected, lymphopenia was a side effect of LX2931, whereas none occurred with CYM5520. Consistent with an osteoanabolic effect, CYM5520 increased osteoblast number, osteoid surface and alkaline phosphatase area 2-3 fold over vehicle. Plasma concentrations of the osteoanabolic marker procollagen I C-terminal propeptide were also elevated by CYM5520 and LX2931. LX2931 but not yet CYM5520 increased cortical thickness and mechanical strength without affecting mineral density. Treatment with a pharmacological S1P2 agonist corrected ovariectomy-induced osteopenia in mice by inducing new bone formation thus constituting a novel osteoanabolic approach to osteoporosis.

Calcimimetics maintain bone turnover in uremic rats despite the concomitant decrease in parathyroid hormone concentration

Calcimimetics decrease parathyroid hormone (PTH) secretion in patients with secondary hyperparathyroidism. The decrease in PTH should cause a reduction in bone turnover; however, the direct effect of calcimimetics on bone cells, which express the calcium-sensing receptor (CaSR), has not been defined. In this study, we evaluated the direct bone effects of CaSR activation by a calcimimetic (AMG 641) invitro and invivo. To create a PTH "clamp," total parathyroidectomy was performed in rats with and without uremia induced by 5/6 nephrectomy, followed by a continuous subcutaneous infusion of PTH. Animals were then treated with either the calcimimetic or vehicle. Calcimimetic administration increased osteoblast number and osteoid volume in normal rats under a PTH clamp. In uremic rats, the elevated PTH concentration led to reduced bone volume and increased bone turnover, and calcimimetic administration decreased plasma PTH. In uremic rats exposed to PTH at 6-fold the usual replacement dose, calcimimetic administration increased osteoblast number, osteoid surface, and bone formation. A 9-fold higher dose of PTH caused an increase in bone turnover that was not altered by the administration of calcimimetic. In an osteosarcoma cell line, the calcimimetic induced Erk1/2 phosphorylation and the expression of osteoblast genes. The addition of a calcilytic resulted in the opposite effect. Moreover, the calcimimetic promoted the osteogenic differentiation and mineralization of human bone marrow mesenchymal stem cells invitro. Thus, calcimimetic administration has a direct anabolic effect on bone that counteracts the decrease in PTH levels.

Vitamin A decreases the anabolic bone response to mechanical loading by suppressing bone formation.

Increased vitamin A consumption is associated with decreased cortical bone mass and increased fracture risk in humans. Rodent studies have demonstrated that hypervitaminosis A increases cortical bone resorption, whereas the importance of the effects on bone formation is less well defined. We used an experimental model of increased bone formation by loading of the tibiae to investigate the effect of vitamin A on bone formation. Control [retinol activity equivalents (RAE) 4.5 µg/g chow] or vitamin A (RAE 60 µg/g chow) diets were given to female C57BL/6N mice for 4 wk, after which the tibiae were subjected to axial loading on alternate days for 2 wk, while the diets were continued. Vitamin A inhibited the loading-induced increase in trabecular and cortical bone volume. This was attributed to inhibition of loading-induced increase in osteoblast number and activity, and expression of osteoblastic genes Sp7, Alpl, and Col1a1 in cortical bone. Vitamin A, loading, and combination thereof also resulted in site-specific effects on bone composition measured by Raman spectroscopy. In summary, a clinically relevant dose of vitamin A suppresses the loading-induced gain of bone mass by decreasing bone formation. These observations may have implications for regulation of bone mass caused by physical activity and the risk of osteoporosis in humans.—Lionikaite, V., Henning, P., Drevinge, C., Shah, F. A., Palmquist, A., Wikström, P., Windahl, S. H., Lerner, U. H. Vitamin A decreases the anabolic bone response to mechanical loading by suppressing bone formation.

Wnt1 is an Lrp5-independent bone-anabolic Wnt ligand.

WNT1 mutations in humans are associated with a new form of osteogenesis imperfecta and with early-onset osteoporosis, suggesting a key role of WNT1 in bone mass regulation. However, the general mode of action and the therapeutic potential of Wnt1 in clinically relevant situations such as aging remain to be established. Here, we report the high prevalence of heterozygous WNT1 mutations in patients with early-onset osteoporosis. We show that inactivation of Wnt1 in osteoblasts causes severe osteoporosis and spontaneous bone fractures in mice. In contrast, conditional Wnt1 expression in osteoblasts promoted rapid bone mass increase in developing young, adult, and aged mice by rapidly increasing osteoblast numbers and function. Contrary to current mechanistic models, loss of Lrp5, the co-receptor thought to transmit extracellular WNT signals during bone mass regulation, did not reduce the bone-anabolic effect of Wnt1, providing direct evidence that Wnt1 function does not require the LRP5 co-receptor. The identification of Wnt1 as a regulator of bone formation and remodeling provides the basis for development of Wnt1-targeting drugs for the treatment of osteoporosis.

Role of Osteocytes in Myeloma Bone Disease: Anti-sclerostin Antibody as New Therapeutic Strategy.

Osteocytes are terminally differentiated cells of the osteoblast lineage. They are involved in the regulation of bone remodeling by increasing osteoclast formation or decreasing bone formation by the secretion of the osteoblast inhibitor sclerostin. Monoclonal antibody anti-sclerostin, Romosozumab, has been developed and tested in clinical trials in patients with osteoporosis. In the last years, the role of osteocytes in the development of osteolytic bone lesions that occurs in multiple myeloma, have been underlined. Myeloma cells increase osteocyte death through the up-regulation of both apoptosis and autophagy that, in turn, triggers osteoclast formation, and activity. When compared to healthy controls, myeloma patients with bone disease have higher osteocyte cell death, but the treatment with proteasome inhibitor bortezomib has been shown to maintain osteocyte viability. In preclinical mouse models of multiple myeloma, treatment with blocking anti-sclerostin antibody increased osteoblast numbers and bone formation rate reducing osteolytic bone lesions. Moreover, the combination of anti-sclerostin antibody and the osteoclast inhibitor zoledronic acid increased bone mass and fracture resistance synergistically. However, anti-sclerostin antibody did not affect tumor burden in vivo or the efficacy of anti-myeloma drugs in vitro. Nevertheless, the combination therapy of anti-sclerostin antibody and the proteasome inhibitor carfilzomib, displayed potent anti-myeloma activity as well as positive effects on bone disease in vivo. In conclusion, all these data suggest that osteocytes are involved in myeloma bone disease and may be considered a novel target for the use of antibody-mediated anti-sclerostin therapy also in multiple myeloma patients.

Parathyroid Hormone (PTH) Increases Skeletal Tumour Growth and Alters Tumour Distribution in an In Vivo Model of Breast Cancer.

Breast cancer cells colonize the skeleton by homing to specific niches, but the involvement of osteoblasts in tumour cell seeding, colonization, and progression is unknown. We used an in vivo model to determine how increasing the number of cells of the osteoblast lineage with parathyroid hormone (PTH) modified subsequent skeletal colonization by breast cancer cells. BALB/c nude mice were injected for five consecutive days with PBS (control) or PTH and then injected with DiD-labelled breast cancer cells via the intra-cardiac route. Effects of PTH on the bone microenvironment and tumour cell colonization and growth was analyzed using bioluminescence imaging, two-photon microscopy, and histological analysis. PTH treatment caused a significant, transient increase in osteoblast numbers compared to control, whereas bone volume/structure in the tibia was unaffected. There were no differences in the number of tumour cells seeding to the tibias, or in the number of tumours in the hind legs, between the control and PTH group. However, animals pre-treated with PTH had a significantly higher number of tumour colonies distributed throughout skeletal sites outside the hind limbs. This is the first demonstration that PTH-induced stimulation of osteoblastic cells may result in alternative skeletal sites becoming available for breast cancer cell colonization.

Absence of complement factor H alters bone architecture and dynamics

Complement system is an important arm of the immune system that promotes inflammation. Complement Factor H (FH) is a critical regulator of the alternative complement pathway. Its absence causes pathology in different organs resulting in diseases such as age related macular degeneration and dense deposit disease. Recent studies suggest that the complement system plays a role in bone development and homeostasis. To determine the role of FH in bone architecture, we studied the FH knockout (fh-/-) mice. 3D reconstructions of femur from 16 week old fh-/- mice reveal significant changes, such as decreased BV/TV (4.5%, p < 0.02), trabecular number (22%, p < 0.01), tissue mineral density (16%, p < 0.04), and increased marrow area (16% p < 0.01), compared to their wild type (WT) counterparts. Kidney function and histology remained normal indicating that bone changes occurred prior to kidney dysfunction. Next we examined cultured osteoblasts and osteoclasts isolated from bone marrow. FH is expressed ubiquitously in the osteoblasts and in the cytoplasm of osteoclasts. The changes caused by absence of FH include: increase in number of osteoblasts (362%) and osteoclasts (342%), increase in RNA (180%) and protein expression of cathepsin K and increased osteoclast function (pit formation, 233%). Actin rearrangement in both osteoblasts and osteoclasts was altered, with a loss of integrity of the F-actin ring at the periphery of the osteoclasts. For the first time our studies demonstrate a direct role of FH in the maintenance of bone structure and function and is highlighted as a promising therapeutic target in bone diseases.

CGMP-dependent protein kinase-2 regulates bone mass and prevents diabetic bone loss.

NO/cGMP signaling is important for bone remodeling in response to mechanical and hormonal stimuli, but the downstream mediator(s) regulating skeletal homeostasis are incompletely defined. We generated transgenic mice expressing a partly-activated, mutant cGMP-dependent protein kinase type 2 (PKG2R242Q) under control of the osteoblast-specific Col1a1 promoter to characterize the role of PKG2 in post-natal bone formation. Primary osteoblasts from these mice showed a two- to three-fold increase in basal and total PKG2 activity; they proliferated faster and were resistant to apoptosis compared to cells from WT mice. Male Col1a1-Prkg2R242Q transgenic mice had increased osteoblast numbers, bone formation rates and Wnt/β-catenin-related gene expression in bone and a higher trabecular bone mass compared to their WT littermates. Streptozotocin-induced type 1 diabetes suppressed bone formation and caused rapid bone loss in WT mice, but male transgenic mice were protected from these effects. Surprisingly, we found no significant difference in bone micro-architecture or Wnt/β-catenin-related gene expression between female WT and transgenic mice; female mice of both genotypes showed higher systemic and osteoblastic NO/cGMP generation compared to their male counterparts, and a higher level of endogenous PKG2 activity may be responsible for masking effects of the PKG2R242Q transgene in females. Our data support sexual dimorphism in Wnt/β-catenin signaling and PKG2 regulation of this crucial pathway in bone homeostasis. This work establishes PKG2 as a key regulator of osteoblast proliferation and post-natal bone formation.

Oxidation-specific epitopes restrain bone formation

Atherosclerosis and osteoporosis are epidemiologically linked and oxidation specific epitopes (OSEs), such as phosphocholine (PC) of oxidized phospholipids (PC-OxPL) and malondialdehyde (MDA), are pathogenic in both. The proatherogenic effects of OSEs are opposed by innate immune antibodies. Here we show that high-fat diet (HFD)-induced bone loss is attenuated in mice expressing a single chain variable region fragment of the IgM E06 (E06-scFv) that neutralizes PC-OxPL, by increasing osteoblast number and stimulating bone formation. Similarly, HFD-induced bone loss is attenuated in mice expressing IK17-scFv, which neutralizes MDA. Notably, E06-scFv also increases bone mass in mice fed a normal diet. Moreover, the levels of anti-PC IgM decrease in aged mice. We conclude that OSEs, whether produced chronically or increased by HFD, restrain bone formation, and that diminished defense against OSEs may contribute to age-related bone loss. Anti-OSEs, therefore, may represent a novel therapeutic approach against osteoporosis and atherosclerosis simultaneously.

Injection of synthetic mesenchymal stem cell mitigates osteoporosis in rats after ovariectomy.

Osteoporosis is a severe skeletal disorder. Patients have a low bone mineral density and bone structural deterioration. Mounting lines of evidence suggest that inappropriate apoptosis of osteoblasts/osteocytes leads to maladaptive bone remodelling in osteoporosis. It has been suggested that transplantation of stem cells, including mesenchymal stem cells, may alter the trajectory of bone remoulding and mitigate osteoporosis in animal models. However, stem cells needed to be carefully stored and characterized before usage. In addition, there is great batch‐to‐batch variation in stem cell production. Here, we fabricated therapeutic polymer microparticles from the secretome and membranes of mesenchymal stem cells (MSCs). These synthetic MSCs contain growth factors secreted by MSCs. In addition, these particles display MSC surface molecules. In vitro, co‐culture with synthetic MSCs increases the viability of osteoblast cells. In a rat model of ovariectomy‐induced osteoporosis, injection of synthetic MSCs mitigated osteoporosis by reducing cell apoptosis and systemic inflammation, but increasing osteoblast numbers. Synthetic MSC offers a promising therapy to manage osteoporosis.

Selective inhibition of Src family kinases by SU6656 increases bone mass by uncoupling bone formation from resorption in mice

Mice deficient in the non-receptor tyrosine kinase Src exhibit high bone mass due to impaired bone resorption and increased bone formation. Although several Src family kinase inhibitors inhibit bone resorption in vivo, they display variable effects on bone formation. SU6656 is a selective Src family kinase inhibitor with weaker activity towards the non-receptor tyrosine kinase Abl and receptor tyrosine kinases which are required for appropriate osteoblast proliferation, differentiation and function. Therefore, we sought to determine whether SU6656 could increase bone mass by inhibiting bone resorption and by stimulating bone formation, and to explore its mechanisms of action. Four-month-old female C57Bl/6J mice received intraperitoneal injections of either 25 mg/kg SU6656 or its vehicle every other day for 12 weeks. SU6656-treated mice exhibited increased bone mineral density, cortical thickness, cancellous bone volume and trabecular thickness. SU6656 inhibited bone resorption in mice as shown by reduced osteoclast number, and diminished expressions of Oscar, Trap5b and CtsK. SU6656 did not affect Rankl or Opg expressions. However, it blocked c-fms signaling, osteoclastogenesis and matrix resorption, and induced osteoclast apoptosis in vitro. In addition, SU6656 stimulated bone formation rates at trabecular, endosteal and periosteal bone envelopes, and increased osteoblast number in trabecular bone. SU6656 did not affect expressions of clastokines favoring bone formation in mice. However, it stimulated osteoblast differentiation and matrix mineralization by specifically facilitating BMP-SMAD signaling pathway in vitro. Knockdown of Src and Yes mimicked the stimulatory effect of SU6656 on osteoblast differentiation. In conclusion, SU6656 uncouples bone formation from resorption by inhibiting osteoclast development, function and survival, and by enhancing BMP-mediated osteoblast differentiation.

IL-6 trans-signalling mediates trabecular, but not cortical, bone loss after ovariectomy.

Bone loss associated with estrogen deficiency occurs due to a high level of bone remodelling, with a greater increase in the level of osteoclast-mediated bone resorption than osteoblast-mediated bone formation. Early studies showed that Interleukin-6 (IL-6) inhibition could prevent the increase in osteoclast numbers associated with ovariectomy. However, IL-6 signals through two possible pathways: classic IL-6 signalling (cis) utilizes a membrane-bound IL-6 receptor (IL-6R), while IL-6 trans-signalling occurs through a soluble IL-6R (sIL-6R). It is not known which of these pathways mediates the bone loss after ovariectomy. We therefore sought to determine whether specific pharmacological inhibition of IL-6 trans-signalling could prevent ovariectomy-induced bone loss in mice. We report that IL-6 trans-signalling inhibition prevented the increase in osteoclasts, and trabecular bone loss, associated with ovariectomy. IL-6 trans-signalling inhibition also reduced bone formation rate, but did not prevent the increase in osteoblast numbers. In contrast, cortical bone loss was not prevented by any IL-6 signalling inhibitor. This suggests that local production of sIL-6R mediates trabecular bone loss in estrogen deficiency, but the increased cortical bone resorption that leads to marrow expansion is independent of IL-6 signalling.