Mouse Bone Tissue Research Articles

Histone H4 Methyltransferase Suv420h2 Maintains Fidelity of Osteoblast Differentiation.

Osteogenic lineage commitment and progression is controlled by multiple signaling pathways (e.g., WNT, BMP, FGF) that converge on bone-related transcription factors. Access of osteogenic transcription factors to chromatin is controlled by epigenetic regulators that generate post-translational modifications of histones ("histone code"), as well as read, edit and/or erase these modifications. Our understanding of the biological role of epigenetic regulators in osteoblast differentiation remains limited. Therefore, we performed next-generation RNA sequencing (RNA-seq) and established which chromatin-related proteins are robustly expressed in mouse bone tissues (e.g., fracture callus, calvarial bone). These studies also revealed that cells with increased osteogenic potential have higher levels of the H4K20 methyl transferase Suv420h2 compared to other methyl transferases (e.g., Suv39h1, Suv39h2, Suv420h1, Ezh1, Ezh2). We find that all six epigenetic regulators are transiently expressed at different stages of osteoblast differentiation in culture, with maximal mRNAs levels of Suv39h1 and Suv39h2 (at day 3) preceding maximal expression of Suv420h1 and Suv420h2 (at day 7) and developmental stages that reflect, respectively, early and later collagen matrix deposition. Loss of function analysis of Suv420h2 by siRNA depletion shows loss of H4K20 methylation and decreased expression of bone biomarkers (e.g., alkaline phosphatase/Alpl) and osteogenic transcription factors (e.g., Sp7/Osterix). Furthermore, Suv420h2 is required for matrix mineralization during osteoblast differentiation. We conclude that Suv420h2 controls the H4K20 methylome of osteoblasts and is critical for normal progression of osteoblastogenesis. J. Cell. Biochem. 118: 1262-1272, 2017. © 2016 Wiley Periodicals, Inc.

Keratin 13 expression reprograms bone and brain metastases of human prostate cancer cells.

Lethal progression of prostate cancer metastasis can be improved by developing animal models that recapitulate the clinical conditions. We report here that cytokeratin 13 (KRT13), an intermediate filament protein, plays a directive role in prostate cancer bone, brain, and soft tissue metastases. KRT13 expression was elevated in bone, brain, and soft tissue metastatic prostate cancer cell lines and in primary and metastatic clinical prostate, lung, and breast cancer specimens. When KRT13 expression was determined at a single cell level in primary tumor tissues of 44 prostate cancer cases, KRT13 level predicted bone metastasis and the overall survival of prostate cancer patients. Genetically enforced KRT13 expression in human prostate cancer cell lines drove metastases toward mouse bone, brain and soft tissues through a RANKL-independent mechanism, as KRT13 altered the expression of genes associated with EMT, stemness, neuroendocrine/neuromimicry, osteomimicry, development, and extracellular matrices, but not receptor activator NF-κB ligand (RANKL) signaling networks in prostate cancer cells. Our results suggest new inhibitors targeting RANKL-independent pathways should be developed for the treatment of prostate cancer bone and soft tissue metastases.

The botanical molecule p-hydroxycinnamic acid as a new osteogenic agent: insight into the treatment of cancer bone metastases.

Bone homeostasis is maintained through a balance between osteoblastic bone formation and osteoclastic bone resorption. Bone loss with aging is induced by decreasing in osteoblastic bone formation and increasing in osteoclastic bone resorption, thereby leading to osteoporosis. Osteoporosis with its accompanying decrease in bone mass is widely recognized as a major public heath problem. Pharmacologic and nutritional factors may play a role in the prevention and treatment of bone loss with aging. p-Hydroxycinnamic acid (HCA), which stimulates bone mineralization in mouse bone tissues in vitro, has been found to be present in the leafstalk of wasabi (Wasabi japonica MATSUM) among various food and plants. Other phenolic acids including cinnamic acid, ferulic acid, caffeic acid and 3,4-dimethoxycinnamic acid did not have osteogenic effects. HCA was demonstrated to stimulate osteoblastic bone formation and suppresses osteoclastic bone resorption in vitro by antagonizing activation of the nuclear factor kappa B. Oral administration of HCA was found to exhibit restorative effects on bone loss induced by ovariectomy and diabetic states, supporting a role in the treatment of osteoporosis. Moreover, HCA was demonstrated to prevent the suppressed osteoblastic mineralization and the enhanced osteoclastogenesis in mouse bone marrow cells cocultured with bone metastatic MDA-MB-231 human breast cancer cells in vitro. The botanical molecule HCA, as a new osteogenic agent, is suggested to play a role in the treatment of cancer bone metastases. This review will discuss an advanced recent finding that HCA may be a useful agent to treat bone metabolic disorder.

Abstract B42: Autocrine/paracrine RANKL-RANK signaling promotes cancer bone metastasis and establishes premetastatic niche recruiting bystander cancer cells to participate in the metastatic process

Abstract RANKL-elicited RANK activity plays critical roles in many biological and pathological conditions, including osteoclast differentiation/bone remodeling, lymph node/thymic development, central thermoregulation and progesterone-driven mammary gland maturation, differentiation and carcinogenesis. RANKL can be derived from osteoblasts, infiltrating inflammatory cells and stromal fibroblasts. We previously showed that malignant prostate cancer (PCa) cells expressed RANKL and that its expression was correlated with clinical PCa progression and bone metastasis. RANKL expression at the single cell level in primary PCa specimens predicts PCa patient survival. We demonstrated RANKL overexpression promoted epithelial-to-mesenchymal transition (EMT), stem cells, and neuroendocrine (NE) phenotypes of PCa cells through transactivation of c-Myc/Max and AP4 via RANK-mediated signaling and conferred PCa bone and soft tissue metastases in mice. Blockade of RANKL-RANK signaling as well as their downstream regulators abolished the metastasis of PCa induced by RANKL. Furthermore, recombinant RANKL protein alone is sufficient to induce bone colonization and growth of RANKL- and non-metastatic PCa and breast cancer cells. Even a few RANKL+ PCa cells are sufficient to initiate the in vivo metastatic cascade by recruiting non-tumorigenic RANKL- PCa cells to participate in the metastatic process via downstream signaling amplification, implicating metastasis-initiating properties of RANKL-expressing PCa cells. Recently, we further demonstrated that RANKL rendered metastatic potential to breast, lung, renal, and liver cancer cells and promote their metastases to bone and other soft tissues in mice. Collectively, these results demonstrated RANKL is a uniform factor that drives bone metastasis of many different cancers, including prostate, breast, lung, renal, and liver cancers. The autocrine/paracrine RANKL-RANK signaling in cancer cells establishes a premetastatic niche through a “vicious cycle”, inducing RANKL and c-Met expression via activation of c-Myc/Max, and this promotes PCa EMT progression, stem and NE cell properties, and bone and soft tissue metastases. RANKL-expressing cancer cells possess metastasis-initiating potential that recruits and reprograms bystander non-tumorigenic cells to participate in the metastatic process. RANKL expression status therefore offers new insights for dissecting the mechanism by which PCa cells exhibit propensity for bone colonization/metastasis. Citation Format: Gina Chia-Yi Chu, Haiyen E. Zhau, Ruoxiang Wang, Leland W.K. Chung. Autocrine/paracrine RANKL-RANK signaling promotes cancer bone metastasis and establishes premetastatic niche recruiting bystander cancer cells to participate in the metastatic process. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B42.

Pim Inhibition Suppresses Osteoclastogenesis and Tumor Growth in Myeloma

Abstract Devastating bone destruction in multiple myeloma (MM) still remains a significant clinical problem. In pursuing factors responsible for MM tumor expansion and bone destruction, we found that the serine/threonine kinase Pim-2 is constitutively over-expressed as an anti-apoptotic mediator, and further up-regulated in MM cells when cocultured with bone marrow stromal cells or osteoclasts (OCs) (Leukemia, 2011). We also demonstrated that Pim inhibition is able to induce bone formation while suppressing MM tumor growth (Leukemia, 2015). However, the impact of Pim inhibition on MM-induced bone resorption remains unknown. Therefore, the present study was undertaken to clarify the role of Pim-2 in osteoclastogenesis enhanced in MM and the therapeutic effects of Pim inhibition on mutual interaction between MM cells and OCs. Pim-2 was highly expressed almost exclusively in cathepsin K-positive mature OCs on the surface of bone but not in other bone marrow cells in normal mouse bone tissues. RANK ligand and TNF-α induced the expression of Pim-2 in monocytes and RAW264.7 preosteoclastic cells at mRNA and protein levels. Inhibitors of the classical NF-κB pathway, SN50 or IMG2001, abolished Pim-2 up-regulation in RAW264.7 cells by RANK ligand or TNF-α, while Pim inhibition marginally affected the nuclear translocation of NF-κB subunits, p50 and p65, as well as the promoter activity of NF-κB, suggesting Pim-2 up-regulation downstream of the NF-κB pathway. Pim-2 appeared to be up-regulated along with c-fos, NFATc1 and cathepsin K during osteoclastogenesis. The Pim inhibitor SMI-16a potently suppressed the RANK ligand-induced expression of c-fos, NFATc1 and cathepsin K in RAW264.7 cells, and abolished osteoclastogenesis and bone resorption enhanced by MM cell conditioned media on hydroxyapatite-coated dishes. Furthermore, the Pim inhibition was found to suppress [Ca2+ ]i oscillation and thereby nuclear translocation of NFATc1, a critical transcription factor for osteoclastogenesis. MM cells and acid-producing OCs are mutually interacted in bone lesions to enhance MM tumor growth and bone destruction while creating an acidic milieu, thereby forming a progressive vicious cycle. Pim-2 was also up-regulated in MM cells when cocultured with OCs as well as bone marrow stromal cells, and to lesser extent merely by acidic conditions. Interestingly, acidic conditions rather preferentially enhanced the cytotoxic effects of the Pim inhibitor SMI-16a on MM cells even in cocultures with OCs or bone marrow stromal cells. Finally, treatment with SMI-16a reduced OC numbers in bone lesions together with tumor reduction and the restoration of bone formation in mouse MM models with intra-tibial injection of murine 5TGM1 MM cells. These results collectively demonstrated that Pim-2 play a critical role in osteoclastogenesis and tumor growth in acidic bone lesions in MM, and further corroborated that Pim-2 is a pivotal therapeutic target for MM bone disease and tumor progression. Disclosures Abe: Novartis Pharma K.K.: Speakers Bureau; Takeda Pharmaceutical Company Limited: Research Funding; Kyowa Hakko Kirin Company, Limited: Research Funding; Astellas Pharma Inc.: Research Funding; Ono Pharmaceutical Co.,Ltd.: Research Funding; GlaxoSmithKline plc: Research Funding.

Identification and Characterization of Lineage(-)CD45(-)Sca-1(+) VSEL Phenotypic Cells Residing in Adult Mouse Bone Tissue.

Murine bone marrow (BM)-derived very small embryonic-like stem cells (BM VSELs), defined by a lineage-negative (Lin(-)), CD45-negative (CD45(-)), Sca-1-positive (Sca-1(+)) immunophenotype, were previously reported as postnatal pluripotent stem cells (SCs). We developed a highly efficient method for isolating Lin(-)CD45(-)Sca-1(+) small cells using enzymatic treatment of murine bone. We designated these cells as bone-derived VSELs (BD VSELs). The incidences of BM VSELs in the BM-derived nucleated cells and that of BD VSELs in bone-derived nucleated cells were 0.002% and 0.15%, respectively. These BD VSELs expressed a variety of hematopoietic stem cell (HSC), mesenchymal stem cell (MSC), and endothelial cell markers. The gene expression profile of the BD VSELs was clearly distinct from those of HSCs, MSCs, and ES cells. In the steady state, the BD VSELs proliferated slowly, however, the number of BD VSELs significantly increased in the bone after acute liver injury. Moreover, green fluorescent protein-mouse derived BD VSELs transplanted via tail vein injection after acute liver injury were detected in the liver parenchyma of recipient mice. Immunohistological analyses suggested that these BD VSELs might transdifferentiate into hepatocytes. This study demonstrated that the majority of the Lin(-)CD45(-)Sca-1(+) VSEL phenotypic cells reside in the bone rather than the BM. However, the immunophenotype and the gene expression profile of BD VSELs were clearly different from those of other types of SCs, including BM VSELs, MSCs, HSCs, and ES cells. Further studies will therefore be required to elucidate their cellular and/or SC characteristics and the potential relationship between BD VSELs and BM VSELs.

Specific amino acid environments of bone marrow are crucial for the maintenance of hematopoietic stem cells

Hematopoietic stem cells (HSCs) have the potential to provide the entire range of blood cells throughout the lifespan, maintaining the hibernation state in specialized bone marrow (BM) microenvironment called niches. Although various types of cells and proteinous factors have been proposed to control hematopoiesis, little is known about environmental factors crucial for the maintenance, survival and function of HSCs in vivo. Here we found that endogenous small molecules such as amino acids are differentially distributed to bone tissues in mice.

Bone marrow ablation demonstrates that estrogen plays an important role in osteogenesis and bone turnover via an antioxidative mechanism

To assess the effect of estrogen deficiency on osteogenesis and bone turnover in vivo, 8-week-old mice were sham-operated or bilaterally ovariectomized (OVX), and after 8weeks, mechanical bone marrow ablation (BMX) was performed and newly formed bone tissue was analyzed from 6days to 2weeks after BMX. Our results demonstrated that OVX mice following BMX displayed 2 reversed phase changes, one phase observed at 6 and 8days after BMX delayed osteogenesis accompanied by a delay in osteoclastogenesis, and the other phase observed at 12 and 14days after BMX increased osteoblastic activity and osteoclastic activity. Furthermore, we asked whether impaired osteogenesis caused by estrogen deficiency was associated with increased oxidative stress, and oxidative stress parameters were examined in bone tissue from sham-operated and OVX mice and OVX mice were administrated with antioxidant N-acetyl-l-cysteine (NAC) or vehicle after BMX. Results demonstrated that estrogen deficiency induced oxidative stress in mouse bone tissue with reduced antioxidase levels and activity, whereas NAC administration almost rescued the abnormalities in osteogenesis and bone turnover caused by OVX. Results from this study indicate that estrogen deficiency resulted in primarily impaired osteogenesis and subsequently accelerated bone turnover by increasing oxidative stress and oxidative stress promises to be an effective target in the process of treatment of postmenopausal osteoporosis.

Bone-specific gene expression patterns and whole bone tissue of female mice are programmed by early life exposure to soy isoflavones and folic acid

Female mice exposed to soy isoflavones (ISO) during early postnatal life have improved bone outcomes at adulthood. Since long-lasting effects may be mediated by DNA methylation, we hypothesized that providing supplemental folic acid (FA), a methyl donor, during early life, would enhance the positive effect of ISO to bone health. Bone-specific gene expression patterns were studied to understand potential mechanisms. CD-1 dams (n=36) were randomized to adequate or supplemental levels of FA (2 or 8 mg/kg diet) during pregnancy and lactation, and offspring received corn oil or ISO (7 mg/kg body weight/d) from postnatal day 1 to 10. From weaning, pups were fed an adequate FA diet and were studied to 4 months of age. Female offspring exposed to supplemental FA+ISO had higher bone mineral density (BMD), trabecular connectivity and peak load at the lumbar spine compared to females exposed to adequate FA. Female offspring exposed to adequate FA+ISO or supplemental FA had higher (P<.05) BMD and greater resistance to fracture at the lumbar spine and the femur; higher trabecular connectivity at the lumbar spine; and lower expression of DNA methyltransferase 3a (Dnmt3a) and neuropeptide Y (NPY) in the femur compared to mice exposed to adequate FA. In addition, only mice exposed to adequate FA+ISO had microstructural improvements at the femur neck and higher serum osteoprotegrin (OPG) and insulin growth factor-I (IGF-I). In summary, exposure to supplemental FA did not enhance the positive effect of ISO in bone. However, exposure to adequate FA+ISO or supplemental FA improved bone at least in part by suppressing Dnmt3a and NPY.

Effect of long‐term voluntary running on bone tissue in mice

BackgroundIt is well known that exercise has positive effects on bone. It has been reported that voluntary exercise increase bone formation and bone density during growing period. However, effects of more long‐term exercise performed until adult on bone tissue are little known.PurposeTo investigate the effects of long‐term voluntary wheel running on histological and mechanical properties of bone tissue in mice.MethodsFour‐week‐old male C57BL/6 mice (n=31) were assigned to 1) a sedentary control (CON) and 2) a voluntary running (VE) groups. Mice in VE group were allowed free access a wheel. Femurs were removed at 4, 28, or 40 weeks of age. Bone volume was measured histologically as the bone volume per tissue volume［BV/TV (%)］, and bone strength was measured using three‐point bending test. Bone strength was normalized to body weight［N/g; maximum breaking force (N) per body weight (g)］.ResultsThe body weight in the CON at 4, 28, 40 weeks of age was 18.4±0.5, 30.1±1.3, 29.8±1.4 g, respectively, while it in VE at 28 and 40 weeks of age was 26.7±0.8, and 29.2±2.4 g. BV/TV (%) significantly decreased with aging from 4 to 28 weeks of age in both groups. There were no significant differences in BV/TV (%) between CON and VE. Also, bone strength significantly increase with aging from 4 to 28 weeks of age in both groups. There were no significant differences between CON and VE.ConclusionOur data suggests that long‐term voluntary running do not effect on histological and mechanical properties of bone tissue in mice.This work was supported by grants from the MEXT‐Supported Program for the Strategic Research Foundation at Private Universities (S1101008).

Phloretin promotes osteoclast apoptosis in murine macrophages and inhibits estrogen deficiency-induced osteoporosis in mice

Bone-remodeling imbalance induced by increased osteoclast formation and bone resorption is known to cause skeletal diseases such as osteoporosis. The reduction of estrogen levels at menopause is one of the strongest risk factors developing postmenopausal osteoporosis. This study investigated osteoprotective effects of the dihydrochalcone phloretin found in apple tree leaves on bone loss in ovariectomized (OVX) C57BL/6 female mice as a model for postmenopausal osteoporosis. OVX demoted bone mineral density (BMD) of mouse femurs, reduced serum 17β-estradiol level and enhanced serum receptor activator of NF-κB ligand (RANKL)/osteoprotegerin ratio with uterine atrophy. Oral administration of 10mg/kg phloretin to OVX mice for 8 weeks improved such effects, compared to sham-operated mice. Phloretin attenuated TRAP activity and cellular expression of β3 integrin and carbonic anhydrase II augmented in femoral bone tissues of OVX mice. This study further examined that osteogenic activity of phloretin in RANKL-differentiated Raw 264.7 macrophages into mature osteoclasts. Phloretin at 1–20μM stimulated Smac expression and capase-3 activation concurrently with nuclear fragmentation of multi-nucleated osteoclasts, indicating that this compound promoted osteoclast apoptosis. Consistently, phloretin enhanced bcl-2 induction but diminished bax expression. Furthermore, phloretin activated ASK-1-diverged JNK and p38 MAPK signaling pathways in mature osteoclasts, whereas it dose-dependently inhibited the RANKL-stimulated activation of ERK. Therefore, phloretin manipulated ASK-1-MAPK signal transduction leading to transcription of apoptotic genes. Phloretin was effective in preventing estrogen deficiency-induced osteoclastogenic resorption.

Curcumin analogue UBS109 prevents bone loss in breast cancer bone metastasis mouse model: involvement in osteoblastogenesis and osteoclastogenesis.

Bone metastasis of breast cancer typically leads to osteolysis, which causes severe pathological bone fractures and hypercalcemia. Bone homeostasis is skillfully regulated through osteoblasts and osteoclasts. Bone loss with bone metastasis of breast cancer may be due to both activation of osteoclastic bone resorption and suppression of osteoblastic bone formation. This study was undertaken to determine whether the novel curcumin analogue UBS109 has preventive effects on bone loss induced by breast cancer cell bone metastasis. Nude mice were inoculated with breast cancer MDA-MB-231 bone metastatic cells (10(6) cells/mouse) into the head of the right and left tibia. One week after inoculation, the mice were treated with control (vehicle), oral administration (p.o.) of UBS109 (50 or 150mg/kg body weight), or intraperitoneal administration (i.p.) of UBS109 (10 or 20mg/kg body weight) once daily for 5days per week for 7weeks. After UBS109 administration for 7weeks, hind limbs were assessed using an X-ray diagnosis system and hematoxylin and eosion staining to determine osteolytic destruction. Bone marrow cells obtained from the femurs and tibias were cultured to estimate osteoblastic mineralization and osteoclastogenesis ex vivo and in vitro. Remarkable bone loss was demonstrated in the tibias of mice inoculated with breast cancer MDA-MB-231 bone metastatic cells. This bone loss was prevented by p.o. administration of UBS109 (50 and 150mg/kg body weight) and i.p. treatment of UBS109 (10 and 20mg/kg) in vivo. Culture of bone marrow cells obtained from the bone tissues of mice with breast cancer cell bone metastasis showed suppressed osteoblastic mineralization and stimulated osteoclastogenesis ex vivo. These changes were not seen after culture of the bone marrow cells obtained from mice treated with UBS109. Moreover, UBS109 was found to stimulate osteoblastic mineralization and suppress lipopolysaccharide (LPS)-induced osteoclastogenesis in bone marrow cells obtained from normal nude mice in vitro. These findings suggest that the novel curcumin analogue UBS109 prevents breast cancer cell bone metastasis-induced bone loss by stimulating osteoblastic mineralization and suppressing osteoclastogenesis.

Effects of pyrophosphate delivery in a peritoneal dialysis solution on bone tissue of apolipoprotein-E knockout mice with chronic kidney disease

Vascular calcification (VC) is a risk factor for cardiovascular mortality in the setting of chronic kidney disease (CKD). Pyrophosphate (PPi), an endogenous molecule that inhibits hydroxyapatite crystal formation, has been shown to prevent the development of VC in animal models of CKD. However, the possibility of harmful effects of exogenous administration of PPi on bone requires further investigation. To this end, we examined by histomorphometry the bone of CKD mice after intraperitoneal PPi administration. After CKD creation or sham surgery, 10-week-old female apolipoprotein-E knockout (apoE(-/-)) mice were randomized to one non-CKD group or 4 CKD groups (n = 10-35/group) treated with placebo or three distinct doses of PPi, and fed with standard diet. Eight weeks later, the animals were killed. Serum and femurs were sampled. Femurs were processed for bone histomorphometry. Placebo-treated CKD mice had significantly higher values of osteoid volume, osteoid surface and bone formation rate than sham-placebo mice with normal renal function. Slightly higher osteoid values were observed in CKD mice in response to very low PPi dose (OV/BV, O.Th and ObS/BS) and, for one parameter measured, to high PPi dose (O.Th), compared to placebo-treated CKD mice. Treatment with PPi did not modify any other structural parameters. Mineral apposition rates, and other parameters of bone formation and resorption were not significantly different among the treated animal groups or control CKD placebo group. In conclusion, PPi does not appear to be deleterious to bone tissue in apoE(-/-) mice with CKD, although a possible stimulatory PPi effect on osteoid formation may be worth further investigation.

Multiscale, Converging Defects of Macro-Porosity, Microstructure and Matrix Mineralization Impact Long Bone Fragility in NF1

Bone fragility due to osteopenia, osteoporosis or debilitating focal skeletal dysplasias is a frequent observation in the Mendelian disease Neurofibromatosis type 1 (NF1). To determine the mechanisms underlying bone fragility in NF1 we analyzed two conditional mouse models, Nf1Prx1 (limb knock-out) and Nf1Col1 (osteoblast specific knock-out), as well as cortical bone samples from individuals with NF1. We examined mouse bone tissue with micro-computed tomography, qualitative and quantitative histology, mechanical tensile analysis, small-angle X-ray scattering (SAXS), energy dispersive X-ray spectroscopy (EDX), and scanning acoustic microscopy (SAM). In cortical bone of Nf1Prx1 mice we detected ectopic blood vessels that were associated with diaphyseal mineralization defects. Defective mineral binding in the proximity of blood vessels was most likely due to impaired bone collagen formation, as these areas were completely devoid of acidic matrix proteins and contained thin collagen fibers. Additionally, we found significantly reduced mechanical strength of the bone material, which was partially caused by increased osteocyte volume. Consistent with these observations, bone samples from individuals with NF1 and tibial dysplasia showed increased osteocyte lacuna volume. Reduced mechanical properties were associated with diminished matrix stiffness, as determined by SAM. In line with these observations, bone tissue from individuals with NF1 and tibial dysplasia showed heterogeneous mineralization and reduced collagen fiber thickness and packaging. Collectively, the data indicate that bone fragility in NF1 tibial dysplasia is partly due to an increased osteocyte-related micro-porosity, hypomineralization, a generalized defect of organic matrix formation, exacerbated in the regions of tensional and bending force integration, and finally persistence of ectopic blood vessels associated with localized macro-porotic bone lesions.

Computer-Automated Static, Dynamic and Cellular Bone Histomorphometry.

Dynamic and cellular histomorphometry of trabeculae is the most biologically relevant way of assessing steady state bone health. Traditional measurement involves manual visual feature identification by a trained and qualified professional. Inherent with this methodology is the time and cost expenditure, as well as the subjectivity that naturally arises under human visual inspection. In this work, we propose a rapidly deployable, automated, and objective method for dynamic histomorphometry. We demonstrate that our method is highly effective in assessing cellular activities in distal femur and vertebra of mice which are injected with calcein and alizarin complexone 7 and 2 days prior to sacrifice. The mineralized bone tissues of mice are cryosectioned using a tape transfer protocol. A sequential workflow is implemented in which endogenous fluorescent signals (bone mineral, green and red mineralization lines), tartrate resistant acid phosphatase identified by ELF-97 and alkaline phosphatase identified by Fast Red are captured as individual tiled images of the section for each fluorescent color. All the images are then submitted to an image analysis pipeline that automates identification of the mineralized regions of bone and selection of a region of interest. The TRAP and AP stained images are aligned to the mineralized image using strategically placed fluorescent registration beads. Fluorescent signals are identified and are related to the trabecular surface within the ROI. Subsequently, the pipelined method computes static measurements, dynamic measurements, and cellular activities of osteoclast and osteoblast related to the trabecular surface. Our method has been applied to the distal femurs and vertebrae of 8 and 16 week old male and female C57Bl/6J mice. The histomorphometric results reveal a significantly greater bone turnover rate in female in contrast to male irrespective of age, validating similar outcomes reported by other studies.

Identification of the Vitamin D Receptor in Osteoblasts and Chondrocytes But Not Osteoclasts in Mouse Bone

Bone is clearly a target of vitamin D and as expected, the vitamin D receptor (VDR) is expressed in osteoblasts. However, the presence of VDR in other cells such as osteocytes, osteoclasts, chondroclasts, and chondrocytes is uncertain. Because of difficulties in obtaining sections of undecalcified adult bone, identification of the site of VDR expression in adult bone tissue has been problematic. In addition, the antibodies to VDR used in previous studies lacked specificity, a property crucial for unambiguous conclusions. In the present study, VDR in the various cells from neonatal and adult mouse bone tissues was identified by a highly specific and sensitive immunohistochemistry method following bone decalcification with EGTA. For accurate evaluation of weak immunosignals, samples from Demay VDR knockout mice were used as negative control. Molecular markers were used to identify cell types. Our results showed that EGTA-decalcification of bone tissue had no detectable effect on the immunoreactivity of VDR. VDR was found in osteoblasts and hypertrophic chondrocytes but not in the multinucleated osteoclasts, chondroclasts, and bone marrow stromal cells. Of interest is the finding that immature osteoblasts contain large amounts of VDR, whereas the levels are low or undetectable in mature osteoblasts including bone lining cells and osteocytes. Proliferating chondrocytes appear devoid of VDR, although low levels were found in the hypertrophic chondrocytes. These data demonstrate that osteoblasts and chondrocytes are major targets of 1α,25-dihydroxyvitamin D, but osteoclasts and chondroclasts are minor targets or not at all. A high level of VDR was found in the immature osteoblasts located in the cancellous bone, indicating that they are major targets of 1α,25-dihydroxyvitamin D. Thus, the immature osteoblasts are perhaps responsible for the vitamin D hormone signaling resulting in calcium mobilization and in osteogenesis.

OP0040 Dynamic in vivo imaging of TH17-mediated control of osteoclast function in live bones by using intravital multiphoton microscopy

BackgroundRheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint synovial inflammation and progressive cartilage/bone destruction. Recently it has been reported that Th17, a CD4+ T cell subset expressing...

Multistep Targeted Nano Drug Delivery System Aiming at Leukemic Stem Cells and Minimal Residual Disease

Refractory leukemia remains the most common therapeutic problem in clinical treatment of leukemia. The key therapy of refractory leukemia is to kill, thoroughly, the minimal residual disease and leukemia stem cells in the highly vascularized red marrow areas. In this study, two new conjugates, alendronate-polyethylene glycol (100) monostearate and folate-polyethylene glycol (100) monostearate, were synthesized to develop a multistep targeting nanostructured lipid carriers by enhancing drug transport to the high bone turnover areas adjacent to the red marrow and targeting the minimal residual disease and leukemia stem cells. This dual targeting system demonstrated a great binding affinity to hydroxyapatite, a model component of bone minerals, and higher cell uptake (in the form of carriers but not drug) and cytotoxicity in the K562 cell line, a leukemia cell line with overexpressed folate receptors, were observed in vitro compared to unmodified carriers, especially when the cells were pretreated and the receptors were up-regulated by all-trans retinoic acid. The comodel test of K562 cells and HA showed that this dual targeting system could desorb from bone surface and be taken up by leukemia cells. For the in vivo study, this dual targeting system exhibited a significant increase in plasma half-life and could specifically accumulate in the bone tissue of rats or mice after intravenous injection. Ex vivo imaging of mice femurs and confocal laser scanning microscope imaging of mice femur slices further confirmed that this dual targeting system could favorably deposit to the osteoblast-enriched areas of high bone turnover in regions of trabecular bone surrounded by red marrow. In vivo antitumor activity in K562/BALB/c-nu leukemia mice showed that the treatment of this dual targeting system significantly reduced the white blood cell (WBC) number in peripheral blood and bone marrow to the normal level. In conclusion, this dual targeting system could precisely target to the regions where the minimal residual disease and leukemia stem cells are located and then be specifically uptaken in large amounts, which is a valuable target for refractory leukemia therapy.

Osteogenic activity of phloretin promoting osteoclast apoptosis in murine macrophages and inhibiting estrogen deficiency‐induced osteoporosis in mice

Bone‐remodeling imbalance is known to cause skeletal diseases such as osteoporosis. Phloretin, a dihydrochalcone and a type of natural phenols, can be found in apple tree leaves. We have previously shown that phloretin inhibits osteoclast differentiation and bone resorption through disturbing TRAF6‐NFATc1‐NF‐κB pathway. RAW 264.7 macrophages incubated with 20μM phloretin and 17β‐estradiol for 5 d in the presence of RANKL stimulated capase‐3 activation, promoting osteoclast apoptosis. RANKL activated Akt and ERK via inducing c‐Src expression, which was attenuated by 1–20 μM phloretin. In addition, phloretin suppressed actin ring formation elevated by RANKL, indicating its inhibition of osteoclast fusion and resorption. This study further investigated inhibitory effects of phloretin on bone loss in ovariectomized (OVX) C57BL/6 female mice. OVX demoted bone mineral density (BMD) of mouse femoral bone tissues, reduced serum 17ßestradiol level and enhanced serum RANKL/osteoprotegerin ratio. The oral administration of 10 mg/kg phloretin to OVX mice for 8 weeks improved such effects, compared to sham‐operated mice. Phloretin attenuated TRAP activity and ß3 integrin expression and carbonic anhydrase II augmented in femoral bone tissues of OVX mice. Therefore, phloretin is effective in antagonizing osteoclastogenic resorption and preventing estrogen deficiency‐induced bone loss.

Maintaining mRNA Integrity during Decalcification of Mineralized Tissues

Biomineralization of the extracellular matrix occurs inappropriately in numerous pathological conditions such as cancer and vascular disease, but during normal mammalian development calcification is restricted to the formation of the skeleton and dentition. The comprehensive study of gene expression in mineralized skeletal tissues has been compromized by the traditional decalcification/fixation methods that result in significant mRNA degradation. In this study we developed a novel RNAlater/EDTA decalcification method that protects the integrity of the mRNA in mature mouse tibial epiphyses. Furthermore, this method preserves the tissue structure to allow histological sectioning and microdissection to determine region-specific gene expression, in addition to immuno- and in situ histology. This method will be widely applicable to the molecular analysis of calcified tissues in various pathological conditions, and will be of particular importance in dissection of the gene expression in mouse bone and joint tissues during development and in important clinical conditions such as arthritis.