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The maintenance of bone is dependent on both osteoclasts, which break down bone, and osteoblasts, which build new bone. Various bone-related disorders, including osteoporosis, can occur as a result of an imbalance between these two cell types. Prolonged use of currently available bone resorption inhibitors may show side effects. Therefore, developing a novel preventive material which effectively inhibits osteoclast differentiation could be beneficial. This study planned to investigate the inhibitory effect of wheat sprout ethanolic extracts (Saegeumgang [SGG] and Arriheuk [ARH]) on the differentiation of osteoclasts induced by RANKL, as well as the mechanisms why fundamental to these effects. The effects of SGG and ARH on bone resorption and osteoclast differentiation were evaluated using RAW 264.7 cells and assessed through TRAP cell count, pit formation, and activity. The expressions of mRNA and protein were accomplished using western blotting, and reverse transcription quantitative polymerase chain reaction analyses were conducted. SGG and ARH were found to suppress osteoclast differentiation in RANKL-stimulated RAW264.7 cells without causing cytotoxic effects. In addition, treatment with SGG and ARH led to a reduction in the number of cells with positive staining for TRAP and TRAP activity. SGG and ARH treatment dose-dependently decreased the pit area in pit formation assays, showing a notable reduction compared to the pit area created by mature osteoclasts. SGG and ARH inhibited osteoclast activity by 84.9% and 95.7% at 200 μg/mL, respectively. In addition, SGG and ARH suppressed the transcriptional activation of various osteoclast-related genes, such as RANK, NFATc1, cathepsin K, c-Fos, TRAP, matrix metallopeptidase-9, dendritic cell-specific transmembrane protein, ATPase H+ transporting v0 subunit d2, and osteoclast-associated receptor in RAW264.7 cells treated with RANKL. SGG and ARH extracts were found to affect the expression of NFATc1 and genes that are specific to osteoclasts during osteoclast differentiation, suggesting their potential use as functional foods or as therapeutic interventions targeting bone health. [ABSTRACT FROM AUTHOR]

Osteoporosis is a progressive metabolic disease characterized by decreased bone mineral density and increased fracture risk. Previous studies have shown that higher intake of vitamin K (VK) correlates with a reduced risk of osteoporosis. However, the effect of menaquinone-4 (MK-4), a specific form of VK, still remains obscure. Therefore, in this study, we investigated the effects of MK-4 on osteoclast differentiation by differentiating RAW 264.7 cells into osteoclasts with the help of receptor activator of nuclear factor-kappa B ligand (RANKL), assessed the mRNA expression of osteoclast-specific genes, and studied the effects of MK-4 in vivo in ovariectomized mice, a postmenopausal osteoporosis murine model. MK-4 inhibited osteoclast differentiation, decreased the mRNA expression of nuclear factor of activated T cells c1 (NFATc1), osteoclast-associated receptor (OSCAR), and cathepsin K (CTSK), and inhibited bone loss in ovariectomized mice. The findings strongly suggest that MK-4 is a therapeutic alternative for postmenopausal osteoporosis. [ABSTRACT FROM AUTHOR]

Ras homologue enriched in brain 1 (Rheb1), an upstream activator of the mechanistic target of rapamycin complex 1 (mTORC1), is known to modulate various cellular processes. However, its impact on bone metabolism in vivo remains unknown. The study aimed at understanding the role of Rheb1 on bone homeostasis. We measured the serum parameters and performed histomorphometry, quantitative real-time polymerase chain reaction, and Western blotting, along with the generation of mouse gene knockout (KO) model, and conducted a microcomputed tomography analysis and tartrate-resistant acid phosphatase staining, to delineate the impacts of Rheb1 on bone homeostasis. In the Rheb1 KO mice, the results showed that Rheb1 KO caused significant damage to the bone microarchitecture, indicating that mTORC1 activity was essential for the regulation of bone homeostasis. Specifically, suppressed mineralization activity in primary osteoblasts and a decreased osteoblast number were observed in the Rheb1 KO mice, demonstrating that loss of Rheb1 led to impaired osteoblastic differentiation. Furthermore, the higher apoptotic ratio in Rheb1-null osteocytes could promote Tnfsf11 expression and lead to an increase in osteoclasts, indicating increased bone resorption activity in the KO mice. The findings confirmed that Rheb1 deletion in osteoblasts/osteocytes led to osteopenia due to impaired bone formation and enhanced bone resorption. [ABSTRACT FROM AUTHOR]

Osteoporosis is one of the most common metabolic skeletal diseases, which affects more than 200 million people worldwide, especially elderly and postmenopausal women. One of the main processes of osteoporosis is attenuated bone formation. Abundant evidence has confirmed that overactivated osteoclasts are responsible for the attenuated bone formation. This study aims at identifying novel methylation-associated biomarkers and therapeutic targets in osteoclasts by integrally analyzing methylation profiles and gene expression data. DNA methylation profile and gene expression data were obtained from the Gene Expression Omnibus (GEO) database. Subsequently, we integrated the two sets of data to screen for differentially expressed genes with differential methylation level (DM-DEGs) between osteoclasts and CD14+ monocytes from donors. Then, Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to uncover the enriched functions and pathways of identified DM-DEGs. In addition, by combining protein-protein interaction analysis and receiver-operator characteristic analysis, we finally identified four hub DM-DEGs. Gene Set Enrichment Analysis was utilized to validate and investigate the potential biological functions of the four hub DM-DEGs. Finally, Real-time quantitative PCR (QPCR) was performed to validate the mRNA expression level of the four identified hub DM-DEGs during osteoclast differentiation. CCRL2, CCL18, C1QB, and SELL were highly correlated with osteoclastic differentiation and osteoporosis phenotype. QPCR revealed that the expression of CCRL2, CCL18, and C1QB was increased during osteoclast differentiation, whereas the expression of SELL was decreased. The present study indicated a connection between gene expression and DNA methylation during osteoclast differentiation and that four hub DM-DEGs in osteoclastogenesis and osteoporosis pathogenesis might be potential candidates for intensive research and therapeutic targets for the treatment of osteoporosis. [ABSTRACT FROM AUTHOR]

Pressure waves from explosions or other traumatic events can damage the neurons of the eye and visual centers of the brain, leading to functional loss of vision. There are currently few treatments for such injuries that can be deployed rapidly to mitigate damage. Brain-derived neurotrophic factor (BDNF) and activation of its receptor tropomycin-related kinase B (TrkB) have neuroprotective effects in a number of degeneration models. Small molecule activators of TrkB, such as N-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-2-oxopiperidine-3-carboxamide (HIOC), cross the blood–brain and blood-retina barriers after systemic administration. We characterize the effects of blast-induced ocular trauma on retinal and visual function. We show that systemic administration of HIOC, a potent small molecule activator of the BDNF/TrkB receptor, preserves visual function in mice exposed to ocular blast injury. The HIOC treatment for one week preserves visual function for at least four months. The HIOC treatment effectively protected vision when the initial dose was administered up to 3 h after blast, but not if the initial treatment was delayed for 24 h. We provide evidence that the therapeutic effect of HIOC is mediated by activation of BDNF/TrkB receptors. The results indicate that HIOC may be useful for managing ocular blast injury and other forms of traumatic optic neuropathy. [ABSTRACT FROM AUTHOR]

Background: Metformin has antitumoral actions in human cancers, including the thyroid, while its effects on metastatic lesions are unclear. Patients with bone metastasis (BM) from thyroid cancers have poor survival. Because metformin inhibits the activation of osteoclasts, which has essential roles in BM, the aim of this study was to investigate the therapeutic effects of metformin on thyroid cancer BM and osteoclast activation in the bone microenvironment. Methods: The anaplastic thyroid cancer (ATC) cell lines FRO and SW1736 were used to test the antitumoral effect of metformin in vitro and in vivo. A murine model of BM was established by intratibial injection of cancer cells. To mimic the BM microenvironment, osteoblasts were treated with conditioned media from the FRO (FRO-CM) and SW1736 (SW1736-CM) cells. Thyroid cancer patients with or without BM were recruited, and the serum receptor activator of nuclear factor kappa-B ligand (RANKL) levels was measured. Results: Metformin treatment significantly reduced the viabilities of the FRO and SW1736 cells in vitro and the tumor growth of SW1736 in vivo. In the murine model of BM, metformin delayed tumor growth in the bone and decreased the numbers of tartrate-resistant acid phosphatase-positive osteoclasts on the bone surface with reduced RANKL in the bone marrow. Furthermore, FRO- or SW1736-CM significantly increased the osteoblastic RANKL productions and activated osteoclast differentiation in whole marrow cultures, which were blocked by metformin treatment. Among 67 thyroid cancer patients, the serum RANKL levels were significantly increased in BM patients compared with patients with lung-only metastasis or no distant metastasis. In addition, the interleukin-6 superfamily in the FRO- or SW1736-CM stimulated STAT3 phosphorylation, which was inhibited by gp130 blocking. Metformin treatment decreased the FRO- or SW1736-CM-induced STAT3 phosphorylation by AMPK phosphorylation. Metformin also inhibited the FRO- or SW1736-CM-induced osteoclastic differentiation of bone marrow-derived monocyte/macrophage by RANK/c-Fos/NFATC1 signaling. Conclusions: In the microenvironment of BM, metformin effectively reduced ATC tumor growth by inhibiting cancer cell viability, blocking cancer cell-induced osteoblastic RANKL production, which further activated osteoclastogenesis, and directly reduced osteoclast differentiation. These multifactorial actions of metformin suggest that it has potential therapeutic effects in thyroid cancer BM. [ABSTRACT FROM AUTHOR]

The pathogenesis of osteoarthritis (OA) is still unclear. It is therefore important to identify relevant diagnostic marker genes for OA. We performed an integrated analysis with multiple microarray data cohorts to identify potential transcriptome markers of OA development. Further, to identify OA diagnostic markers, we established gene regulatory networks based on the protein–protein interaction network involved in these differentially expressed genes (DEGs). Using support vector machine (SVM) pattern recognition, a diagnostic model for OA prediction and prevention was established. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that 190 DEGs were mainly enriched in pathways like the tumor necrosis factor signaling pathway, interleukin-17 signaling pathway, mitogen-activated protein kinase signaling pathway, nuclear factor kappa-light-chain-enhancer of activated B cells signaling pathway, and osteoclast differentiation. Eight hub genes (POSTN, MMP2, CTSG, ELANE, COL3A1, MPO, COL1A1, and COL1A2) were considered potential diagnostic biomarkers for OA, the area under curve (AUC) was >0.95, which showed high accuracy. The sensitivity and specificity of the SVM model of OA based on these eight genes reached 100% in multiple external verification cohorts. Our research provides a theoretical basis for OA diagnosis for clinicians. [ABSTRACT FROM AUTHOR]

Aims: Bone is the most frequent site of prostate cancer (PCa) metastasis. Tumor cells interact with the bone microenvironment interrupting tissue balance. Heme oxygenase-1 (HO-1; encoded by Hmox1) appears as a potential target in PCa maintaining the cellular homeostasis. Our hypothesis is that HO-1 is implicated in bone physiology and modulates the communication with PCa cells. Here we aimed at (i) assessing the physiological impact of Hmox1 gene knockout (KO) on bone metabolism in vivo and (ii) determining the alterations of the transcriptional landscape associated with tumorigenesis and bone remodeling in cells growing in coculture (PCa cells with primary mouse osteoblasts [PMOs] from BALB/c Hmox1+/+, Hmox1+/−, and Hmox1−/− mice). Results: Histomorphometric analysis of Hmox1−/− mice bones exhibited significantly decreased bone density with reduced remodeling parameters. A positive correlation between Hmox1 expression and Runx2, Col1a1, Csf1, and Opg genes was observed in PMOs. Flow cytometry studies revealed two populations of PMOs with different reactive oxygen species (ROS) levels. The high ROS population was increased in PMOs Hmox1+/− compared with Hmox1+/+, but was significantly reduced in PMOs Hmox1−/−, suggesting restrained ROS tolerance in KO cells. Gene expression was altered in PMOs upon coculture with PCa cells, showing a pro-osteoclastic profile. Moreover, HO-1 induction in PCa cells growing in coculture with PMOs resulted in a significant modulation of key bone markers such as PTHrP and OPG. Innovation and Conclusion: We here demonstrate the direct implications of HO-1 expression in bone remodeling and how it participates in the alterations in the communication between bone and prostate tumor cells. [ABSTRACT FROM AUTHOR]

Increasing evidence has announced the emerging roles of long noncoding RNAs (lncRNAs) in modulating bone homeostasis due to their potential regulating effects on bone-related cells' proliferation, migration, differentiation and apoptosis. Thus, lncRNAs have been considered as a promising gene tool to facilitate the bone regeneration process and then to predict and cure bone-related diseases such as osteosarcoma, osteoporosis, and osteoarthritis. In this review, we first enumerated several kinds of dysregulated lncRNAs and concisely summarized their regulating role in bone formation as well as resorption process. The related mechanisms were also discussed, respectively. Then, the positive or negative behavior of these lncRNAs in bone-related diseases was elucidated. This review provides an in-depth sight about the lncRNA's clinical values and limitations, which is conducive to explore new gene targets and further establish new therapeutic strategies for bone-related disease. [ABSTRACT FROM AUTHOR]

Bone is maintained by an intricate balance between bone formation and bone resorption. The presence of inflammation can contribute to an imbalance in bone homeostasis by enhancing differentiation and activity of osteoclasts, the cells that participate in the breakdown of bone. Polyphenols such as flavonoids found in plant-derived foods have been shown to have anti-inflammatory effects in various tissues. Tart cherries are a rich source of such polyphenolic compounds. Using mouse macrophage cells (RAW 264.7), we examined whether tart cherry polyphenols could dose dependently inhibit the proliferation and activity of receptor activator of nuclear factor kappa-B ligand (RANKL) differentiated osteoclasts under inflammatory conditions. Tartrate-resistant acid phosphatase (TRAP) activity and staining of TRAP-positive multinucleated cells, used as indicators of osteoclast differentiation and activity, tended to decrease with tart cherry polyphenols treatment. Osteoprotegerin expression by osteoclasts was decreased in a similar manner. A significant increase in nitrite concentration was observed with the lower doses of tart cherry polyphenols of 50 and 100 μg/mL (P < .05). However, higher doses of tart cherry polyphenols (200 and 300 μg/mL) reduced nitrite concentrations below that of the control that received no tart cherry polyphenols treatment (P < .05). Western blot analyses showed that protein expression of cyclooxygenase-2 (COX-2) followed a similar trend, although results were not statistically significant. On the other hand, tart cherry polyphenols treatments dose dependently increased inducible nitric oxide synthase protein expression, with statistical significance noted at doses of 200 and 300 μg/mL. Overall, our findings suggest that the polyphenols associated with tart cherries potentially inhibit osteoclast differentiation and activity, which may be beneficial to bone health. [ABSTRACT FROM AUTHOR]

Approximately half of the unexplained recurrent spontaneous abortions remain unexplained (URSAs). We aimed to provide novel insights into the biological characteristics and related pathways of differentially expressed genes (DE-genes), DE-methylated genes, and DE-miRNAs in URSA, and construct a molecular miRNAs–mRNAs network. Four data sets (GSE22490, GSE121950, GSE73025, and GSE43256) were gained from GEO data sets. We identified the DE-genes, DE-methylated genes, and DE-miRNAs using the LIMMA package in R software. Function and enrichment analyses were conducted using DAVID. A protein–protein network was performed by STRING. We predicted the target genes of DE-miRNA using DIANA-microT-CDS. Then, we constructed miRNAs–mRNAs network. There were 137 genes that overlapped in two expression profile data sets (GSE121950 and GSE22490). We found 10 overlapping DE-methylated genes and DE-genes with opposite expression alteration trends. All those 10 genes were hypermethylated lowly expressed genes. Pathway analysis illustrated that DE-genes were enriched in osteoclast differentiation, leishmaniasis, NF-kappa B signaling pathway, Toll-like receptor signaling pathway, and tuberculosis. Based on protein–protein interaction analysis, TLR8, TLR2, CD86, TLR4, IL10, CD163, FCGR1A, CXCL8, FCGR3A, HCK, PLEK, and MNDA were identified as hub genes for DE-genes. We screened out 47 DE-miRNAs and 42 overlapping DE-genes between predicted target genes of DE-miRNAs and the 137 DE-genes. We then constructed miRNAs–mRNAs network. This study identified several genes and miRNAs involved in the development and progression of URSA, including FCGR1A, FCGR3A, CXCL8, HCK, PLEK, IL10, hsa-miR-498, and hsa-miR-4530. Although further in vivo and in vitro validations are required, our results may provide a theoretical basis for future studies. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are endogenous noncoding small molecule RNAs that regulate cell proliferation, differentiation, fat metabolism, and hormone secretion. Studies have shown that miRNAs regulate the processes related to osteoporosis, including the differentiation of osteoblasts, osteoclasts, and chondrocytes, and are one of the important regulatory factors of some bone metabolic diseases. In our previous study, it has been revealed that natural compound Polygonatum sibiricum polysaccharide (PSP) can promote osteoblast formation and block osteoclastogenesis through Wnt/β-catenin signaling pathway. This study was designed to investigate whether PSP can inhibit expression of osteoclast-related genes by Hippo signaling pathway, which was prevented by effectively blocking the expression of miR-1224. This study showed that there were 27 differentially expressed miRNAs when PSP inhibits osteoclastogenesis, the most notable of which was miR-1224. Furthermore, the study showed that PSP increased the level of Limd1, which was the target gene of miR-1224. In conclusion, these findings demonstrate that PSP suppressed osteoclastogenesis in vitro through the Hippo signaling pathway based on miR-1224. This study may aid in the development of a therapeutic approach utilizing PSP for the enhancement of bone health and prevention of osteoporosis. [ABSTRACT FROM AUTHOR]

Objective: The goal of this research was to evaluate c(RGDyK) conjugated to phosphonate-based cross-bridged chelators using Cu-free click chemistry in the 4T1 mouse mammary tumor bone metastasis model in comparison with 64Cu-CB-TE2A-c(RGDyK), which previously showed selective binding to integrin αvβ3 on osteoclasts.Experimental: Two phosphonate-based cross-bridged chelators (CB-TE1A1P and CB-TE1K1P) were conjugated to c(RGDyK) through bio-orthogonal strain-promoted alkyne-azide cycloaddition. In vitro and in vivo evaluation of the 64Cu-labeled TE1A1P-DBCO-c(RGDyK) (AP-c(RGDyK)), TE1K1P-PEG4-DBCO-c(RGDyK) (KP-c(RGDyK)), and CB-TE2A-c(RGDyK) were compared in the 4T1 mouse model of bone metastasis. The affinities of the unconjugated and chelator-c(RGDyK) analogs for αvβ3 integrin were determined using a competitive-binding assay. For in vivo evaluation, BALB/c mice were injected with 1 × 105 4T1/Luc cells in the left ventricle. Formation of metastases was monitored by bioluminescence imaging (BLI) followed by small-animal PET/CT 2 h postinjection of radiotracers.Results: The chelator-peptide conjugates showed similar affinity to integrin αvβ3, in the low nM range. PET imaging demonstrated a higher uptake in bones having metastases for all 64Cu-labeled c(RGDyK) analogs compared with bones in nontumor-bearing mice. The correlation between uptake of 64Cu-AP-c(RGDyK) and 64Cu-KP-c(RGDyK) in bones with metastases based on PET/CT imaging, and osteoclast number based on histomorphometry, was improved over the previously investigated 64Cu-CB-TE2A-c(RGDyK).Conclusion: These data suggest that the phosphonate chelator conjugates of c(RDGyK) peptides are promising PET tracers suitable for imaging tumor-associated osteoclasts in bone metastases. [ABSTRACT FROM AUTHOR]

Aims: Lipopolysaccharide (LPS) is considered a prominent pathogenic factor in inflammatory bone diseases. LPS challenge contributes to the production of reactive oxygen species (ROS) in diverse inflammatory diseases. However, its mechanism remains to be clarified in bone. Thus, we investigated the critical mechanism of ROS in LPS-induced osteoclastogenesis and bone loss. Results: Antioxidant prevented LPS-induced osteoclast formation via inhibition of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and c-Fos expression in preosteoclasts. Moreover, LPS-induced osteoclast formation via ROS was attenuated by treatment with c-Jun N-terminal protein kinase (JNK) inhibitor. Interestingly, LPS also activated signal transducer and activator of transcription 3 (STAT3), which is suppressed by antioxidants. We found that knockdown of STAT3 or use of a STAT3 inhibitor resulted in a significant reduction in interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and nitric oxide (NO) production, followed by decreased osteoclast formation by LPS. Peroxiredoxin II (PrxII) is a member of the antioxidant enzyme family, and it plays a protective role against oxidative damage caused by ROS. In our study, ROS production and osteoclast formation by LPS was significantly enhanced in PrxII−/− cells. Moreover, JNK-mediated c-Fos and NFATc1 expression was promoted in PrxII−/− cells. Furthermore, STAT3 activation and accompanying IL-1β, IL-6, and NO production was also increased in PrxII−/− cells. Consistent with the in vitro result, PrxII-deficient mice showed increased osteoclast formation and bone loss by LPS challenge compared with wild-type mice. Innovation: For the first time, we showed that LPS-induced ROS signaling is dependent on the coordinated mechanism of JNK and STAT3 during osteoclastogenesis, which is negatively regulated by PrxII. Conclusion: We suggest that PrxII could be useful in the development of a novel target for inflammatory bone loss. Antioxid. Redox Signal. 22, 63-77. [ABSTRACT FROM AUTHOR]

Physiological primary teeth exfoliation is a normal phenomenon during teeth development. However, retained primary teeth can often be observed in the patients with cleidocranial dysplasia (CCD) caused by mutation of Runx2. The potential regulative mechanism is still unknown. In the present study, periodontal ligament stem cells (PDLSCs) were derived from different resorbed stages of primary teeth and permanent teeth from normal patients and primary teeth from CCD patient. The proliferative, osteogenic and osteoclast-inductive capacities of PDLSCs from each group were detected. We demonstrated here that the proliferative ability of PDLSCs was reduced while the osteogenic and the osteoclast-inductive capacity of PDLSCs were enhanced during root resorption. The results also showed that PDLSCs from permanent teeth and CCD patient expressed low level of Runx2 and RANKL while high level of OPG. However, expression of Runx2 and RANKL were increased while expression of OPG was decreased in PDLSCs derived from resorbed teeth. Furthermore, Runx2 regulating the expression of RANKL and OPG and the osteoclast-inductive capacity of PDLSCs were confirmed by gain or loss of function assay. These data suggest that PDLSCs promote osteoclast differentiation via Runx2 upregulating RANKL and downregulating OPG, leading to enhanced root resorption that results in physiological exfoliation of primary teeth. [ABSTRACT FROM AUTHOR]

Background: We have shown that thyroid-stimulating hormone (TSH) has a direct inhibitory effect on osteoclastic bone resorption and that TSH receptor (TSHR) null mice display osteoporosis. To determine the stage of osteoclast development at which TSH may exert its effect, we examined the influence of TSH and agonist TSHR antibodies (TSHR-Ab) on osteoclast differentiation from murine embryonic stem (ES) cells to gain insight into bone remodeling in hyperthyroid Graves' disease. Methods: Osteoclast differentiation was initiated in murine ES cell cultures through exposure to macrophage colony stimulation factor, receptor activator of nuclear factor кB ligand, vitamin D, and dexamethasone. Results: Tartrate resistant acid phosphatase (TRAP)-positive osteoclasts formed in ∼12 days. This coincided with the expected downregulation of known markers of self renewal and pluripotency (including Oct4, Sox2, and REX1). Both TSH and TSHR-Abs inhibited osteoclastogenesis as evidenced by decreased development of TRAP-positive cells (∼40%-50% reduction, p = 0.0047), and by decreased expression, in a concentration-dependent manner, of osteoclast differentiation markers (including the calcitonin receptor, TRAP, cathepsin K, matrix metallo-proteinase-9, and carbonic anhydrase II). Similar data were obtained using serum immunoglobulin-Gs (IgGs) from patients with hyperthyroid Graves' disease and known TSHR-Abs. TSHR stimulators inhibited tumor necrosis factor-alpha mRNA and protein expression, but increased the expression of osteoprotegerin (OPG), an antiosteoclastogenic human soluble receptor activator of nuclear factor кB ligand receptor. Neutralizing antibody to OPG reversed the inhibitory effect of TSH on osteoclast differentiation evidencing that the TSH effect was at least in part mediated by increased OPG. Conclusion: These data establish ES-derived osteoclastogenesis as an effective model system to study the regulation of osteoclast differentiation in early development. The results support the observations that TSH has a bone protective action by negatively regulating osteoclastogenesis. Further, our results implicate TSHR-Abs in offering skeletal protection in hyperthyroid Graves' disease, even in the face of high thyroid hormone and low TSH levels. [ABSTRACT FROM AUTHOR]

High maternal blood glucose levels caused by diabetes mellitus can irreversibly lead to maldevelopment of the growing fetus with specific effects on the skeleton. To date, it remains controversial at which stage embryonic development is affected. Specifically during embryonic bone development, it is unclear whether diminished bone mineral density is caused by reduced osteoblast or rather enhanced osteoclast function. Therefore, the aim of this study was to characterize the growth as well as the skeletal differentiation capability of pluripotent embryonic stem cells (ESCs), which may serve as an in vitro model for all stages of embryonic development, when cultured in diabetic levels of D-glucose (4.5 g/L) versus physiological levels (1.0 g/L). Results showed that cells cultivated in physiological glucose gave rise to a higher number of colonies with an undifferentiated character as compared to cells grown in diabetic glucose concentrations. In contrast, these cultures were characterized by slightly decreased expression of proteins associated with the stem cell state. Furthermore, differentiation of ESCs into osteoblasts and osteoclasts was favored in physiological glucose concentrations, demonstrated by an increased matrix calcification, enhanced expression of cell-type-specific mRNAs, as well as activity of the cell-type-specific enzymes, alkaline, and tartrate resistant acidic phosphatase. In fact, this pattern was noted in murine as well as in primate ESCs. Our study suggests that an interplay between both the osteoblast and the osteoclast lineage is needed for proper skeletal development to occur, which seems impaired in hyperglycemic conditions. [ABSTRACT FROM AUTHOR]

AbstractObjective:The purpose of this in vivostudy was to determine the effect of two low-intensity laser therapy (LILT) protocols on macroscopic and microscopic parameters of experimental tooth movement. Materials and Methods:To induce experimental tooth movement in rats, 40 cN of orthodontic force was applied to the left first molars. Next, a gallium–aluminum–arsenide (Ga-Al-As) diode laser with a wavelength of 830 nm and power output of 100 mW was applied with fluence of 6000 J/cm2on the area around the moved tooth. Two different application protocols were used in the experimental groups: one with daily irradiation and another with irradiation during early stages. Macroscopic and microscopic analyses were performed at days 2 and 7 of tooth movement. The amount of tooth movement was measured with a caliper, and tartrate-resistant acid phosphatase and picrosirius staining were used to enable identification of osteoclasts and immature collagen, respectively. Results:The amount of tooth movement did not differ between the irradiated and nonirradiated groups on days 2 and 7 of the experiment. On day 2, no difference was observed in the number of osteoclasts or the percentage of immature collagen. On day 7, there was an increase in the number of osteoclasts after daily applications of LILT, while two applications produced no significant difference from control. The amount of immature collagen on the tension side significantly increased in the nonirradiated group and when LILT was applied for only 2 d, whereas it was shown to be inhibited by daily LILT applications (p< 0.05). Conclusion:The tested LILT protocols were unable to accelerate tooth movement. Even though the number of osteoclasts increased when LILT was applied daily, the repair at the tension zone was inhibited. [ABSTRACT FROM AUTHOR]

AbstractObjective:The aim of this study was to assess by light microscopy changes in alveolar bone during orthodontic movement in rats. Background:Orthodontic movement causes both removal and deposition of bone tissue. The use of laser phototherapy (LPT) is considered an enhancement factor for bone repair. Methods:Thirty Wistar rats were divided into two groups (n= 15) and subdivided according to animal death (7,13, and 19 days). Half of the animals in each group were treated with LPT during orthodontic movement. After animal death, specimens were processed and underwent histological and semi-quantitative analyses (HE and Sirius red). Results:LPT-irradiated specimens showed significantly higher numbers of osteoclasts when compared with controls at both 7 (p= 0.015) and 19 (p= 0.007) days, as well as significant increases in the number of osteoblasts (p= 0.015) between days 7 and 13. The amount of collagen matrix was significantly reduced between days 7 and 13 at both pressure and tension sites in controls (p= 0.015) but not in LPT-treated animals. LPT-treated subjects showed significantly greater deposition of collagen matrix at the pressure site at both the thirteenth (p= 0.007) and nineteenth days (p= 0.001). At the tension site, a significant increase in the amount of collagen matrix was observed in non-irradiated specimens (p= 0.048) between days 7 and 19. Conclusions:LPT caused significant histological changes in the alveolar bone during induced tooth movement, including alterations in the number of both osteoclasts and osteoblasts and in collagen deposition in both pressure and tension areas. [ABSTRACT FROM AUTHOR]

Cyclooxygenase-2 (COX-2) inhibitors suppress bone repair and bone formation by suppressing angiogenesis as well as potentially interfering with osteoblast and osteoclast functions. In spite of these reports, there is a controversy over the exact effects of COX-2 inhibitors on bone formation processes itself. This work was designed to investigate the effect of COX-2 inhibitor on osteogenesis of human bone marrow-derived mesenchymal stem cells (MSC). MSCs in osteogenesis were treated with COX-2 inhibitor (celecoxib and naproxen) in the absence or presence of interleukin-1β (IL-1β), which was used to induce inflammation. Following differentiation, alkaline phosphatase (ALP) and calcium contents of IL-1β-treated MSC were significantly reduced by high doses of COX-2 inhibitors compared with the low-dose group. However, in non-inflammatory-conditioned MSCs, ALP and calcium contents were not reduced by COX-2 inhibitors. The mRNA expression of Runx2/Cbfα1, Dlx5, and osteocalcinwas also decreased by COX-2 inhibitors in inflammatory-conditioned MSCs and showed a significant decrease for the high dose while they remained constant in the non-inflammatory-conditioned MSCs. These data indicate that the osteogenic potential of MSC is inhibited/delayed by the treatment of high-dose NSAIDs under inflammatory conditions. [ABSTRACT FROM AUTHOR]

Background and Aims: Paget's disease of bone (PDB) is a focal bone disorder affecting the skeleton segmentally. The disease affects osteoclasts which increase in size, number, and activity. One of the etiopathogenic hypotheses is that the disease is genetic. It has been reported that Rho GEF Vav3 is an essential factor in the regulation of osteoclast function, and alteration of the VAV3 gene could influence the development of the disease. The aim of our study was to perform an association study between variants of the VAV3 gene and the risk of developing Paget's disease of bone. Patients and Methods: The genotypic and allelic distribution of the VAV3 c.892A>T/p.T298S (rs7528153) polymorphism was compared between a cohort of 238 Spanish subjects with PDB and a cohort of 253 healthy subjects. Results: Our results indicated that individuals carrying the VAV3 rs7528153 TT genotype were at a significantly increased risk of developing PDB (p < 0.001, odds ratio [OR] = 3.15, 95% confidence interval [95% CI] = 1.77-5.61). Conclusions: These results suggest that inheriting the VAV3 rs7528153 polymorphism is a likely susceptibility factor for developing Paget's disease of bone. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) are characterized by their hematopoiesis-supporting and immunosuppressive capacity, while osteoclasts are main cell components in the endosteal hematopoietic stem cell niche and pivotal players in osteoimmunology. To clarify the association of these 2 kinds of cells, mouse CD11bmonocytes were cultured onto MSC layers in the presence or absence of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-B ligand (RANKL). The results showed that MSCs independently supported osteoclast development and this effect was enhanced by M-CSF and RANKL. Interestingly, tumor necrosis factor- (TNF-)–stimulated MSCs turned to inhibit osteoclast formation and protect tusk slices from osteoclastic resorption. Real-time PCR and ELISA assays demonstrated that osteoprotegerin expression at both mRNA and protein levels in TNF-–stimulated MSCs was up-regulated, at least partially by activating the mitogen-activated protein kinase pathway. Furthermore, TNF-–stimulated MSCs maintained their immunophenotypic, multipotential differentiation and immunosuppressive characteristics. Moreover, MSCs treated with synovial fluid from rheumatoid arthritis patients modulated osteoclast generation in close relation with the TNF- levels. This study suggests that MSCs exhibit dual modulatory function on osteoclasts and the result might shed light on understanding the involvement of MSCs in the inflammatory diseases. [ABSTRACT FROM AUTHOR]

AbstractNuclear factor-kappa B (NF-κB) is involved in osteoclast differentiation and activation. Thus, the blockade of the NF-κB pathway might be a novel therapeutic strategy for treating bone metabolic diseases. Periodontitis is subgingival inflammation caused by bacterial infection; this disease also is thought to be a chronic focal point responsible for systemic diseases. In this study, NF-κB decoy oligodeoxynucleotides (ODNs) were topically applied for experimental periodontitis in a debris-accumulation model and wound healing in a bone-defect model of beagle dogs to investigate the effect of decoy ODN on bone metabolism. Application of NF-κB decoy ODN significantly reduced interleukin-6 activity in crevicular fluid and improved alveolar bone loss in the analysis of dental radiographs and DEXA. Direct measurement of exposed root that lost alveolar bone support revealed that NF-κB decoy treatment dramatically protected bone from loss. In a bone-defect model, NF-κB decoy ODN promoted the healing process as compared with control scrambled decoy in micro-CT analysis. Overall, inhibition of NF-κB by decoy strategy prevented the progression of bone loss in periodontitis and promoted the wound healing in bone defects through the inhibition of osteoclastic bone resorption. Targeting of NF-κB might be a potential therapy in various bone metabolic diseases. Antioxid. Redox Signal.11, 2065–2075. [ABSTRACT FROM AUTHOR]

AbstractObjective:This study aimed to investigate the effect of low-intensity pulsed laser (LIPL; 650 nm, 2 mW) irradiation on mRNA expression of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) in rat calvarial cells. Materials and Methods:Cultured cells were treated with LIPL irradiation of 1.14 J/cm2(group A) or 2.28 J/cm2(group B), and non-irradiated cells (group C) were used as controls. The changes in cell numbers, alkaline phosphatase (ALP) activity, RANKL, and OPG mRNA expression in the three study groups was determined using MTT, UV/VIS spectrophotometry, and RT-PCR analyses. Results:The cell numbers in groups A and B increased significantly (7.52 and 8.80, respectively), as did ALP activity (71.95 and 88.20, respectively), compared with group C (p< 0.001). Meanwhile, RANKL and OPG mRNA expression in group A were 51.06 lower and 3.35 times higher, respectively, than those seen in the controls (p< 0.05), and the RANKL:OPG mRNA ratio in group A was 81.82 lower than that in group C (p< 0.005). Conclusion:LIPL irradiation may directly promote osteoblast proliferation and differentiation, and indirectly inhibit osteoclast differentiation, by downregulating the RANKL:OPG mRNA ratio in osteoblasts. Thus LIPL irradiation may play an important role in bone remodeling, and should be valuable for the treatment of bone diseases such as osteoporosis. [ABSTRACT FROM AUTHOR]

Objective:Recently it was found that thyrotropin (TSH) receptors are present both in osteoclast and osteoblast and that TSH can modulate bone remodeling independent of thyroid hormones. The aim of this study was, firstly, to evaluate the effects of acute administration of TSH on bone remodeling markers both in men and in women and, secondly, to evaluate if these effects are mediated by variations in serum osteoprotegerin (OPG) and receptor activator of nuclear factor-KB ligand (RANKL).Design:We studied 30 thyroidectomized patients (10 premenopausal and 10 postmenopausal women, 10 men) affected by thyroid carcinoma on l-thyroxine therapy. Eighty age- and sex-matched subjects were used as controls. A blood sample was drawn from each patient at baseline and 3 and 5 days after recombinant human TSH (rhTSH) administration, in preparation for 131I whole body scan, to assess serum bone markers and serum OPG and RANKL levels.Main outcome:At baseline, postmenopausal women and men had significantly higher values of bone turnover markers and serum OPG compared to control subjects. In all thyroidectomized patients serum RANKL was lower than in controls. After rhTSH administration, serum N-terminal propeptide of type-I procollagen (PINP), a marker of bone formation, increased significantly in postmenopausal women, while serum RANKL significantly increased after 3 days in postmenopausal patients and men returning to baseline values at day 5. Serum OPG levels did not change significantly.Conclusions:The low serum TSH observed in thyroidectomized patients on l-thyroxine therapy is associated with an increase of bone turnover in postmenopausal women and men that is associated with an increase of OPG and a decrease of serum RANKL levels. The acute TSH administration results in an increase of PINP, an index of osteoblastic activity, associated with an increase of serum RANKL. The lack of this response in premenopausal women suggests an influence of estrogen status on bone reactivity to TSH. [ABSTRACT FROM AUTHOR]

Estrogen deficiency arising with the menopause promotes marked acceleration of bone resorption, which can be restored by hormone replacement therapy. The inhibitory effects of estrogen seem to involve indirect cytokine- mediated effects via supporting bone marrow cells, but direct estrogen–receptor mediated effects on the bone-resorbing osteoclasts have also been proposed. Little information is available on whether estrogens modulate human osteoclastogenesis or merely inhibit the functional activity of osteoclasts. To clarify whether estrogens directly modulate osteoclastic activities human CD14+ monocytes were cultured in the presence of M-CSF and RANKL to induce osteoclast differentiation. Addition of 0.1–10 nM 17 β-estradiol to differentiating osteoclasts resulted in a dose-dependent reduction in tartrate resistant acid phosphatase (TRACP) activity reaching 60% at 0.1 nM. In addition, 17 β-estradiol inhibited bone resorption, as measured by the release of the C-terminal crosslinked telopeptide (CTX), by 60% at 0.1 nM, but had no effect on the overall cell viability. In contrast to the results obtained with differentiating osteoclasts, addition of 17 β-estradiol (0.001–10 nM) to mature osteoclasts did not affect bone resorption or TRACP activity. We investigated expression of the estrogen receptors, using immunocytochemistry and Western blotting. We found that ER- α is expressed in osteoclast precursors, whereas ER- β is expressed at all stages, indicating that the inhibitory effect of estrogen on osteoclastogenesis is mediated by ER- α for the major part. In conclusion, these results suggest that the in vivo effects of estrogen are mediated by reduction of osteoclastogenesis rather than direct inhibition of the resorptive activity of mature osteoclasts. [ABSTRACT FROM AUTHOR]

Bone homeostasis is maintained by the balanced action of bone-forming osteoblasts and bone-resorbing osteoclasts. Multinucleated, mature osteoclasts develop from hematopoietic stem cells via the monocyte–macrophage lineage, which also give rise to macrophages and dendritic cells. Despite their distinct physiologic roles in bone and the immune system, these cell types share many molecular and biochemical features. To provide insights into how osteoclasts differentiate and function to control bone metabolism, we employed a systematic approach to profile patterns of osteoclast-specific gene expression by combining suppression subtractive hybridization (SSH) and cDNA microarray analysis. Here we examined how gene expression profiles of mature osteoclast differ from macrophage or dendritic cells, how gene expression profiles change during osteoclast differentiation, and how Mitf, a transcription factor critical for osteoclast maturation, affects the gene expression profile. This approach revealed a set of genes coordinately regulated for osteoclast function, some of which have previously been implicated in several bone diseases in humans. [ABSTRACT FROM AUTHOR]