Osteoclast Surface Research Articles

SO07118F-SODIUM FLUORIDE POSITRON EMISSION TOMOGRAPHY AND BONE HISTOMORPHOMETRY - COMPARISON BETWEEN ONLY BONE TURNOVER -BASED AND EXPERT EVALUATION -BASED CLASSIFICATION OF RENAL OSTEODYSTROPHY

Abstract Background and Aims The diagnosis and the differentiation of renal osteodystrophy (ROD) are challenging. Bone biopsy is the golden standard, but it is invasive and not available in every center. Bone turnover rate is defined by bone formation rate and/or activation frequency. Adynamic bone disease is defined as low turnover bone with reduced osteoblast- and osteoclast activities. Hyperparahyreoid bone disease or osteitis fibrosa is defined as high turnover bone with osteoclast- and osteoblast activities and fibrosis. 18F- Sodium Fluoride positron emission tomography (18F-NaF PET) is a noninvasive imaging technique that allows assessment of regional bone turnover. The aim was to assess how well bone turnover –based classification of ROD correlates with the classification determined by an expert histomorphometrist (HK), and how these correlate with 18F-NaF PET analysis Method A total of 24 dialysis patients underwent a 18F-NaF PET scan. Fluoride activity was measured at the anterior iliac crest and in the lumbar region. An iliac crest bone biopsy was obtained within 4 weeks from the PET-scan. The diagnosis of bone histomorphometry was determined based on turnover-mineralization-volume (TMV) classification. Firstly, bone turnover was assessed using bone formation rate and activation frequency. Secondly, also other histomorphometric parameters (eg. osteoid volume, osteoid surface, resorption surface, mineralized surface, osteoblast and osteoclast surfaces and peritrabecular fibrosis) were also taking into account for classification of ROD by a histomorphometrist. Results Based on bone turnover parameters only, 12% of the patients had high turnover and 64% low turnover. When the diagnosis of renal osteodystrophy was made by a histomorphometrist, 40% had hyperparathyreoid bone/osteitis fibrosa and 24% adynamic bone disease or ostemalasia. 18F-NaF PET´s sensitivity to recognize hyperparathyreoid bone disease was 80% end specificity 100% (cut-of value 0.055).18F-NaF PET´s sensitivity to recognize adynamic bone disease was 100% and specificity 61% (cut-of value of fluoride-activity 0.038) Conclusion 18F-NaF PET works well as a diagnostic tool, when the diagnosis of ROD is based on the histopathological evaluation. It remains unknown how variations in normal bone turnover rate can be detected in CKD patients by 18F-NaF PET and if treatment decisions of ROD can be made only based on bone turnover.

Quantitative histomorphometric analysis of halved iliac crest bone biopsies yield comparable ROD diagnosis as full 7.5mm wide samples.

Histomorphometric analysis of a transiliac bone biopsy is the gold standard for the diagnosis of renal osteodystrophy (ROD). This procedure is costly, invasive and usually performed with a trephine with an internal diameter of 7.5mm. Our objective was to evaluate the accuracy of ROD diagnosis on halved histological bone sections to determine if they are comparable to the standard 7.5mm samples. We included 68 bone biopsies performed in CKD patients for diagnostic purposes with a 7.5mm diameter trephine. Quantitative histomorphometric analysis of the whole bone samples was performed including assessment of bone mineralization, turnover and volume. Each histological section (representing the whole 7.5mm diameter biopsy) was then divided lengthwise in two hemisections (representing the 3.5mm diameter biopsy). Histomorphometric analysis was repeated this time on the two hemibiopsies for each sample, blinded from initial results. Diagnoses were classified as osteitis fibrosa, adynamic bone disease, mixed uremic bone disease, osteomalacia or other. Correlations between the whole sample and the hemibiopsies for each parameter were studied. Concordance between the various bone parameters and final ROD diagnosis obtained from the whole section versus the two hemi sections was evaluated. Highly significant correlations were found between parameters measured on the whole section and the corresponding hemisections, with r coefficient of 0.98 for osteoid surface and thickness and bone formation rate, 0.97 for osteoclast surface, and 0.96 for bone volume (p<0.001). Final diagnosis was in full accordance between the whole biopsy and the two corresponding hemi-biopsies in 91% of cases. Accurate diagnosis of ROD type was obtained by evaluation of bone surface areas of 3mm diameter. These data suggest that small invasive bone biopsies might provide accurate ROD diagnostics while decreasing both invasiveness and cost of the procedure.

Classifications in Brief: The Dorr Classification of Femoral Bone.

Classifications in Brief: The Dorr Classification of Femoral Bone.

Effects of mild hyperbaric oxygen on osteoporosis induced by hindlimb unloading in rats.

Disuse-induced bone loss is caused by a suppression of osteoblastic bone formation and an increase in osteoclastic bone resorption. There are few data available for the effects of environmental conditions, i.e., atmospheric pressure and/or oxygen concentration, on osteoporosis. This study examined the effects of mild hyperbaric oxygen at 1317hPa with 40% oxygen on unloading-induced osteoporosis. Eighteen 8-week old male Wistar rats were randomly divided into three groups: the control for 21days without unloading and mild hyperbaric oxygen (NOR, n = 6), the unloading for 21days and recovery for 10days without mild hyperbaric oxygen (HU + NOR, n = 6), and the unloading for 21days and recovery for 10days with mild hyperbaric oxygen (HU + MHO, n = 6). The cortical thickness and trabecular bone surface area were decreased in the HU + NOR group compared to the NOR group. There were no differences between the NOR and HU + MHO groups. Osteoclast surface area and Sclerostin (Sost) mRNA expression levels were decreased in the HU + MHO group compared to the HU + NOR group. These results suggested that the loss of the cortical and trabecular bone is inhibited by mild hyperbaric oxygen, because of an inhibition of osteoclasts and enhancement of bone formation with decreased Sost expression. We conclude that exposure to mild hyperbaric oxygen partially protects from the osteoporosis induced by hindlimb unloading.

The old sheep: a convenient and suitable model for senile osteopenia.

Existing osteoporosis models in sheep exhibit some disadvantages, e.g., challenging surgical procedures, serious ethical concerns, failure of reliable induction of substantial bone loss, or lack of comparability to the human condition. This study aimed to compare bone morphological and mechanical properties of old and young sheep, and to evaluate the suitability of the old sheep as a model for senile osteopenia. The lumbar vertebral body L3 of female merino sheep with two age ranges, i.e., old animals (6-10years; n = 41) and young animals (2-4years; n = 40), was analyzed concerning its morphological and mechanical properties by bone densitometry, quantitative histomorphometry, and biomechanical testing of the corticalis and/or central spongious region. In comparison with young sheep, old animals showed only marginally diminished bone mineral density of the vertebral bodies, but significantly decreased structural (bone volume, - 15.1%; ventral cortical thickness, - 11.8%; lateral cortical thickness, - 12.2%) and bone formation parameters (osteoid volume, osteoid surface, osteoid thickness, osteoblast surface, all - 100.0%), as well as significantly increased bone erosion (eroded surface, osteoclast surface). This resulted in numerically decreased biomechanical properties (compressive strength; - 6.4%). Old sheep may represent a suitable model of senile osteopenia with markedly diminished bone structure and formation, and substantially augmented bone erosion. The underlying physiological aging concept reduces challenging surgical procedures and ethical concerns and, due to complex alteration of different facets of bone turnover, may be well representative of the human condition.

The Protective Role of Green Tea against Ethanol-mediated Bone Alterations in Adult Male Rabbits: Biochemical, Histological and Morphometrical Study

Background: Chronic alcohol abuse is a widespread clinical disorder. It leads to bone alterations, mainly osteoporosis. Next to water, tea (green or black) is the most widely consumed beverage in the world. Bone mineral density is affected by several compounds contained in tea extracts.Aim of the Work: Our work aimed to investigate the effects of alcohol on adult male rabbit's compact bone and the possible protective effect of green tea extract. (GTE)Material and Methods: Twenty-four young adult male rabbits were used. They were divided into equal 4 groups: normal control, green tea, ethanol (EtOH) and EtOH – green tea. After 120 days of treatment, all animals were anaesthetized and sacrificed. Blood ethanol and serum ionized calcium (iCa), phosphorus (P) and alkaline phosphatase (ALP) levels were estimated. Serum creatinine, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were estimated to determine renal and hepatic integrity. Femurs of each rabbit were processed for histological and morphometrical study.Results: Chronic alcohol ingestion resulted in a non_significant decrease in serum iCa level and a significant decrease in serum ALP activity. EtOH -treated rabbits did not differ from the controls in mean serum levels of ALT, AST, creatinine or phosphorus. Bone sections from EtOH- treated rabbits revealed multiple areas of degeneration in the thin cartilaginous plate. The trabeculae were thinner, shorter and fewer than those from controls. There was a significant decrease in osteoblast surface and a significant increase in osteoclast surface. Green tea supplementation resulted in marked improving of biochemical, histological and all histomorphometric parameters under investigation.Conclusion: From the foregoing, dangerous effects of alcohol on the bone became clear. So it is better to restrict its consumption. Natural products as green tea are able to protect against bone deterioration.

Indigenous Microbiota Protects against Inflammation-Induced Osteonecrosis

Medication-related osteonecrosis of the jaw (MRONJ) is a rare intraoral lesion that occurs in patients undergoing long-term and/or high-dose therapy with nitrogen-containing bisphosphonates, a RANKL inhibitor, antiangiogenic agents, or mTOR inhibitors. The presence of pathogenic bacteria is highly associated with advanced stages of MRONJ lesions; however, the exact role of indigenous microbes in MRONJ development is unknown. Here, we report that the normal oral flora in mice protects against inflammation-induced osteonecrosis. In mice that developed osteonecrosis following tooth extraction, there was increased bacterial infiltration when compared with healed controls. Antibiotic-mediated oral dysbiosis led to a local inhibition of bone resorption in the presence of ligature-induced periodontitis (LIP). There was no significant difference in empty lacunae, necrotic bone formation, osteoclast number, and surface area in antibiotic-treated as compared with conventionally colonized mice following extraction of healthy teeth after zoledronic acid infusions. However, extraction of LIP teeth led to increased empty lacunae, necrotic bone, and osteoclast surface area in antibiotic- and zoledronic acid–treated mice as compared with conventionally colonized mice. Our findings suggest that the presence of the indigenous microbiota protects against LIP-induced osteonecrosis.

Moderate Nrf2 Activation by Genetic Disruption of Keap1 Has Sex-Specific Effects on Bone Mass in Mice

Keap1 is a negative controller of the transcription factor Nrf2 for its activity. The Keap1/Nrf2 signaling pathway has been considered as a master regulator of cytoprotective genes, and exists in many cell types including osteoblasts and osteoclasts. Our previous study shows Nrf2 deletion decreases bone formation. Recent studies show hyperactivation of Nrf2 causes osteopenia in Keap1−/− mice, and Keap1−/− osteoblasts have significantly less proliferative potential than Keap1+/− osteoblasts. We aimed to examine if moderate Nrf2 activation by disruption of Keap1 impacts bone metabolism. We examined bone phenotype of Keap1 heterozygotic mice (Ht) in comparison with Keap1 wild type (WT) mice. Deletion or knockdown of Keap1 enhanced the gene expression of Nrf2, ALP and wnt5a in cultured primary osteoblasts compared to WT control. In male mice, compared with their age-matched littermate WT controls, Keap1 Ht mice showed significant increase in bone formation rate (+30.7%, P = 0.0029), but did not change the ultimate force (P < 0.01). The osteoclast cell numbers (−32.45%, P = 0.01) and surface (−32.58%, P = 0.03) were significantly reduced by Keap1 deficiency in male mice. Compared to male WT mice, serum bone resorption marker in male Keap1 Ht mice was significantly decreased. Our data suggest that moderate Nrf2 activation by disruption of Keap1 improved bone mass by regulating bone remodeling in male mice.

Conditional deletion of Adrb2 in mesenchymal stem cells attenuates osteoarthritis-like defects in temporomandibular joint.

β2-adrenergic signal transduction in mesenchymal stem cells (MSCs) induces subchondral bone loss in osteoarthritis (OA) of temporomandibular joints (TMJs). However, whether conditional deletion of β2-adrenergic receptor (Adrb2) in nestin+ MSCs can alleviate TMJ-OA development remains unknown. In this study, nestin-Cre mice were crossed with Adrb2 flox mice to generate mice lacking Adrb2 expression specifically in the nestin+ MSCs (Adrb2-/-), and TMJ-OA development in such mice was investigated. Adrb2 flox mice (Adrb2+/+) and Adrb2-/- mice were subjected to unilateral anterior crossbite (UAC), while mice in the control group were subjected to sham operation. Adrb2+/+ and Adrb2-/- mice in the control group showed no distinguishable phenotypic changes in body weight and length, mandibular condylar size, and other histomorphological parameters of the condylar subchondral bone. A significant increase in subchondral bone loss and cartilage degradation was observed in Adrb2+/+ UAC mice; the former was characterized by decreased bone mineral density, bone volume fraction, and trabecular plate thickness, and increased trabecular separation, osteoclast number and osteoclast surface, and pro-osteoclastic factor expression; the latter was characterized by decreased cartilage thickness, chondrocyte density, proteoglycan area, and collagen II and aggrecan expression, but increased matrix metalloproteinase and alkaline phosphatase expression and percentage area of calcified cartilage. Adrb2 deletion in nestin+ MSCs largely attenuated UAC-induced increase in condylar subchondral bone loss, cartilage degradation, and aberrant calcification at the osteochondral interface. Thus, Adrb2-expressing MSCs in the condylar subchondral bone play an important role in TMJ-OA progression and may serve as novel therapeutic targets for TMJ-OA.

Atherogenic diet-induced bone loss is primarily due to increased osteoclastogenesis in mice

Atherogenic diet (AD) decreased bone density and increased serum cholesterol level in male mice, implying that cholesterol participates in bone loss. The aim of the present study was to identify the cells responsible for bone loss and evaluate the involved mechanism. AD resulted in increased number and surface of osteoclasts (OCs) with in vivo tartrate-resistant acid phosphatase (TRAP) staining, suggesting a critical role of OCs in cholesterol-induced bone loss. In vitro, cholesterol loading by oxidized low-density lipoprotein (oxLDL) increased the size and number of OCs as well as bone resorption activity, suggesting that cholesterol loading affects the number and activity of OCs. In contrast, cholesterol depletion by simvastatin decreased osteoclastogenesis and bone resorption. oxLDL stimulated osteoblasts (OBs) to increase expression of receptor activator of nuclear factor kappa-Β ligand (RANKL), resulting in increased OC formation when OBs were co-cultured with bone marrow derived macrophages. oxLDL increased expression of CD36 and liver X receptors (LXRα) in OCs as well as low density lipoprotein receptor (LDLR) and LXRα in OBs. These results suggest that CD36 and LXRα mediate the effect of oxLDL in OCs, whereas LDLR and LXRα mediate the effect of oxLDL in OBs. These findings demonstrate cholesterol-induced bone loss with increasing number and activity of OCs in mice, suggesting another harmful effect of cholesterol, a major cause of atherosclerosis.

The deleterious effects of smoking in bone mineralization and fibrillar matrix composition

IntroductionThis study aimed to verify the effects of cigarette smoke exposure in bone mineralization and fibrillar matrix composition as well as in bone healing after tibial fracture induction. MethodsC57Bl/6 Mice were assigned according to exposure and surgery: C room air; F room air and tibia open osteotomy; CS cigarette smoke; FCS cigarette smoke and tibia open osteotomy. In order to study fracture healing we performed, under anesthesia, a bone injury through a tibial shaft osteotomy. Bone samples were obtained to evaluate bone histomorphometry, trabecular morphology and volume, trabecular collagen types composition and presence of inflammatory cytokines and growth factors. ResultsCS exposure significantly reduced the thickness of bone trabeculae associated with decrease in mineralizing surface and mineral deposition rate, leading a lower bone formation rate and longer mineralization time. Resorption surface and osteoclastic surface were greater in the CS group, attesting increased resorptive action. There was a decrease in type I collagen deposition and genes expression in the CS and FCS groups compared to C group and in contrast there was an increase in type V collagen deposition and genes expression in the CS, FC and FSC groups compared to C group. Also, CS exposure induced a decrease in bone forming cytokines and an increase in inflammatory associated cytokines, and these changes were intensified under fracture conditions. ConclusionCigarette smoke exposure alters bone matrix composition and worsens bone mineralization, leading to bone fragility by increasing collagen V synthesis and deposition and impairing collagen I fibril forming and assembling. And these deleterious effects contributed to the worsening in fracture healing after tibia osteotomy.

Pisidium coreanum Inhibits Multinucleated Osteoclast Formation and Prevents Estrogen-Deficient Osteoporosis.

Mollusks have served as important sources of human food and medicine for a long time. Raw Pisidium coreanum, a freshwater bivalve of the phylum Mollusca, is used in traditional therapies in parts of Asia. However, the therapeutic effects of Pisidium coreanum on bone diseases are not known. We investigated the functional roles of Pisidium coreanum in osteoporotic bone diseases. Pisidium coreanum inhibited the differentiation of bone marrow-derived monocytic cells into mature osteoclasts in vitro. The ovariectomized mice that received oral administration of Pisidium coreanum showed improvements in both trabecular and cortical bones. This preventive activity of Pisidium coreanum against bone loss was due to limited osteoclast maturation with reduced osteoclast surface extent in trabecular bone tissue. The formation of large multinucleated osteoclasts in vitro was significantly decreased in response to Pisidium coreanum, consistent with the reduced expression levels of osteoclast markers and fusion-related genes, such as NFATc1, p65, integrin αvβ3, DC-STAMP, OC-STAMP, Atp6v0d2, FAK, CD44, and MFR. These data suggest that Pisidium coreanum inhibits osteoclast differentiation by negatively regulating the fusion of mononuclear osteoclast precursors. Thus, our data demonstrate the ability of Pisidium coreanum to effectively prevent estrogen-deficient osteoporosis through inhibition of multinucleated osteoclast formation.

DSS-induced colitis produces inflammation-induced bone loss while irisin treatment mitigates the inflammatory state in both gut and bone

Chronic pediatric inflammatory bowel disease (IBD) leads to lack of bone accrual, bone loss, and increased fractures. Presently there is no cure, and many IBD treatments incur negative side effects. We previously discovered treatment with exogenous irisin resolved inflammatory changes in the colon, gut lymphatics, and bone in a mild IBD rodent model. Here we assess irisin treatment in severe IBD induced via dextran sodium sulfate (DSS). Male Sprague Dawley rats (2-mo-old) were untreated (Con) or given 2% DSS in drinking water. In week two, half of each group (Con + Ir and DSS + Ir) received injections of recombinant irisin (i.p., 2x/wk). After 4 weeks, gut inflammation was associated with declines in bone mineral density and cancellous bone volume. Furthermore, elevated osteocyte TNF-α, interleukin-6, RANKL, OPG, and sclerostin corresponded with higher osteoclast surfaces and lower bone formation rate in DSS animals as well as lower ultimate load. While irisin treatment improved colon inflammation, there were no improvements in bone density or bone mechanical properties; however, irisin elevated bone formation rate, decreased osteoclast surfaces, and reduced osteocyte pro-inflammatory factors. These data highlight the negative impact of chronic gut inflammation on bone as well as the therapeutic potential of irisin as an anti-inflammatory treatment.

Impact of a Long-Term High Fat Diet on Bone Microarchitecture and Muscle Structure in Adult Male and Female Normal Mice

Background and Hypothesis: Hyperglycemia is a major source of disease and morbidity among the adult population. Prior studies correlate long-term high fat diet (HFD) mediated hyperglycemia with bone fragility and muscle weakness. Furthermore, the mechanism driving hyperglycemia between sexes are unknown. Our group previously showed that HFDs induced insulin resistance in male mice and glucose intolerance in female mice. This establishes the need to study the impact of long-term HFDs on the bones and muscles using an older cohort of both male and female mice. For that, we hypothesized a long-term HFD mediated hyperglycemia will change bone and muscle structures and impair their functions in adult male and female mice.&#x0D; Experimental Design or Project Methods: 22-week C57Bl6 mice were fed either a HFD or low fat diet (LFD) for 25 weeks. After euthanasia, bones and muscles were harvested and evaluated using MicroCT, histology, and mechanical testing. Statistical analysis was performed using GraphPad Prism with p&lt;0.05 considered significant.&#x0D; Results: MicoCT data saw significant reductions to cortical thickness (p&lt;0.05), bone mineral density (p&lt;0.001), and increases to medullary area (p&lt;0.05) among HFD males and females compared to LFD. HFD-males also experienced significant increase in cortical porosity (P&lt;0.001) whereas no changes were noted in HFDfemales. Trabecular bone volume was relatively unchanged. HFD increased cortical osteoclast surface (p&lt;0.001) for both sexes. Bone histology saw increased marrow adiposity among HFD-females (p&lt;0.05). Muscle histology exhibited HFD-related reductions in myofiber diameter (p&lt;0.001) for both sexes. Mechanical testing demonstrated reduced young’s modulus (p&lt;0.05) and yield stress (p&lt;0.05) among HFD mice, despite non-significant differences in ultimate strength.&#x0D; Conclusion and Potential Impact: The changes associated with a long-term HFD differed between sexes but still led to functional impairments of bone and muscle for both sexes, emphasizing the importance of looking further into the mechanisms responsible for these changes. This can potentially translate to the clinic in the treatment of musculoskeletal complications associated with HFDs.

MicroRNA-29a represses osteoclast formation and protects against osteoporosis by regulating PCAF-mediated RANKL and CXCL12

Osteoporosis deteriorates bone mass and biomechanical strength, becoming a life-threatening cause to the elderly. MicroRNA is known to regulate tissue remodeling; however, its role in the development of osteoporosis remains elusive. In this study, we uncovered that silencing miR-29a expression decreased mineralized matrix production in osteogenic cells, whereas osteoclast differentiation and pit formation were upregulated in bone marrow macrophages as co-incubated with the osteogenic cells in transwell plates. In vivo, decreased miR-29a expression occurred in ovariectomy-mediated osteoporotic skeletons. Mice overexpressing miR-29a in osteoblasts driven by osteocalcin promoter (miR-29aTg/OCN) displayed higher bone mineral density, trabecular volume and mineral acquisition than wild-type mice. The estrogen deficiency-induced loss of bone mass, trabecular morphometry, mechanical properties, mineral accretion and osteogenesis of bone marrow mesenchymal cells were compromised in miR-29aTg/OCN mice. miR-29a overexpression also attenuated the estrogen loss-mediated excessive osteoclast surface histopathology, osteoclast formation of bone marrow macrophages, receptor activator nuclear factor-κ ligand (RANKL) and C–X–C motif chemokine ligand 12 (CXCL12) expression. Treatment with miR-29a precursor improved the ovariectomy-mediated skeletal deterioration and biomechanical property loss. Mechanistically, miR-29a inhibited RANKL secretion in osteoblasts through binding to 3′-UTR of RANKL. It also suppressed the histone acetyltransferase PCAF-mediated acetylation of lysine 27 in histone 3 (H3K27ac) and decreased the H3K27ac enrichment in CXCL12 promoters. Taken together, miR-29a signaling in osteogenic cells protects bone tissue from osteoporosis through repressing osteoclast regulators RANKL and CXCL12 to reduce osteoclastogenic differentiation. Arrays of analyses shed new light on the miR-29a regulation of crosstalk between osteogenic and osteoclastogenic cells. We also highlight that increasing miR-29a function in osteoblasts is beneficial for bone anabolism to fend off estrogen deficiency-induced excessive osteoclastic resorption and osteoporosis.

The Hajdu Cheney mutation sensitizes mice to the osteolytic actions of tumor necrosis factor α

Hajdu Cheney syndrome (HCS) is characterized by craniofacial developmental abnormalities, acro-osteolysis, and osteoporosis and is associated with gain-of-NOTCH2 function mutations. A mouse model of HCS termed Notch2tm1.1Ecan harboring a mutation in exon 34 of Notch2 replicating the one found in HCS was used to determine whether the HCS mutation sensitizes the skeleton to the osteolytic effects of tumor necrosis factor α (TNFα). TNFα injected over the calvarial vault caused a greater increase in osteoclast number, osteoclast surface, and eroded surface in Notch2tm1.1Ecan mice compared with littermate WT controls. Accordingly, the effect of TNFα on osteoclastogenesis was greatly enhanced in cultures of bone marrow-derived macrophages (BMMs) from Notch2tm1.1Ecan mice when compared with the activity of TNFα in control cultures. TNFα induced the expression of Notch2 and Notch2 mutant mRNA by ∼2-fold, possibly amplifying the NOTCH2-dependent induction of osteoclastogenesis. The effect of TNFα on osteoclastogenesis in Notch2tm1.1Ecan mutants depended on NOTCH2 activation because it was reversed by anti-NOTCH2 negative regulatory region and anti-jagged 1 antibodies. The inactivation of Hes1 prevented the TNFα effect on osteoclastogenesis in the context of the Notch2tm1.1Ecan mutation. In addition, the induction of Il1b, but not of Tnfa and Il6, mRNA by TNFα was greater in Notch2tm1.1Ecan BMMs than in control cells, possibly contributing to the actions of TNFα and NOTCH2 on osteoclastogenesis. In conclusion, the HCS mutation enhances TNFα-induced osteoclastogenesis and the inflammatory bone-resorptive response possibly explaining the acro-osteolysis observed in affected individuals.

Up‐regulated CST5 inhibits bone resorption and activation of osteoclasts in rat models of osteoporosis via suppression of the NF‐κB pathway

Here, we aim at exploring the effect of CST5 on bone resorption and activation of osteoclasts in osteoporosis (OP) rats through the NF‐κB pathway. Microarray analysis was used to screen the OP‐related differentially expressed genes. Osteoporosis was induced in rats by intragastric retinoic acid administration. The serum levels of tartrate‐resistant acid phosphatase (TRAP), bone alkaline phosphatase (BALP) and osteocalcin (OC) and the expression of CD61 on the surface of osteoclasts were examined. The number of osteoclasts and the number and area of resorption pits were detected. Besides, the pathological changes and bone mineral density in bone tissues of rats were assessed. Also, the relationship between CST5 and the NF‐κB pathway was identified through determining the expression of CST5, RANKL, RANK, OPG, p65 and IKB. Poorly expressed CST5 was indicated to affect the OP. CST5 elevation and inhibition of the NF‐κB pathway decreased serum levels of TRAP, BALP and OC and expression of CD61 in vivo and in vitro. In OP rats, CST5 overexpression increased trabecular bones and bone mineral density of bone tissues, but decreased trabecular separation, fat within the bone marrow cavities and the number of osteoclasts through inhibiting the NF‐κB pathway. In vivo experiments showed that CST5 elevation inhibited growth in number and area of osteoclastic resorption pits and restrained osteoclastic bone absorption by inhibiting the NF‐κB pathway. In summary, overexpression of CST5 suppresses the activation and bone resorption of osteoclasts by inhibiting the activation of the NF‐κB pathway.

The Effects of Annatto Tocotrienol Supplementation on Cartilage and Subchondral Bone in an Animal Model of Osteoarthritis Induced by Monosodium Iodoacetate.

Osteoarthritis is a degenerative joint disease which primarily affects the articular cartilage and subchondral bones. Since there is an underlying localized inflammatory component in the pathogenesis of osteoarthritis, compounds like tocotrienol with anti-inflammatory properties may be able to retard its progression. This study aimed to determine the effects of oral tocotrienol supplementation on the articular cartilage and subchondral bone in a rat model of osteoarthritis induced by monosodium iodoacetate (MIA). Thirty male Sprague-Dawley rats (three-month-old) were randomized into five groups. Four groups were induced with osteoarthritis (single injection of MIA at week 0) and another served as the sham group. Three of the four groups with osteoarthritis were supplemented with annatto tocotrienol at 50, 100 and 150 mg/kg/day orally for five weeks. At week 5, all rats were sacrificed, and their tibial-femoral joints were harvested for analysis. The results indicated that the groups which received annatto tocotrienol at 100 and 150 mg/kg/day had lower histological scores and cartilage remodeling markers. Annatto tocotrienol at 150 mg/kg/day significantly lowered the osteocalcin levels and osteoclast surface of subchondral bone. In conclusion, annatto tocotrienol may potentially retard the progression of osteoarthritis. Future studies to confirm its mechanism of joint protection should be performed.

Anomalous bone changes in ovariectomized type 2 diabetic rats: inappropriately low bone turnover with bone loss in an estrogen-deficient condition.

Estrogen deprivation accelerates bone resorption, leading to imbalance of bone remodeling and osteoporosis in postmenopausal women. In the elderly, type 2 diabetes mellitus (T2DM) frequently coexists as an independent factor of bone loss. However, little is known about the skeletal changes in a combined condition of estrogen deficiency and T2DM. Herein, we performed ovariectomy (OVX) in nonobese Goto-Kakizaki (GK) T2DM rats to examine changes associated with calcium and phosphate metabolism and bone microstructures and strength. As expected, wild-type (WT) rats subjected to ovariectomy (OVX-WT) had low trabecular bone volume and serum calcium with increased dynamic histomorphometric and serum bone markers, consistent with the high turnover state. T2DM in GK rats also led to low trabecular volume and serum calcium. However, the dynamic histomorphometric markers of bone remodeling were unaffected in these GK rats, indicating the distinct mechanism of T2DM-induced bone loss. Interestingly, OVX-GK rats were found to have anomalous and unique changes in bone turnover-related parameters, i.e., decreased osteoblast and osteoclast surfaces with lower COOH-terminal telopeptide of type I collagen levels compared with OVX-WT rats. Furthermore, the levels of calciotropic hormones, i.e., parathyroid hormone and 1,25(OH)2D3, were significantly decreased in OVX-GK rats. Although the OVX-induced bone loss did not further worsen in GK rats, a three-point bending test indicated that OVX-GK bones exhibited a decrease in bone elasticity. In conclusion, T2DM and estrogen deficiency both led to microstructural bone loss, the appearance of which did not differ from each factor alone. Nevertheless, the combination worsened the integrity and suppressed the turnover, which might eventually result in adynamic bone disease.

Effects of rigosertib on the osteo-hematopoietic niche in myelodysplastic syndromes

Rigosertib is a novel multi-kinase inhibitor, which has clinical activity towards leukemic progenitor cells of patients with high-risk myelodysplastic syndromes (MDS) after failure or progression on hypomethylating agents. Since the bone marrow microenvironment plays an important role in MDS pathogenesis, we investigated the impact of rigosertib on cellular compartments within the osteo-hematopoietic niche. Healthy C57BL/6J mice treated with rigosertib for 3weeks showed a mild suppression of hematopoiesis (hemoglobin and red blood cells, both - 16%, p < 0.01; white blood cells, - 34%, p < 0.05; platelets, - 38%, p < 0.05), whereas there was no difference in the number of hematopoietic stem cells in the bone marrow. Trabecular bone mass of the spine was reduced by rigosertib (- 16%, p = 0.05). This was accompanied by a lower trabecular number and thickness (- 6% and - 10%, respectively, p < 0.05), partly explained by the increase in osteoclast number and surface (p < 0.01). Milder effects of rigosertib on bone mass were detected in an MDS mouse model system (NHD13). However, rigosertib did not further aggravate MDS-associated cytopenia in NHD13 mice. Finally, we tested the effects of rigosertib on human mesenchymal stromal cells (MSC) in vitro and demonstrated reduced cell viability at nanomolar concentrations. Deterioration of the hematopoietic supportive capacity of MDS-MSC after rigosertib pretreatment demonstrated by decreased number of colony-forming units, especially in the monocytic lineage, further supports the idea of disturbed crosstalk within the osteo-hematopoietic niche mediated by rigosertib. Thus, rigosertib exerts inhibitory effects on the stromal components of the osteo-hematopoietic niche which may explain the dissociation between anti-leukemic activity and the absence of hematological improvement.