Influence Bone Remodeling Research Articles

Expression of Wnt-Inhibitors and SDF-1 in Whole Bone Marrow Biopsies in Association to the Osteolytic Bone Disease of Multiple Myeloma.

AbstractAbstract 2922Osteolytic bone disease (OBD) in multiple myeloma (MM) is caused by a combination of osteoclast hyperactivation and osteoblast inhibition. Several studies have shown that the Wnt-inhibitor DKK1 is one of the factors involved in osteoblast inhibition (Tian, NEJM, 2003), while the role of other Wnt-pathway inhibitors, such as secreted frizzled related protein-2 (SFRP2), SFRP3, sclerostin and Wnt inhibitory factor 1 (WIF1) are less studied. In addition stromal derived factor 1 (SDF-1/CXCL12) that interacts with CXCR4 in bone-marrow (BM) is a potential factor in OBD, although conflicting data has been reported. SDF-1has in mesenchymal stromal cells (MSC) been found downregulated in MM patients compared to healthy volunteers (HV) and also downregulated in MM plasma cells vs. normal plasma cells, while SDF-1 serum levels has been correlated to the extent of OBD in MM.Until now, gene expression studies in MM have been performed on isolated MM plasma cells or bone marrow aspirates, which are not completely representative of the cell composition in the BM micro-environment. We used a novel strategy whereby BM biopsies are snap-frozen and subsequently gene expression is analyzed. Using this strategy we have examined the gene-expression of Wnt inhibitors and SDF1. In addition, we have evaluated the protein levels of DKK1 and SDF-1 in matched BM plasma samples.An additional BM core biopsy obtained during the diagnostic procedure of MM patients was snap-frozen. Biopsies were cut, homogenized and RNA was purified and analyzed by qRT-PCR using low density arrays (Applied Biosystems). The relative quantitative gene expression was calculated using 3 internal reference genes (ABL, GAPDH and GUS). OBD was evaluated using standard radiographs. All patients were untreated and did not receive medicine that could influence bone remodeling. We examined 10 healthy volunteers (HV), 35 monoclonal gammopathy of unknown significance (MGUS) and 65 untreated MM patients, which according to radiographic findings were divided into NO/LOW and advanced OBD, i.e. OBD in ≥2 regions.ELISA was performed on a total of 31 matched BM plasma samples of HV, MGUS and MM obtained at the same time point as the biopsies. In addition, extra samples without gene data (N=52) were analyzed. Commercial kits for DKK-1 (RnD, Quantikine) and SDF-1 (RnD, Quantikine) were run in duplicates according to manufacturer's instructions.Gene expression of DKK1 and SFRP3 were significantly (p<0.05) associated with OBD with increasing gene-expression with increasing OBD, while SDF-1 were significantly associated with decreasing gene-expression with increasing OBD. Gene expression of SFRP2, sclerostin and WIF1 did not associate with the extent of OBD. When comparing HV, MGUS and MM independent of OBD, sclerostin showed a tendency to be higher expressed in MM vs. MGUS and HV (p=0.08).A significant correlation between gene expression levels and protein expression levels was found for DKK-1 (Spearman's rho= 0.57, p=0.0006,) while the correlation of gene and protein expression levels of SDF-1 did not reach significance (Spearman's rho =0.31, p=0.074).The protein level of DKK1 in BM plasma was upregulated in MM and associated significantly with the OBD level (p<0.05). The protein level of SDF1 did not correlate to the extent of OBD.Our gene expression study of MM with minimal manipulation of the samples showed that the Wnt-inhibitors DKK1 and SFRP3 were significantly associated to the extent of OBD, while SDF1 gene expression inversely correlated with the extent of OBD. The report of simultaneous measurement of not only DKK1 but also SFRP3 and other Wnt-inhibitors is novel. The use of whole snap-frozen BM biopsies is a new method to evaluate gene expression in MM that makes it possible to include the tumor micro-environment and investigate patients independent of degree of MM plasma cell infiltration, and we were able to reproduce the DKK1 findings by others. The gene expression results were complemented by protein data for DKK1 and SDF-1. Our study shows for the first time the difference between SDF1 gene and protein levels in matched MM patient samples, and the difference measured incites us to further investigate the post-translational regulation of SDF-1, and the possibility of SDF1 protein being less bound to CXCR4 in MM. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Expression of Factors in the Hepatocyte Growth Factor (HGF) Pathway in Whole Bone Marrow Biopsies in Association to the Osteolytic Bone Disease of Multiple Myeloma

AbstractAbstract 3977Osteolytic bone disease (OBD) in multiple myeloma (MM) is known to be caused by a combination of osteoclast hyperactivation and osteoblast inhibition. One of the pathways known to be involved in osteoblast inhibition from in vitro studies is the HGF pathway consisting of HGF, its receptor MET, the co-receptor Syndecan-1 (SDC-1), the partial MET antagonist Decorin and HGF activator responsible for HGF processing to its active form. So far, gene expression studies in MM have been performed on isolated MM plasma cells or bone marrow (BM) aspirates, which are not completely representative of the cell composition in the BM micro-environment. We used a novel strategy, whereby gene expression of factors associated with the HGF pathway was evaluated in snap-frozen BM biopsies, and moreover we determined the protein levels in matched BM plasma samples.An additional BM core biopsy obtained during the diagnostic procedure of MM patients was snap-frozen. Biopsies were cut, homogenized and RNA was purified and analyzed by qRT-PCR using low density arrays (Applied Biosystems). The relative quantitative gene expression was calculated using 3 internal reference genes (ABL, GAPDH and GUS). OBD was evaluated using standard radiographs. All patients were untreated and did not receive medicine that could influence bone remodeling. We examined 10 healthy volunteers (HV), 35 monoclonal gammopathy of unknown significance (MGUS) and 65 untreated MM patients, which according to radiographic findings were divided into NO/LOW and advanced OBD, i.e. OBD in ≥2 regions.ELISA was performed on a total of 31 matched BM plasma samples of HV, MGUS and MM obtained at the same time point as the biopsies. In addition, extra samples without gene data (N=52) were analyzed. Commercial kits for SDC-1 (Diaclone), HGF (RnD, Quantikine) and Decorin (RnD, Duoset) were run in duplicates according to manufacturer's instructions.Gene expression of HGF, SDC1, and MET were significantly different comparing HV, MGUS, no/low and advanced OBD (p<0.05) (For HGF, see figure 1). Decorin was not associated to OBD. HGF activator was not expressed in any of our samples, but only in the positive control.A significant correlation between gene and protein expression levels measured by ELISA was found for SDC-1 (Spearman's rho= 0.463, p=0.0058) and HGF (Spearman's rho=0.45, p=0.01). No correlation was found between Decorin gene levels and BM plasma levels (Spearman's rho =-0.24, p=0.22).The protein level of SDC-1 and HGF in BM plasma were both upregulated in MM and associated significantly to OBD level (p<0.05), while Decorin were significantly downregulated in MGUS and MM samples compared to HVs (p<0.05). A significant difference in HGF BM plasma levels were found between patients with no/limited OBD (median: 1.7ng/mL) and advanced OBD (median: 6.2ng/mL) in BM plasma.In our expression study reflecting the in vivo situation in MM patients, genes in the HGF pathway and proteins were significantly associated to OBD. The use of whole snap-frozen BM biopsies is a novel strategy for evaluation of gene expression in MM making it possible to investigate patients independent of degree of MM plasma cell infiltration. In addition to the dys-regulated gene expression levels alteration of SDC-1 and HGF was also observed at protein level, supporting the gene expression findings, and underscoring the usefulness of BM biopsies for gene expression studies in MM. Furthermore, our study for the first time shows up regulation of HGF in association with OBD at both gene and protein level in a large clinical material. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

“INTEGRINating” the connexin hemichannel function in bone osteocytes through the action of integrin α5

Mechanical loading influences skeletal structural integrity and bone remodeling. Application of a mechanical stimulus such as fluid flow shear stress to the bone osteocytes activates the cascade of mechanotransduction mediated by multiple signaling molecules. Hemichannels formed by connexin molecules are emerging as a candidate mechanosensor. Connexin 43 (Cx43) hemichannels open in response to mechanical stimulation to release bone modulators which influence bone remodeling. Our study identified a direct interaction between integrin α5 and Cx43 which was essential for hemichannels to open. Uncoupling the interaction blocked the hemichannels and shear stress enhanced the interaction between the two proteins to promote channel opening. More importantly, integrin α5, independent of its association with fibronectin, was activated upon shear stress through a PI3K signaling pathway. These results suggest a critical regulatory mechanism for Cx43 hemichannel opening through the association of integrin α5, resulting in release of bone anabolic factors required for bone development.

New insights into osteoporosis: the bone–fat connection

Osteoporosis and obesity are chronic disorders that are both increasing in prevalence. The pathophysiology of these conditions is multifactorial and includes genetic, environmental and hormonal determinants. Although it has long been considered that these are distinct disorders rarely found in the same individual, emerging evidence from basic and clinical studies support an important interaction between adipose tissue and the skeleton. It is proposed that adiposity may influence bone remodelling through three mechanisms: (i) secretion of cytokines that directly target bone, (ii) production of adipokines that influence the central nervous system thereby changing sympathetic impulses to bone and (iii) paracrine influences on adjacent skeletal cells. Here we focus on the current understanding of bone-fat interactions and the clinical implications of recent studies linking obesity to osteoporosis.

Dasatinib as a Bone-Modifying Agent: Anabolic and Anti-Resorptive Effects

BackgroundBone loss, in malignant or non-malignant diseases, is caused by increased osteoclast resorption and/or reduced osteoblast bone formation, and is commonly associated with skeletal complications. Thus, there is a need to identify new agents capable of influencing bone remodeling. We aimed to further pre-clinically evaluate the effects of dasatinib (BMS-354825), a multitargeted tyrosine kinase inhibitor, on osteoblast and osteoclast differentiation and function.MethodsFor studies on osteoblasts, primary human bone marrow mensenchymal stem cells (hMSCs) together with the hMSC-TERT and the MG-63 cell lines were employed. Osteoclasts were generated from peripheral blood mononuclear cells (PBMC) of healthy volunteers. Skeletally-immature CD1 mice were used in the in vivo model.ResultsDasatinib inhibited the platelet derived growth factor receptor-β (PDGFR-β), c-Src and c-Kit phosphorylation in hMSC-TERT and MG-63 cell lines, which was associated with decreased cell proliferation and activation of canonical Wnt signaling. Treatment of MSCs from healthy donors, but also from multiple myeloma patients with low doses of dasatinib (2–5 nM), promoted its osteogenic differentiation and matrix mineralization. The bone anabolic effect of dasatinib was also observed in vivo by targeting endogenous osteoprogenitors, as assessed by elevated serum levels of bone formation markers, and increased trabecular microarchitecture and number of osteoblast-like cells. By in vitro exposure of hemopoietic progenitors to a similar range of dasatinib concentrations (1–2 nM), novel biological sequelae relative to inhibition of osteoclast formation and resorptive function were identified, including F-actin ring disruption, reduced levels of c-Fos and of nuclear factor of activated T cells 1 (NFATc1) in the nucleus, together with lowered cathepsin K, αVβ3 integrin and CCR1 expression.ConclusionsLow dasatinib concentrations show convergent bone anabolic and reduced bone resorption effects, which suggests its potential use for the treatment of bone diseases such as osteoporosis, osteolytic bone metastasis and myeloma bone disease.

An Isolated Cryptic Peptide Influences Osteogenesis and Bone Remodeling in an Adult Mammalian Model of Digit Amputation

Biologic scaffolds composed of extracellular matrix (ECM) have been used successfully in preclinical models and humans for constructive remodeling of functional, site-appropriate tissue after injury. The mechanisms underlying ECM-mediated constructive remodeling are not completely understood, but scaffold degradation and site-directed recruitment of progenitor cells are thought to play critical roles. Previous studies have identified a cryptic peptide derived from the C-terminal telopeptide of collagen IIIα that has chemotactic activity for progenitor cells. The present study characterized the osteogenic activity of the same peptide in vitro and in vivo in an adult murine model of digit amputation. The present study showed that the cryptic peptide increased calcium deposition, alkaline phosphatase activity, and osteogenic gene expression in human perivascular stem cells in vitro. Treatment with the cryptic peptide in a murine model of mid-second phalanx digit amputation led to the formation of a bone nodule at the site of amputation. In addition to potential therapeutic implications for the treatment of bone injuries and facilitation of reconstructive surgical procedures, cryptic peptides with the ability to alter stem cell recruitment and differentiation at a site of injury may serve as powerful new tools for influencing stem cell fate in the local injury microenvironment.

Ameloblastin is not implicated in bone remodelling and repair

Ameloblastin (AMBN) is an enamel matrix protein produced by ameloblasts. It has been suggested that AMBN might also be implicated in craniofacial bone formation. Our objective was to determine whether AMBN has an effect on osteogenic mineralisation and influences bone remodelling and repair. MC3T3-E1 cells were screened for endogenous expression of enamel proteins using real time PCR. Various osteogenic cells were infected with lentivirus encoding for AMBN and protein expression was verified using immunochemistry. Cultures were stained with alizarin red and mineralisation was quantified. Healing bone was probed for expression of AMBN by DNA microarray analysis. Tooth extraction, experimental tooth movement (ETM), and creation of a non-critical size bone defect in the tibia (BDT) were carried out in wild type and AMBN(Δ5-6) mutant mice. Tissues were processed for immunolabelling of AMBN and Bril, an osteoblast specific protein associated with active bone formation. MC3T3-E1 cells and healing bone showed no significant expression of AMBN. Overexpression of AMBN in osteogenic cultures induced no noticeable changes in mineralisation. In wild type mice, AMBN was immunodetected in ameloblasts and enamel, but not in normal bone, and at sites where bone remodelling and repair were induced. Bone remodelling during ETM and BDT repair in AMBN(Δ5-6) mice were not significantly different from that in wild type animals. Our results suggest that AMBN does not influence osteogenic activity in vitro under the conditions used, and does not participate in craniofacial bone remodelling under mechanical stress and in repair of non-critical size bone defects.

Effect of anorganic bovine bone to autogenous cortical bone ratio upon bone remodeling patterns following maxillary sinus augmentation

Maxillary sinus augmentation is a predictable implant site development technique, although several local and systemic factors may influence outcomes. The aim of this study was to evaluate healing patterns and bone remodeling activity following the use of two different graft mixtures for maxillary sinus augmentation. Patients in need of maxillary sinus augmentation were randomly assigned to two different groups. A graft mixture using a 50% autologous bone (AB) to 50% anorganic bovine bone (ABB) ratio was used in group 1, while a 20% AB to 80% ABB ratio was utilized for group 2. After a 6-month healing period, bone core biopsies were harvested for histological, histomorphometrical, and immunohistochemical analyses. Twenty-eight subjects participated in this study. No statistically significant differences were found between groups in regards to vital bone and non-mineralized tissue proportions. Higher number of osteoid lines (18.05 ± 10.06 in group 1 vs. 9.01 ± 7.53 in group 2; P = 0.023) and higher cellularity, particularly regarding the number of osteocytes (631.85 ± 607.98 in group 1 vs. 219.08 ± 103.26 in group 2; P = 0.002), were observed in specimens from group 1. Differences in expression patterns of osteopontin and tartrate-resistant acid phosphatase were also detected between groups. AB to ABB ratio appears to influence bone remodeling patterns and cell content following maxillary sinus augmentation procedures. Similar proportion of vital bone was found in specimens obtained from both groups. More cellular presence was observed in samples containing higher proportions of AB.

FSHR gene polymorphisms influence bone mineral density and bone turnover in postmenopausal women

FSH, via its receptor (FSHR), influences bone remodeling and osteoclast proliferation and activity. The aim of this study was to evaluate the influence of two single nucleotide polymorphisms (SNPs) of the FSHR gene on bone mineral density (BMD) and bone turnover markers (bone alkaline phosphatase and type I collagen C-telopeptides) in postmenopausal women. Two hundred and eighty-nine unrelated postmenopausal women were genotyped for the SNPs rs1394205 and rs6166. BMD was estimated using dual-energy X-ray absorptiometry and quantitative ultrasound (QUS) methodologies. AA rs6166 women showed a lower BMD (femoral neck and total body), lower stiffness index (calcaneal QUS), and higher serum levels of bone turnover markers compared to GG rs6166 women. The prevalence of osteoporosis was significantly higher in AA rs6166 women compared with GG rs6166 women. These results were not influenced by circulating levels of FSH and estrogens. The SNP rs6166 of the FSHR gene significantly influences BMD in postmenopausal women. In particular, AA rs6166 women are at increased risk of postmenopausal osteoporosis compared with GG rs6166 women, independently of circulating levels of FSH and estrogens. Previous studies have demonstrated that this SNP influences cell and tissue response to hyperstimulation of FSHR in vivo and in vitro. Our study results appear in agreement with these experimental data and with known biological actions of FSH/FSHR system in bone homeostasis.

Role of chemerin/CMKLR1 signaling in adipogenesis and osteoblastogenesis of bone marrow stem cells

Maintenance of healthy bone mass requires a well-coordinated balance between the ongoing processes of bone formation and bone resorption. Bone-forming osteoblasts derive from resident adult stem cells within bone marrow called bone marrow stromal cells (BMSCs). These BMSCs are multipotent and also can give rise to adipocytes, which do not contribute directly to bone formation but may influence bone remodeling through the release of bioactive signaling molecules. Chemerin is a novel adipocyte-derived signaling molecule that promotes adipocyte differentiation. In this study we examined the role of chemerin and the cognate receptors CMKLR1 and CCRL2 as determinants of osteoblast and adipocyte differentiation of the preosteoblast 7F2 cell line and of primary BMSCs. Expression and secretion of chemerin increased dramatically with adipocyte differentiation of these cells. Functionally, knockdown of chemerin or CMKLR1 expression using RNA interference abrogated adipocyte differentiation, clonal expansion, and basal proliferation of BMSCs. In contrast, knockdown of either gene was associated with increased osteoblast marker gene expression and mineralization in response to osteoblastogenic stimuli. Forced expression of the adipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) induced chemerin expression and partially rescued the loss of adipogenesis associated with chemerin or CMKLR1 knockdown in BMSCs. Taken together, these data support a novel role for chemerin/CMKLR1 signaling in regulating adipogenesis and osteoblastogenesis of bone marrow-derived precursor cells. These data reveal a potential role for this signaling pathway as a modulator of bone mass.

Moderate Ingestion of Alcohol Is Associated With Acute Ethanol-Induced Suppression of Circulating CTX in a PTH-Independent Fashion

The "J shape" curve linking the risk of poor bone health to alcohol intake is now well recognized from epidemiological studies. Ethanol and nonethanol components of alcoholic beverages could influence bone remodeling. However, in the absence of a solid underlying mechanism, the positive association between moderate alcoholic intake and BMD remains questionable because of confounding associated social factors. The objective of this work was to characterize the short-term effects of moderate alcohol consumption on circulating bone markers, especially those involved in bone resorption. Two sequential blood-sampling studies were undertaken in fasted healthy volunteers (age, 20-47 yr) over a 6-h period using beer of different alcohol levels (<0.05-4.6%), solutions of ethanol or orthosilicic acid (two major components of beer), and water +/- calcium chloride (positive and negative controls, respectively). Study 1 (24 subjects) assessed the effects of the different solutions, whereas study 2 (26 subjects) focused on ethanol/beer dose. Using all data in a "mixed effect model," we identified the contributions of the individual components of beer, namely ethanol, energy, low-dose calcium, and high-dose orthosilicic acid, on acute bone resorption. Markers of bone formation were unchanged throughout the study for all solutions investigated. In contrast, the bone resorption marker, serum carboxy terminal telopeptide of type I collagen (CTX), was significantly reduced after ingestion of a 0.6 liters of ethanol solution (>2% ethanol; p <or= 0.01, RM-ANOVA), 0.6 liters of beer (<0.05-4.6% ethanol; p < 0.02), or a solution of calcium (180 mg calcium; p < 0.001), but only after calcium ingestion was the reduction in CTX preceded by a significant fall in serum PTH (p < 0.001). Orthosilicic acid had no acute effect. Similar reductions in CTX, from baseline, were measured in urine after ingestion of the test solutions; however, the biological variability in urine CTX was greater compared with serum CTX. Modeling indicated that the major, acute suppressive effects of moderate beer ingestion (0.6 liters) on CTX were caused by energy intake in the early phase (approximately 0-3 h) and a "nonenergy" ethanol component in the later phase (approximately 3 to >6 h). The early effect on bone resorption is well described after the intake of energy, mediated by glucagon-like peptide-2, but the late effect of moderate alcohol ingestion is novel, seems to be ethanol specific, and is mediated in a non-calcitonin- and a non-PTH-dependent fashion, thus providing a mechanism for the positive association between moderate alcohol ingestion and BMD.

Rho GTPase signaling and PTH 3–34, but not PTH 1–34, maintain the actin cytoskeleton and antagonize bisphosphonate effects in mouse osteoblastic MC3T3-E1 cells

Cytoskeletal elements are critical for cell morphology and signal transduction, and are involved in many cellular processes including motility, intracellular transport, and differentiation. Small GTP-binding proteins (G proteins) of the Ras family, such as RhoA, influence various elements of the cytoskeleton. RhoA stabilizes the actin cytoskeleton and promotes formation of focal adhesions. We found previously that RhoA is expressed in osteoblastic cells and is translocated to the plasma membrane and activated by PTH 1–34 as well as by Nleu8,18 Tyr34 PTH 3–34 amide, a PTH analog that does not increase cAMP. We therefore investigated effects of manipulating RhoA on the actin cytoskeleton of osteoblastic MC3T3-E1 cells. Three inhibitors were used: 1) GGTI-2166, a geranylgeranyl transferase I inhibitor that prevents the isoprenylation and membrane translocation of RhoA, 2) Y-27632, a Rho kinase inhibitor, and 3) alendronate, a nitrogen (N)-containing bisphosphonate that reduces intracellular geranylgeranylpyrophosphate through inhibiting farnesyl pyrophosphate synthase. To increase RhoA activity, we used the geranylgeranyl group donor geranylgeraniol (GGOH), and a constitutively active RhoA. The F-actin cytoskeleton and focal adhesions (FA) were visualized with rhodamine–phalloidin and fluorescent anti-vinculin antibodies, respectively. Cells were imaged with confocal microscopy. Actin stress fiber density, edge actin bundle density, focal adhesion density, cellular area and circularity (a morphological descriptor relating area and perimeter) were quantified by a program developed with Matlab® software. GGTI-2166, Y-27632, and alendronate reduced actin stress fibers, FA density, and FA size, but had no effect on edge actin bundle density, cellular area, or circularity. GGOH completely antagonized the effects of alendronate, but did not significantly affect responses to GGTI-2166 or Y-27632. Constitutively active RhoA antagonized the effects of alendronate and GGTI-2166, but not those of Y-27632. The effects of alendronate were also antagonized by Nleu8,18 Tyr34 PTH 3–34 amide, but not by PTH 1–34. The results indicate that RhoA is involved in the maintenance of stress fibers and focal adhesions in osteoblastic cells, that PTH can affect this pathway independently of cAMP, and that a N-containing bisphosphonate can affect the actin cytoskeleton and focal adhesions through actions on geranylgeranyl groups and potentially through RhoA. In view of the importance of the actin cytoskeleton, the findings constitute evidence that N-containing bisphosphonates, when they attain certain concentrations, have effects on osteoblasts that could influence bone remodeling.

Effect of running and swimming exercise training on skeletal growth and bone microstructure of the American alligator (Alligator mississippiensis) with and without the cardiac shunt

Exercise affects the vertebrate skeleton via biomechanical and biochemical stress. Exercise‐induced strains can elicit increased primary bone formation and/or secondary remodelling of limb bones. Exhaustive exercise results in high lactic acid loads, which may require buffering by bone mineral and thus influence bone remodelling. Cardiac shunting in crocodylians is thought to divert protons to the enteric circulation and reduce acidity of systemic arterial blood in exercise‐stressed animals. We subjected juvenile alligators to exhaustive exercise (running or swimming) every other day for 17 months, and compared their skeletons with those of sedentary controls. Prior to exercise training, half the animals in each group had their left aorta surgically occluded, thus rendering them incapable of cardiac shunting. Alligators were fed twice weekly, and given injections of fluorescent dyes to determine bone deposition rates. Preliminary data show that bone formation rate at the femoral midshaft correlates with whole‐body growth rate in all groups, but is significantly higher in treadmill runners. This suggests that terrestrial, but not aquatic, exercise stimulates bone growth. Loss of cardiac shunting did not affect bone formation or remodelling, which suggests cardiac shunting does not protect skeletal integrity in alligators. Funded by NSF IOB 04445680 to JWH. TO and JMB supported by NIH 2T32AR047752.

A study of bone marrow and subcutaneous fatty acid composition in subjects of varying bone mineral density

Osteoporosis is associated with an increase in marrow fat. Fats, particularly polyunsaturated fats, either in co-cultures or diet, have been shown to significantly influence bone remodeling. Whether the increase in marrow fat seen in osteoporosis is also associated with a change in fatty acid composition is not known. This study was undertaken to investigate the fatty acid composition in subjects of varying bone mineral density (BMD). Samples of marrow fat and subcutaneous fat from 126 subjects (98 females, 34 males, mean age 69.7 ± 10.5 years) undergoing orthopedic surgery were analyzed for fatty acid composition by gas chromatography. These results were correlated with BMD assessed by DXA. A total of 22 fatty acids were identified in marrow and subcutaneous fat. Significant differences in fatty acid composition existed between marrow and subcutaneous fat as well as between marrow fat samples obtained from the proximal femur and proximal tibia. Other than cis-7-hexadecenoic acid [C16:1 ( n = 9)] and docosanoic acid [C22:0], no difference in marrow fatty acid composition was evident between subject groups of varying BMD (normal, low bone mass, and osteoporosis). In conclusion, there exists a wide range of individual fatty acids in marrow fat. Marrow fatty acid composition differs from that of subcutaneous fat and varies between predominantly erythropoetic and fatty marrow sites. Other than cis-7-hexadecenoic acid [C16:1 ( n = 9)] and docosanoic acid [C22:0], no difference in marrow fatty acid composition was evident between subjects of varying BMD.

Dissociation of the neuronal regulation of bone mass and energy metabolism by leptin in vivo

The leptin regulation of bone remodeling, which has been documented through studies of loss-of-function mutations of this hormone or of its receptor in mice and humans, still raised several unanswered questions. For instance, it has been assumed but not formally demonstrated that this regulation occurs through neuronal means. Likewise, it has not been possible until now to dissociate the influence leptin exerts on appetite and energy expenditure from this function. We show here through mouse genetic studies that a deletion of the leptin receptor in neurons results in an increase in bone formation and bone resorption, resulting in a high bone mass as seen in leptin-deficient mice. In contrast, the same deletion in osteoblasts only does not influence bone remodeling. Furthermore, through the use of l/l mice, a model of gain of function in leptin signaling harboring a Y985L substitution in the leptin receptor, we show that leptin signaling inhibits bone mass accrual by up-regulating sympathetic activity independently of any change in appetite or energy expenditure. This work establishes that in vivo leptin regulates bone mass accrual by acting through neuronal means and provides a direct demonstration that this function of leptin can occur independently of its regulation of energy metabolism.

Experimental osteoporosis induced in female albino rats and its antagonism by Indian black scorpion ( Heterometrus bengalensis C.L.Koch) venom

The present study was designed to explore the antiosteoporosis activity of the Indian black scorpion ( Heterometrus bengalensis) venom on experimental osteoporosis female albino rats. Sham operated control rats were designated as Gr I, Gr II animals served as osteoporosis control, Gr III osteoporosis rats were treated with SV (1/25th of MLD), Gr IV osteoporosis rats were treated with 1/50th of MLD of SV and Gr V osteoporosis rats were treated with standard (calcium and vit-D3). As compared with the Gr I rats, the Gr II rats showed typical osteoporosis changes in increased of urinary Ca 2+, PO 4 3−, CRE, OH-P levels, serum/plasma Ca 2+, PO 4 3−, TRAP, IL1, IL6, TNFα and PTH level, bone Ca 2+, PO 4 3−, Mg 2+, Zn 2+ and decreased level of serum/plasma ALP, EST and PTH, bone Na +. In Gr III, Gr IV and Gr V rats, the osteoporosis changes of urine, serum and bone, were significantly restored as compared with the Gr II rats. The bone dimensions, morphology and histological changes observed in Gr II rats were restored in Gr III, Gr IV and Gr V rats. This study confirms that the Indian black scorpion venom may influence bone remodeling process by stimulating bone formation and reducing bone resorption process of osteogenesis.

Effects of α/β-androstenediol immune regulating hormones on bone remodeling and apoptosis in osteoblasts

A large body of evidence suggests that the immune system directly impacts bone physiology. We tested whether immune regulating hormones (IRH), 17β-androstenediol (β-AED), 7β,17β-androstenetriol (β-AET) or the 17α-androstenediol (α-AED), and 7α,17β-androstenetriol (α-AET) metabolites could directly influence bone remodeling in vitro using human fetal osteoblasts (FOB-9). The impact on bone remodeling was examined by comparing the ratio of RANKL/OPG gene expression in response to AED and AET compounds. The α-AED was found to significantly increase in the ratio of RANKL/OPG gene expression and altering the morphology of RANKL stained FOB-9 cells. Cell viability was assessed using a Live/Dead assay. Again α-AED was unique in its ability to reduce the proportion of viable cells, and to induce mild apoptosis of FOB-9 cells. Treatment of FOB-9 cells with WY14643, an activator of PPAR-α and -γ, also significantly elevated the percentage of dead cells. This increase was abolished by co-treatment with GW9962, a specific inhibitor of PPAR-γ. Analysis of PPAR-γ mRNA by Quantitative RT-PCR and its activation by DNA binding demonstrated that α-AED increased PPAR-γ activation by 19%, while β-AED conferred a 37% decrease in PPAR-γ activation. In conclusion, α-AED opposed β-AED by elevating a bone resorption scenario in osteoblast cells. The increase in RANKL/OPG is modulated by an activation of PPAR-γ that in turn caused mild apoptosis of FOB-9 cells.

Are there biological markers of wear?

Potential systemic markers of implant wear include products of the wear process (particles and ions) and mediators of the inflammatory reaction that can be induced by wear. Ions from polymers used in arthroplasty are not specific, but high metal ion levels may help identify patients with unexpectedly high wear of metal-on-metal implants. The kinetics of ion production, transport, and excretion are complex, however, so it is currently difficult to interpret the significance of mild elevations in metal ions. Indices of bone turnover (eg, collagen fragments) and mediators involved in the inflammatory reaction to particles (eg, osteoprotegerin, RANKL, interleukins) may be associated with osteolysis, but systemic disorders (eg, osteoarthritis) and the use of medications that influence bone remodeling limit the predictive value of these analytes with respect to the consequences of implant wear. Using genomic and proteomic methods to measure multiple analytes offers promise, but the challenge is to identify markers specifically associated with wear that are not elevated by other conditions that often coexist in this patient population.

Increased Osteoprotegerin Levels in Response to Increased Bone Resorption in AL Amyloidosis: Comparisons with Bone Remodeling of Multiple Myeloma.

Primary systemic amyloidosis (AL) and multiple myeloma (MM) are both clonal plasma cell disorders with distinct clinical characteristics. Bone disease is a hallmark of symptomatic MM in contrast to AL where lytic bone lesions are not found. Myeloma bone disease is characterized by an imbalance between osteoblastic and osteolytic activity induced by plasma cell-stroma interaction resulting in deregulation of numerous cytokines involved in bone remodeling. There are no data about bone metabolism in AL and the similarities or differences with MM. Levels of bone remodeling indices were measured in stored serum samples, collected from 62 previously untreated patients with AL amyloidosis. These included:i.bone resorption markers [C- and N- telopeptide of type-I collagen (CTX and NTX, respectively), and tartrate resistant acid phosphatase type-5b (TRACP-5b)],ii.bone formation markers [bone-alkaline phosphatase (bALP), and osteocalcin (OC)], andiii.osteoclast regulators [soluble receptor activator of nuclear factor-kB ligand (sRANKL), osteoprotegerin (OPG), macrophage inflammatory protein-1 alpha (MIP-1a), interleukin-6 (IL-6) and osteopontin (OPN)], as previously described.These markers were also measured in serum collected from 35 age-matched healthy controls and 35 newly diagnosed, untreated, symptomatic myeloma patients. Median age of AL patients at diagnosis was 61 years (range 39–80 years); heart was involved in 60% and kidneys in 66% of patients, while in 64% ≥2 organs were involved. In 16% of AL patients creatinine was ≥2 mg/dl. Median b2-microglobulin was 1.8 mg/l (range 0.5–28 mg/l); 64% of patients had >10% bone marrow infiltration by plasma cells. None of AL patients had lytic bone lesions in plain radiography or other features suggestive of myeloma such as hypercalcemia, significant anemia unrelated to renal impairment or predominant Bence-Jones proteinuria. The levels of sRANKL and OPG were both increased in AL patients compared to controls (p=0.07 and p<0.001). OPG was significantly higher in AL than in MM patients (p<0.001), but not sRANKL; thus sRANKL/OPG ratio was not different between AL and controls but it was significantly lower than in MM patients (p=0.009). Other osteoclast stimulators (MIP-1a, OPN, IL-6) were also increased in AL compared to controls (p<0.02). Bone resorption markers (CTX, NTX, TRACP-5b) were elevated in both AL and MM (p=0.008, p<0.001 and p<0.001 for AL vs. controls, respectively, and p=0.006, p<0.001 and p<0.001 for MM vs. controls). On the contrary, bone formation markers were not different between AL patients and controls, while in MM both bALP and OC had lower values compared to controls. Renal involvement did not affect bone markers but in AL patients with a creatinine level of >2 mg/dl CTX and NTX were higher than in patients with a creatinine level of <2mg/dl (p<0.001 and p=0.016, respectively). Involvement of other organs or the number of involved organs did not appear to influence bone remodeling. This study suggests that bone metabolism in AL is characterized by increased bone resorption with a pattern similar to that found in MM. Increased OPG levels in AL indicate more effective compensation of osteoclast activation compared to MM, probably explaining the lack of lytic disease in AL and possibly reflecting differences in the pathophysiology of plasma cell-stroma interaction between AL and MM.

Bone Remodeling is Different in Metaphyseal and Diaphyseal-fit Uncemented Hip Stems

Femoral component stability in uncemented total hip arthroplasties depends on periprosthetic bone remodeling. Stem design is an important factor influencing bone remodeling, however the design that promotes the most bone remodeling is unclear. We examined metaphyseal and diaphyseal-fit stems to determine the effect of stem design on bone remodeling and stability. Twenty-three patients who had total hip arthroplasties (28 hips) with metaphyseal-fit stems were matched with 27 patients (32 hips) who had uncemented total hip arthroplasties with diaphyseal-fit stems. We assessed preoperative radiographs for canal fill, canal shape, and bone quality. We then assessed postoperative radiographs for periprosthetic bone remodeling including spot welds, cortical hypertrophy, and pedestal formation. Patients were examined clinically using a modified Harris hip score. Patients with metaphyseal stems had increased cortical hypertrophy 1 year postoperatively. However, there was no functional difference 2 years postoperatively. Both stem designs resulted in bone remodeling by 2 years postoperatively with similar clinical results.