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Simple Summary: Multiple myeloma is a plasma cell cancer with bone destruction and is still considered an incurable disease despite advancements in its treatment. Over 90% of patients experience bone destruction during the disease's course. NIPEP-OSS has a targeted osteogenic activity with a broad safety margin. Therefore, we repurposed NIPEP-OSS for multiple myeloma bone disease (MMBD). The present study demonstrated that NIPEP-OSS delays mouse myeloma progression via bone formation in MMBD mouse models. Multiple myeloma (MM) is the second most common hematological malignancy. It is a clonal B-cell disorder characterized by the proliferation of malignant plasma cells in the bone marrow, the presence of monoclonal serum immunoglobulin, and osteolytic lesions. An increasing amount of evidence shows that the interactions of MM cells and the bone microenvironment play a significant role, suggesting that these interactions may be good targets for therapy. The osteopontin-derived collagen-binding motif-bearing peptide NIPEP-OSS stimulates biomineralization and enhances bone remodeling dynamics. Due to its unique targeted osteogenic activity with a broad safety margin, we evaluated the potential of NIPEP-OSS for anti-myeloma activity using MM bone disease (MMBD) animal models. In a 5TGM1-engrafted NSG model, the survival rates of the control and treated groups were significantly different (p = 0.0014), with median survival times of 45 and 57 days, respectively. The bioluminescence analyses showed that myeloma slowly developed in the treated mice compared to the control mice in both models. NIPEP-OSS enhanced bone formation by increasing biomineralization in the bone. We also tested NIPEP-OSS in a well-established 5TGM1-engrafted C57BL/KaLwRij model. Similar to the previous model, the median survival times of the control and treated groups were significantly different (p = 0.0057), with 46 and 63 days, respectively. In comparison with the control, an increase in p1NP was found in the treated mice. We concluded that NIPEP-OSS delays mouse myeloma progression via bone formation in MMBD mouse models. [ABSTRACT FROM AUTHOR]

During human skeletal growth, bone is formed via different processes. Two of them are: new bone formation by depositing bone at the periosteal (outer) surface and bone remodeling corresponding to a local renewal of tissue. Since in remodeling formation is preceded by resorption, we hypothesize that modeling and remodeling could require radically different transport paths for ionic precursors of mineralization. While remodeling may recycle locally resorbed mineral, modeling implies the transport over large distances to the site of bone apposition. Therefore, we searched for potential differences of size, arrangement and chemical composition of mineral particles just below surfaces of modeling and remodeling sites in femur midshaft cross-sections from healthy children. These bone sites were mapped using scanning synchrotron X-ray scattering, Raman microspectroscopy, energy dispersive X-ray analysis and quantitative backscattered electron microscopy. The results show clear differences in mineral particle size and composition between the sites, which cannot be explained by a change in the rate of mineral apposition or accumulation. At periosteal modeling sites, mineral crystals are distinctly larger, display higher crystallinity and exhibit a lower calcium to phosphorus ratio and elevated Na and Mg content. The latter may originate from Mg used for phase stabilization of mineral precursors and therefore indicate different time periods for mineral transport. We conclude that the mineralization process is distinctively different between modeling and remodeling sites due to varying requirements for the transport distance and, therefore, the stability of non-crystalline ionic precursors, resulting in distinct compositions of the deposited mineral phase. In growing children new bone is formed either due to apposition of bone tissue e.g. at the outer ridge of long bones to allow growth in thickness (bone modeling), or in cavities inside the mineralized matrix when replacing tissue (bone remodeling). We demonstrate that mineral crystal shape and composition are not the same between these two sites, which is indicative of differences in mineralization precursors. We suggest that this may be due to a longer mineral transport distance to sites of new bone formation as compared to remodeling where mineral can be locally recycled. Image, graphical abstract [ABSTRACT FROM AUTHOR]

The objective of this study was to access/identify the effects of phosphorus (P) restriction as a result of non-inclusion of monocalcium phosphate on broiler's diet after 21 d of age and the use of high doses of phytase (PHY). Broilers were divided into 4 treatments: positive control (PC), with phosphate inclusion; with phytase (WPHY), a reduction of 0.15% of available P and calcium (Ca) with the inclusion of 1,000 FYT; and 2 treatments with P restriction starting at 21 d of age: Without phosphate (WoP) and without phosphate supplemented with 4,000 FYT PHY (WoP+4000 FYT). Broilers performance (WG, FI, and FCR) and bone characteristics (bone weight, mineral residue, and Ca and P ratio) were assessed at 41 d of age. In relation to performance, the exclusion of monocalcium phosphate from the diet resulted in worse WG and FCR. However, the inclusion of higher PHY dose (WoP+4000 FYT) overcame the mineral deficiency, and performance was similar to treatments that met bird's mineral requirements (PC and WPHY). The treatment with mineral restriction (WoP) had the worst results in every bone quality assessment. However, broilers of WoP+4000 FYT group had bone parameters similar to PC and WPHY. Therefore, removing phosphate in grower and finishing phases has negative impacts on broiler's performance and bone conformation, but adding high phytase dosage (4,000 FYT) overcome this damages. [ABSTRACT FROM AUTHOR]

There has been discussion regarding microbial phytase replacing inorganic phosphorus (P) supplementation in broiler diets. Therefore, an experiment was conducted to examine the effect of phytase supplementation on diets low in inorganic P. Ross 308 broilers (n = 288) were fed one of 6 experimental diets in 4 phases. The control diet had 16.20, 10.90, 9.40, and 6.10 g/kg inorganic P in the Starter, Grower 1, Grower 2 and Finisher phase respectively. The remaining diets had 10.50 g/kg inorganic P in the Starter phase. Two of the diets had graded reductions in inorganic P of 5.10, 3.60, and 0.60 g/kg or 2.00, 0.50, and 0.60 g/kg for the Grower 1, Grower 2 and Finisher phase respectively, plus 500 FTU phytase. Three of the diets had inorganic P levels of 0.40, 0.50, and 0.60 g/kg for the Grower 1, Grower 2 and Finisher diets respectively and either 500, 750, or 1,000 FTU phytase. Broiler performance was analyzed at d 10, 20, 26, and 35. On d 35, ileal calcium (Ca) and P digestibility and tibia bone strength, mineralization, and mineral content were analyzed. There were no significant differences between the control diet and diet containing 1,000 FTU phytase and low inorganic P in the grower or finisher diets based on bird performance, tibia strength, and Ca and P digestibility. Birds fed the control diet had significantly higher BWG (P = 0.001), bone strength (P < 0.001) and ash content (P < 0.001) compared to birds fed the diets with 500 FTU or 750 FTU phytase and low inorganic P in the grower and finisher stages. This may be due to incomplete dephosphorylation of the inositol ring of phytate with these doses of phytase, but with 1,000 FTU phytase there was almost complete phosphate hydrolysis of each phytate. This study showed that relying on phytase alone to ensure full supply of P in broiler diets is viable in finisher diets but is not recommended in grower diets unless phytase is supplied at doses of 1,000 FTU or greater. [ABSTRACT FROM AUTHOR]

Two experiments were conducted to investigate the effect of high dietary calcium (Ca) level on growth performance, Ca and phosphorus (P) metabolism, and nutrient utilization in ducklings subjected to normal and low P levels in diets. A completely randomized design was used with a factorial arrangement of 2 total dietary P levels [normal-P (0.60%) and low-P (0.45%) groups] x 4 dietary Ca levels [low-Ca (0.55%), normal-Ca (0.75%), medium-Ca (0.95%) and high-Ca (1.15%) groups)]. Compared to normal-P group, low-P group had lower (P < 0.05) final body weight (BW), average daily gain (ADG), and average daily feed intake (ADFI) and reduced (P < 0.05) serum Ca and P levels, bone Ca, P, and ash content, and bone mineral density in ducklings during the starter period. Under the low-P group, birds from high-Ca group had lower (P < 0.05) final BW, ADG, ADFI, bone ash content, bone mineral density, and the utilization of energy, Ca, and P than those from low-Ca, normal-Ca, and medium-Ca groups. Our results indicate that high-Ca diet induced greater growth suppression and bone mineralization loss in ducklings fed a low-P diet. The aggravated negative effect of high dietary Ca level with a low P level might be related to the elevated serum alkaline phosphatase activity and the reduced utilization of energy, Ca, and P. [ABSTRACT FROM AUTHOR]

The Fto gene locus has been linked to increased body weight and obesity in human population studies, but the role of the actual FTO protein in adiposity has remained controversial. Complete loss of FTO protein in mouse and of FTO function in human patients has multiple and variable effects. To determine which effects are due to the ability of FTO to demethylate mRNA, we genetically engineered a mouse with a catalytically inactive form of FTO. Our results demonstrate that FTO catalytic activity is not required for normal body composition although it is required for normal body size and viability. Strikingly, it is also essential for normal bone growth and mineralization, a previously unreported FTO function. [ABSTRACT FROM AUTHOR]

Phosphorus (P) is an essential mineral for the development and maintenance of the vertebrate skeletal system. Modulation of P levels is believed to influence metabolism and the physiological responses of gene expression. In this study, we investigated the influence of dietary P on skeletal deformities and osteocalcin gene expression in zebrafish (Danio rerio), and sought to determine appropriate levels in a diet. We analyzed a total of 450 zebrafish within 31 days of hatching. Animals were distributed in a completely randomized experimental design that consisted of five replications. After an eight-week experiment, fish were diaphanized to evaluate cranial and spinal bone deformities. Increases in dietary phosphorus were inversely proportional to the occurrence of partial spine fusions, the absence of spine fusions, absence of parallelism between spines, intervertebral spacing, vertebral compression, scoliosis, lordosis, ankylosis, fin caudal insertion, and craniofacial deformities. Additionally, osteocalcin expression was inversely correlated to P levels, suggesting a physiological recovery response for bone mineralization deficiency. Our data showed that dietary P concentration was a critical factor in the occurrence of zebrafish skeletal abnormalities. We concluded that 1.55% P in the diet significantly reduces the appearance of skeletal deformities and favors adequate bone mineralization through the adjustment of osteocalcin expression. [ABSTRACT FROM AUTHOR]

Seven proprotein convertases cleave the basic amino acid consensus sequence K/R-X n -K/R ↓ (where n = 0, 2, 4 or 6 variable amino acids) to activate precursor proteins. Despite similarities in substrate specificity, basic amino acid-specific proprotein convertases have a distinct tissue distribution allowing for enzymatic actions on tissue-resident substrates. Proprotein convertase 5/6 (PC5/6) has two splice variants – soluble PC5/6A and membrane-bound PC5/6B – and is expressed during mouse development in many tissues including bone and tooth, but little is known about the substrates for PC5/6 therein. Osteopontin (OPN) is an abundant bone extracellular matrix protein with roles in mineralization, cell adhesion and cell migration, and it has putative consensus sequence sites for cleavage by PC5/6, which may modify its function in bone. Since PC5/6-knockout mouse embryos show developmental abnormalities, and reduced overall mineralization, we designed this study to determine whether OPN is a substrate of PC5/6. In silico analysis of OPN protein sequences identified four potential PC5/6 consensus cleavage sites in human OPN, and three sites – including a noncanonical sequence – in mouse OPN. Ex vivo co-transfections with human OPN revealed complete OPN cleavage reducing full-length OPN (~ 70 kDa) to an N-terminal fragment migrating at ~ 50 kDa and two C-terminal fragments at ~ 18 kDa and ~ 16 kDa. Direct cleavage of OPN by PC5/6A – the predominant isoform expressed in human osteoblast cells – was confirmed by cell-free enzyme-substrate assays and by mass spectrometry. The latter was also used to investigate potential cleavage sites. Co-transfections of PC5/6 and mouse OPN showed partial cleavage of OPN into a C-terminal OPN fragment migrating at ~ 30 kDa and an N-terminal fragment migrating at ~ 29 kDa. Micro-computed tomography of PC5/6-knockout embryos at E18.5 confirmed a reduction in mineralized bone, and in situ hybridization performed on cryo-sections of normal mouse bone using Pcsk5 and Opn anti-sense and control-sense cRNA probes indicated the co-localization of the expression of these genes in bone cells. This mRNA expression profile was supported by semi-quantitative RT-PCR using osteoblast primary cultures, and cultured MC3T3-E1 osteoblast and MLO-Y4 osteocyte cell lines. Immunoblotting for OPN from mouse bone extracts showed altered OPN processing in PC5/6-knockout mice compared to wildtype mice. OPN fragments migrated at ~ 25 kDa and ~ 16 kDa in wildtype bone and were not present in PC5/6-deficient bone. In conclusion, this study demonstrates that Pcsk5 is expressed in bone-forming cells, and that OPN is a novel substrate for PC5/6. Cleavage of OPN by PC5/6 may modify the function of OPN in bone and/or modulate other enzymatic cleavages of OPN, leading to alterations in the bone phenotype. [ABSTRACT FROM AUTHOR]

Objective Purinergic 2X7 receptor (P2X7R) activation modulates in vitro mineralization by primary rat and human osteoblasts. However, the detailed mechanism of how P2X7R activation affects primary human osteoblasts remains unclear. The aim of this study was to investigate the effect of P2X7R activation on human mandibular-derived osteoblast (hMOB) differentiation. Design Primary human osteoblasts were obtained from non-pathologic mandibular bone from healthy patients. The hMOBs were cultured in osteogenic medium with or without 0.5–5 μM 2′(3′)- O -(4-benzoyl) benzoyl-ATP (BzATP), a selective P2X7R agonist. The mRNA expression of osteogenic differentiation markers and WNT-signaling molecules was investigated by quantitative real time polymerase chain reaction. In vitro mineral deposition was determined by Alizarin Red S staining. Transfection of small interfering RNA was performed to confirm the effect of P2X7R activation. WNT/β-catenin signaling was detected by immunofluorescence staining for β-catenin. Results BzATP inhibited osteogenic medium-induced RUNX2 and OSX mRNA expression in hMOBs. Moreover, BzATP significantly retarded in vitro mineralization. These findings indicated that BzATP/P2X7R activation inhibited hMOB differentiation. Interestingly, reduced WNT3A mRNA expression and blockage of osteogenic medium-induced β-catenin nuclear translocation were also found. These data suggested that WNT signaling might be a target of P2X7R-regulated osteogenic differentiation. Furthermore, when recombinant human WNT3A was added to the BzATP-treated group, it rescued the reduced RUNX2 and OSX expression, and in vitro mineralization. Conclusion Our results demonstrate that P2X7R activation by BzATP inhibits hMOB differentiation. This inhibitory effect was associated with inhibition of the WNT/β-catenin signaling pathway. [ABSTRACT FROM AUTHOR]

“Broiler-type” chickens are fast-grow-ing, heavy-bodied birds with high demands on bone quality. Phenamil increased mineralization in cultured murine mesenchymal stem cells. Phenamil effects were tested in 2 groups of weight and gender matched day-old broiler chickens (n = 13). Oral administration of 30 mg phenamil/kg body weight d 1 to 13 reduced growth of chicks d 5 to 14 (P = 0.002); with phenamil-treated (PT) chick body weight being 84% of vehicle-treated (VT) chicks’ body weight on d 14. Tissues collected on d 15 showed that femur lengths and widths did not differ, but tibias from PT chicks were 6% shorter (P = 0.002) and 13% narrower (P = 0.012) with 18% thinner tibial cross-sections (P < 0.008) than in VT chicks. Angles of the caudal aspect of the anterior surface of keeled-sternums were 166° in PT chicks, flatter than the 148° found in VT chicks (P = 0.000). Total mineral content of both tibia and femur were lower in PT chicks (P = 0.005 for both). Bone Ca, P, and Mg (ppm) in ash were similar, but Ca:P was lower (1.70 vs 1.75) in PT versus VT chicks (P < 0.05). Osteocalcin was ∼20% lower (P = 0.020), PINP was ∼45% higher (P = 0.000) in PT chicks. Carboxy-terminal telopeptide type I collagen (ICTP) and cross-linked N-telopeptide of type I collagen (NTX1) were similar in the 2 groups. Phenamil had unexpected and detrimental effects on bone formation in growing broiler chicks, reducing linear skeletal growth and markedly changing bone architecture. [ABSTRACT FROM AUTHOR]

A study was conducted to examine the effects of different dietary levels of available phosphorus (aP) on P excretion, bone mineralization, performance and the mRNA expression of sodium-dependent P transporters in growing pigs. Sixty-day old growing pigs (n= 54) with an average initial BW of 19.50 ± 1.11 kg were randomly allocated to a control diet (C) containing 0.23% available phosphorus (aP), T1 containing 0.17% aP and T2 containing 0.11% aP. There were 6 pens per treatment with 3 pigs per pen. Body weight and feed intake were measured weekly. At the end of each week, one pig from each pen was housed in a metabolic crate for 24 h to collect fecal and urine samples and then sacrificed to obtain third metacarpal (MC3) bones and jejunal and kidney samples. Bones were scanned by Dual Energy X-ray Absorptiometry (DEXA). Fecal and urine samples were sub-sampled and analyzed for P content. The expression of P transporter mRNA in jejunum and kidney samples was measured using quantitative real-time polymerase chain reaction (qRT-PCR). Data were analyzed using GLM procedure of the Statistical Analysis System (SAS Institute version 9.2). Pigs fed the T2 diet had reduced (P< 0.05) average daily gain (ADG) and gain to feed (G:F) compared to those fed the C diet during week 2. Overall, ADG and G:F were also reduced (P< 0.05) in pigs fed the T2 diet compared to those fed the C and T1 diets. Bone mineral density (BMD) and bone mineral content (BMC) were reduced (P< 0.05) in pigs fed the T2 diet compared to those fed the C diet throughout the experiment. At week 1, jejunal mRNA expression of Na (+)-dependent phosphate transporter 2 (SLC34A2) was increased (P< 0.01) in pigs fed the T2 diet compared to C diet. Renal mRNA expression of Na(+)-dependent phosphate transporter 1 (SLC34A1) and SLC34A3 were increased (P< 0.05) in pigs fed the T2 diet compared to those fed the C diet at week 2 and was accompanied by lower (P< 0.05) urinary P in pigs fed the T2 diet during week 2 and week 3. In conclusion, growing pigs are highly sensitive to low dietary P as shown by reduced ADG, bone mineralization and urinary P level, but moderate reduction in dietary P up to 0.17% aP in the diet has the potential to reduce environmental pollution by reducing P concentration in swine manure and without compromising performance. [ABSTRACT FROM AUTHOR]

Feeding poultry litter biochar (PLB), a product resulting from litter gasification, could decrease diet cost, maintain broiler growth performance, and decrease land application of manure. Past research observed improved pellet quality and mineral availability with dietary inclusions of 6 to 7% PLB; however, broiler performance was decreased, likely due from toxic-heavymetal content (e.g., 99 ppm arsenic) and increased digesta viscosity of the particular PLB. The objective of the current study was to assess lower dietary inclusions of PLB, with decreased toxic-heavy-metal content, on descriptive feed manufacture, broiler performance, digesta viscosity, bone mineralization, and apparent ileal amino acid digestibility (AIAAD). Treatments were arranged as a Diet Formulation x Phytase Addition factorial in a randomized complete block design with 8 replications per treatment. Four dietary formulations were produced: positive control (PC) with 0.45% non phytate phosphorus (nPP), negative control with 0.23% nPP, and 2 or 4% PLBwith 0.45% nPP. Phytasewas either included at 9,500 ftu/kg or withheld. There were 8 treatments total. Contrasts comparing PC and 2 or 4% PLB also were conducted. Diets were pelleted and crumbled prior to feeding. Positive and negative control formulations and phytase addition affected bird performance as expected. Birds fed 2% PLB had increased FCR and birds fed 4% PLB had decreased LWGwhen compared to PC (P<0.05). However, these performance deficiencies were corrected with phytase inclusion. Birds fed diets with PLB had similar tibia measures compared to the PC. No significant main effects, interactions, or contrast comparisons were observed for viscosity data. The incorporation of PLB into formulations without phytase increased diet AIAAD of some amino acids relative to the corresponding PC (P<0.05). A lower toxic-heavy-metal PLB product (e.g., 22 ppm arsenic) provided available minerals and amino acids to diets, but performance similar to PC necessitated the addition of phytase. [ABSTRACT FROM AUTHOR]

A pig trial was performed to analyze the effect of different dietary phytase levels on bone mineralization and ileal phytate degradation in 2 different growing stages. Fifty-six postweaned piglets were fed 4 diets differing in P, Ca, Na, and phytase contents. Diets contained either recommended (positive control [PC]) or reduced (negative control [NC]) mineral levels and phytase was added to NC diets at 500 or 2,000 phytase units/kg feed (NC500 and NC2000, respectively). At d 42 and 98, samples (n = 7) were taken for analyses in rib bones (Ca, P, Fe, Mg, Mn, Se, and Zn) and in ileal digesta (myo-inositol phosphates [myo-inositol hexakisphosphate {InsP6}, myo-inositol pentakisphosphate {InsP5}, myo-inositol tetrakisphosphate, and myo-inositol trisphosphate] were analyzed by high-pressure ion chromatography and inositol was analyzed by HPLC). Data were analyzed by ANOVA and means were separated by the Tukey's test with significance set at P < 0.05. Dietary mineral reduction reduced bone mineral content, except in ribs collected at d 42 (P < 0.05). The NC500 diet compared with the NC improved all minerals except Se in ribs (d 98; P < 0.05). The higher phytase dose (NC2000) restored nearly all bone minerals to the level of PC pigs (P > 0.05). Phytate degradation up to the proximal ileum was not influenced by dietary mineral level (PC compared with NC, P > 0.05) whereas phytase reduced InsP6 and InsP5 levels in ileal digesta (P < 0.05 for NC and PC at both phytase application rates) with the lowest levels seen in NC2000 (d 98; P < 0.05). The efficient phytate degradation could explain the improvements in bone mineralization, especially at high phytase application level, probably due to improved mineral availability. [ABSTRACT FROM AUTHOR]

Study Design: A study examining the clinical protocol of scoliosis wound irrigation, demonstrating povidone-iodine's (PVI) effect on human osteoblast cells. Primary and immortal cell line osteoblasts were treated with 0.35% PVI for 3 minutes, and analyzed for proliferation rate, oxidative capacity, and mineralization.Objective: To model spinal wound irrigation with dilute PVI in vitro, in order to investigate the effect of PVI on osteoblast proliferation, metabolism, and bone mineralization.Summary Of Background Data: Previously PVI irrigation has been proposed as a safe and effective practice to avoid bacterial growth after spinal surgery. However, recent evidence in multiple cell types suggests that PVI has a deleterious effect on cellular viability and cellular function.Methods: Primary and immortal human osteoblast cells were exposed to either phosphate buffered saline control or with 0.35% PVI for 3 minutes. Cellular proliferation was measured over the duration of 7 days by MTS assay. Oxygen consumption rate, extracellular acidification rate, and proton production rate were analyzed using a Seahorse XF24 Bioanalyzer. Protein expression of the electron transport chain subunits CII-SDHB, CIII-UQRCR2, and CV-ATP5A was measured via Western blotting. Mineralized bone nodules were stained with alizarin red.Results: Expressed as a percentage of normal osteoblast proliferation, osteoblasts exposed to 0.35% PVI exhibited a significant 24% decrease in proliferation after 24 hours. This was a sustained response, resulting in a 72% decline in cellular proliferation at 1 week. There was a significant reduction in oxygen consumption rate, extracellular acidification rate, and proton production rate (P < 0.05), in osteoblasts that had been exposed to 0.35% PVI for 3 minutes, coupled with a marked reduction in the protein expression of CII-SDHB. Osteoblasts exposed to 0.35% PVI exhibited reduced bone nodule mineralization compared to control phosphate buffered saline exposed osteoblasts (P < 0.01).Conclusion: PVI has a rapid and detrimental effect on human osteoblast cellular proliferation, metabolic function, and bone nodule mineralization.Level Of Evidence: NA. [ABSTRACT FROM AUTHOR]

Biocompatibility is a key issue in the development of new implant materials. In this context, a novel class of biodegrading Mg implants exhibits promising properties with regard to inflammatory response and mechanical properties. The interaction between Mg degradation products and the nanoscale structure and mineralization of bone, however, is not yet sufficiently understood. Investigations by synchrotron microbeam x-ray fluorescence (μXRF), small angle x-ray scattering (μSAXS) and x-ray diffraction (μXRD) have shown the impact of degradation speed on the sites of Mg accumulation in the bone, which are around blood vessels, lacunae and the bone marrow. Only at the highest degradation rates was Mg found at the implant–bone interface. The Mg inclusion into the bone matrix appeared to be non-permanent as the Mg-level decreased after completed implant degradation. μSAXS and μXRD showed that Mg influences the hydroxyl apatite (HAP) crystallite structure, because markedly shorter and thinner HAP crystallites were found in zones of high Mg concentration. These zones also exhibited a contraction of the HAP lattice and lower crystalline order. [ABSTRACT FROM AUTHOR]

An attractive strategy for the regeneration of tissues has been the use of extracellular matrix analogous biomaterials. Peptide-based fibrillar hydrogels have been shown to mimic the structure of extracellular matrix offering cells a niche to undertake their physiological functions. In this study, the capability of an ionic-complementary peptide FEFEFKFK (F, E, and K are phenylalanine, glutamic acid, and lysine, respectively) hydrogel to host human mesenchymal stem cells in three dimensions and induce their osteogenic differentiation is demonstrated. Assays showed sustained cell viability and proliferation throughout the hydrogel over 12 days of culture and these human mesenchymal stem cells differentiated into osteoblasts simply upon addition of osteogenic stimulation. Differentiated osteoblasts synthesized key bone proteins, including collagen-1 (Col-1), osteocalcin, and alkaline phosphatase. Moreover, mineralization occurred within the hydrogel. The peptide hydrogel is a naturally biodegradable material as shown by oscillatory rheology and reversed-phase high-performance liquid chromatography, where both viscoelastic properties and the degradation of the hydrogel were monitored over time, respectively. These findings demonstrate that a biodegradable octapeptide hydrogel can host and induce the differentiation of stem cells and has the potential for the regeneration of hard tissues such as alveolar bone. [ABSTRACT FROM AUTHOR]

A study was conducted to examine the effects of 25-hydroxyvitamin D3 (25-(OH)D3) on fecal Ca and P excretion, bone mineralization, performance and the mRNA expression of intestinal transporter genes in growing female pigs. Sixty-day old gilts (n= 24) with an average initial BW of 23.13 ± 1.49 kg were randomly allocated to a control diet (diet 1) containing wheat/corn/soybean meal and 150 IU kg−1of Vitamin D3, diet 1 + 50 μg of 25-(OH)D3 kg−1(diet 2) and diet 1 + 100 μg of 25-(OH)D3 kg−1(diet 3). The pigs were housed in an individual pen and had ad libitum access to feed and water for 42 days, and BWG and feed intake were measured weekly. Measures of bone mineralization and expression of Ca and P transporters mRNA were analyzed using Dual Energy X-Ray Absortiometry (DEXA) and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Data were analyzed using GLM procedure of the Statistical Analysis System (SAS Institute version 9.2). Fecal Ca and P concentration were significantly reduced (P≤ 0.05) in pigs fed diets 2 and 3 compared with the control diet. Supplementation of 25-(OH)D3 did not significantly improve bone mineralization, animal performance and intestinal transporters mRNA expression except for SLC34A1, a sodium-dependent phosphate transporter 1. In conclusion, supplementation of 25-(OH)D3 in swine nutrition may not improve animal performance but has the potential to reduce environmental pollution by increasing dietary Ca and P retention while reducing their excretion. [ABSTRACT FROM AUTHOR]

Eldecalcitol (ELD), an active form of vitamin D analog approved for the treatment of osteoporosis in Japan, increases lumbar spine bone mineral density (BMD), suppresses bone turnover markers, and reduces fracture risk in patients with osteoporosis. We have previously reported that treatment with ELD for 6 months improved the mechanical properties of the lumbar spine in ovariectomized (OVX) cynomolgus monkeys. ELD treatment increased lumbar BMD, suppressed bone turnover markers, and reduced histomorphometric parameters of both bone formation and resorption in vertebral trabecular bone. In this study, we elucidated the effects of ELD on bone quality (namely, mineralization, microarchitecture, microdamage, and bone collagen crosslinks) in OVX cynomolgus monkeys in comparison with OVX-vehicle control monkeys. Density fractionation of bone powder prepared from lumbar vertebrae revealed that ELD treatment shifted the distribution profile of bone mineralization to a higher density, and backscattered electron microscopic imaging showed improved trabecular bone connectivity in the ELD-treated groups. Higher doses of ELD more significantly reduced the amount of microdamage compared to OVX-vehicle controls. The fractionated bone powder samples were divided according to their density, and analyzed for collagen crosslinks. Enzymatic crosslinks were higher in both the high-density (≥ 2.0 mg/mL) and low-density (< 2.0 mg/mL) fractions from the ELD-treated groups than in the corresponding fractions in the OVX-vehicle control groups. On the other hand, non-enzymatic crosslinks were lower in both the high- and low-density fractions. These observations indicated that ELD treatment stimulated the enzymatic reaction of collagen crosslinks and bone mineralization, but prevented non-enzymatic reaction of collagen crosslinks and accumulation of bone microdamage. Bone anti-resorptive agents such as bisphosphonates slow down bone remodeling so that bone mineralization, bone microdamage, and non-enzymatic collagen crosslinks all increase. Bone anabolic agents such as parathyroid hormone decrease bone mineralization and bone microdamage by stimulating bone remodeling. ELD did not fit into either category. Histological analysis indicated that the ELD treatment strongly suppressed bone resorption by reducing the number of osteoclasts, while also stimulating focal bone formation without prior bone resorption (bone minimodeling). These bidirectional activities of ELD may account for its unique effects on bone quality. [ABSTRACT FROM AUTHOR]

The regulators affecting skeletal tissue formation and its maintenance include a wide array of molecules with very diverse functions. More recently, sphingolipids have been added to this growing list of regulatory molecules in the skeletal tissues. Sphingolipids are integral parts of various lipid membranes present in the cells and organelles. For a long time, these macromolecules were considered as inert structural elements. This view, however, has radically changed in recent years as sphingolipids are now recognized as important second messengers for signal-transduction pathways that affect cell growth, differentiation, stress responses and programmed death. In the current review, we discuss the available data showing the roles of various sphingolipids in three different skeletal cell types-chondrocytes in cartilage and osteoblasts and osteoclasts in bone. We provide an overview of the biology of sphingomyelin phosphodiesterase 3 (SMPD3), an important regulator of sphingolipid metabolism in the skeleton. SMPD3 is localized in the plasma membrane and has been shown to cleave sphingomyelin to generate ceramide, a bioactive lipid second messenger, and phosphocholine, an essential nutrient. SMPD3 deficiency in mice impairs the mineralization in both cartilage and bone extracellular matrices leading to severe skeletal deformities. A detailed understanding of SMPD3 function may provide a novel insight on the role of sphingolipids in the skeletal tissues. [ABSTRACT FROM AUTHOR]

Background: Lead exposure during pregnancy may impair skeletal development. Oxidative stress is one of the important mechanisms for lead toxicity effects. The aim of this study was to investigate ascorbic acid and garlic effects on bone mineralization in lead exposed pregnant rats. Materials and Methods: In this experimental study, 50 pregnant Wistar rats were randomly divided into 5 groups; group (L) exposed to lead acetate, group (L+C) exposed to lead acetate and ascorbic acid (vitamin C), group (L+G) exposed to lead acetate and garlic juice, sham group treated with tap water plus 0.4 mL/L normal hydrogen chloride (HCl) and 0.5 mg/L sugar, control group without any intervention. All treatments were done during pregnancy. After birth, blood and bone lead levels were measured and then all neonates were sacrificed, and their right tibia bone processed for alizarin red and Alcian blue staining. Results: Blood lead levels in L group increased significantly in both mothers and their neonate compared to control animals. In addition, the neonates born to L group showed markedly higher lead concentrations in their bone than that of controls. In contrast, we found no significant changes in blood and bone lead levels in lead exposed neonates that received ascorbic acid and garlic. Bone formation in neonates of L group was clearly disrupted. Interestingly, both ascorbic acid and garlic treatments could apparently improve bone formation during pregnancy in lead exposed neonates. Conclusion: Ascorbic acid and garlic consumption during pregnancy may improve the deleterious effects of lead exposure on bone mineralization. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate the effects of corn naturally contaminated with low levels of aflatoxins (AF) on performance, calcium (Ca) and phosphorus (P) metabolism, and bone mineralization in broilers. Four hundred and fifty 1-d-old male broiler chicks were randomly allotted to control (no AF), and the diet with the corn contaminated with AF as a substitute for 50% or 100% of the corn in control (HAC or AAC). The broilers in HAC and AAC were administered with 6.5-8.0 and 13.0-16.0 μg AF/kg BW per day respectively in overall period (1-42 d). Ten birds per treatment were selected at d 43 to explore the apparent digestibility of Ca and P using all excreta collection method after 2 days adaptation. The AAC diet reduced (P<0.05) the performance of birds in overall period, whereas the HAC diet temporarily decreased (P<0.05) the performance of birds in starter phase (1-21 d). The corn contaminated with AF had no effect (P>0.24) on the apparent digestibility of Ca and P. The increasing AF intake content linearly increased (P<0.001) serum parathyroid hormone (PTH) level, and decreased (P>0.05) serum 1,25-dihydroxychocalciferol and P concentrations on d 42. On d 21, the AAC diet reduced (P<0.05) tibia breaking strength (TBS) when compared to control or HAC group, however, it did not affect (P>0.10) the percentages of ash, Ca and P in tibia. On d 42, the increasing AF intake level linearly decreased (P<0.05) TBS, and the percentages of Ca and P in tibia. These results indicated that the low level of AF intake had negative effects on the P metabolism and bone mineralization of broilers at exposure of 42 days, which might relate to the changes in vitamin D and PTH metabolism. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effects of graded levels of montmorillonite, a constituent of clay, on performance, hematological parameters and bone mineralization in weaned pigs. One hundred and twenty, 35-d-old crossbred pigs (Duroc×Large White×Landrace, 10.50±1.20 kg) were used in a 28-d experiment and fed either an unsupplemented corn-soybean meal basal diet or similar diets supplemented with 0.5, 1.0, 2.5 or 5.0% montmorillonite added at the expense of wheat bran. Each treatment was replicated six times with four pigs (two barrows and two gilts) per replicate. Feed intake declined (linear and quadratic effect, p< 0.01) with increasing level of montmorillonite while feed conversion was improved (linear and quadratic effect, p<0.01). Daily gain was unaffected by dietary treatment. Plasma myeloperoxidase declined linearly (p = 0.03) with increasing dietary level of montmorillonite. Plasma malondialdehyde and nitric oxide levels were quadratically affected (p<0.01) by montmorillonite with increases observed for pigs fed the 0.5 and 1.0% levels which then declined for pigs fed the 2.5 and 5.0% treatments. In bone, the content of potassium, sodium, copper, iron, manganese and magnesium were decreased (linear and quadratic effect, p<0.01) in response to an increase of dietary montmorillonite. These results suggest that dietary inclusion of montmorillonite at levels as high as 5.0% does not result in overt toxicity but could induce potential oxidative damage and reduce bone mineralization in pigs. [ABSTRACT FROM AUTHOR]

Objectives To deliver the efficacy and safety of Ch- GNPs (Chitosan gold nanoparticles) conjugated anti-inflammatory molecules peroxisome proliferator activated receptor gamma ( PPARγ) on implant surface titanium (Ti) to reduce implant-induced inflammation. Materials and methods The Ch-GNPs were conjugated with the PPARγ cDNA through a coacervation process. Conjugation was cast over Ti surfaces by dipping, and cells were seeded on different sizes (6 × 6 × 0.1 cm and 1 × 1 × 0.1 cm; n = 3) of Ti surfaces. The size of Ch- GNPs and surface characterization of Ti was performed using UV-vis spectroscopy, TEM (Transmission electron microscopy) and EDX (energy-dispersive X-ray). The DNA conjugation and transfection capacity of Ch- GNPs were simultaneously confirmed by agarose gel electrophoresis, β-galactosidase staining, and immunoblotting. Results The Ch- GNPs were well dispersed and spherical in shape, with average size around 10-20 nm. Ti surfaces coated with Ch- GNPs/LacZ, as transfection efficacy molecule, showed strong β-galactosidase staining in MC-3T3 E1 cells. Cells cultured on Ch- GNPs/ PPARγ-coated Ti surfaces were able to inhibit implant-induced inflammation by simultaneously suppressing the expression of tumor necrosis factor- alpha ( TNF-α), interleukin-1 beta ( IL-1β), inducible nitric oxide synthase ( iNOS), cyclooxygenase-2 ( COX-2), and matrix metalloproteinase-2 ( MMP-2). The inhibition mechanism of Ch- GNPs/ PPARγ was due to inhibition of both reactive oxygen species ( ROS) and nitric oxide ( NO) secretion ( n = 3; P < 0.05). In addition, Ch-GNPs/PPARγ was able to increase expression of bone morphogenetic protein (BMP-7) and runt-related transcription factor-2 ( RUNX-2). Furthermore, alkaline phosphatase activity ( ALP) was also increased than that in control ( n = 3; P < 0.01). Whereas, expression of receptor activator of NF-κB ligand ( RANKL) was decreased. Conclusions The novel gene delivery materials, like Ch- GNPs, can carry the PPARγ cDNA into the required areas of the implant surfaces, thus aiding to inhibit inflammation and promote osteoblast function. Thus, the PPARγ on implant surfaces may promote its clinical application on peri-implantitis or periodontitis like diseases. [ABSTRACT FROM AUTHOR]

The objectives of this study were to deter-mine the ileal digestibility of P in potassium phosphate, phytase-related ileal digestible P release, bone-mineral-ization-based ileal digestible P equivalency of phytase, and phytase-related efficiency of ileal digestible P uti-lization for bone mineralization in broiler chickens at 2 dietary concentrations of highly soluble Ca (HSC). Birds were sorted by BW at d 15 posthatch and as-signed to 8 cages per diet with 8 birds per cage. Twelve diets were arranged in a 2 x 6 factorial of HSC at 5 or 6 g/kg and P supply treatment at 6 levels consisting of 4 added P levels (P from KH2P04 added at 0, 0.7, 1.4, or 2.1 g/kg of diet) or 2 added phytase levels (500 or 1,000 phytase units). On d 24 posthatch, ileal digesta were collected for ileal P digestibility (IPD) determina-tion and the left tibia was collected from the 4 heaviest birds in each cage for bone ash determination. Weight gain, G:F, and tibia ash were higher (P < 0.05) at 5 than at 6 g of HSC/kg. Added P from KH2P04 or add-ed phytase linearly increased (P < 0.001) weight gain, G:F, tibia ash, and IPD. The IPD of KH2P04 derived from multiple linear regressions of digestible on total P intake for the diets without added phytase showed a reduction (P < 0.05) from 89.5 to 84.5% with increased HSC from 5 to 6 g/kg. Polynomial regressions of digest-ible P intake on phytase intake indicated that 1,000 units of added phytase released 1.701 or 1.561 g of di-gestible P in diets containing 5 or 6 g of added HSC/ kg, respectively Polynomial regressions of tibia ash on digestible P or phytase intake in diets containing 5 or 6 g of added HSC/kg at 1,000 phytase units gave di-gestible P equivalency of 1.487 or 1.448 g, respectively. Thus, phytase-related efficiency of ileal digestible P uti-lization for bone mineralization was 87.4 and 92.8% in diets containing 5 or 6 g of added HSC/kg, respectively [ABSTRACT FROM AUTHOR]

Hypophosphatasia (HPP) is a heterogeneous rare inborn error of bone and mineral metabolism caused by mutations in the ALPL gene encoding the isoenzyme, tissue-nonspecific alkaline phosphatase (TNAP). These mutations result in a decreased level of TNAP activity and increased levels of its substrates, including inorganic pyrophosphate, pyridoxal-5′-phosphate and phosphoethanolamine. Clinical presentations are highly variable, ranging from stillbirth and absence of mineralization in severe disease to mild dental problems or osteopenia in adulthood. Further clinical symptoms include defective bone mineralization with bone deformities, recurrent fractures, chronic non-bacterial osteomyelitis, craniosynostosis, neonatal seizures, nephrocalcinosis, muscular hypotonia, failure to thrive and dental abnormalities with premature exfoliation of teeth and caries. Prognosis is very poor in severe perinatal forms with most patients dying from pulmonary complications of their skeletal disease but patients with mild phenotypes (adult form or Odonto-HPP) usually do not have a limitation in their life expectancy. Although TNAP is a ubiquitous enzyme, mostly known for its crucial role during mineralization of bone and teeth, its exact biological role in different human organs is still unclear, and the pathophysiology of symptoms due to TNAP deficiency in HPP are not understood in detail. Since inflammation and tissue destruction of the musculoskeletal system may occur in HPP, TNAP may also play an important role in controlling inflammatory processes. Recent investigations provide evidence that TNAP is also essentially involved in the development of the central nervous system and might contribute to multiple functions of the human brain. HPP can be diagnosed on clinical, biochemical and radiological criteria, and genetic testing confirms the diagnosis and is useful for genetic counseling. Since clinical symptoms are highly variable, patients should be followed up by a multidisciplinary team having experience in HPP treatment. Up to now, no curative treatment of HPP is available. Therefore, symptomatic treatment in particular with regard to pain, seizures and other metabolic phenomena is most important. However, recently, enzyme replacement therapy with a bone-targeted recombinant human TNAP molecule has been reported to improve bone mineralization, respiratory function and physical activity in severely affected infants with HPP, and further clinical trials are ongoing. Hopefully, this and other new therapeutic strategies may improve the prognosis and quality of life of patients with HPP and may contribute to our understanding of bone metabolism in general. [ABSTRACT FROM AUTHOR]

Objectives: The purpose of this study was to examine the ability of CT to assess the relative difference of degree of bone mineralization (grey level) parameters in a human mandible. Methods: Ten mandibular sections from cadavers (81.5 ± 12.1 years) were scanned using micro-CT with 27.2 µm voxel size and cone beam CT (CBCT) with 200 µm, 300 µm, and 400 µm voxel sizes. In addition, 15 clinical CBCT images from young patients (mean age 18.9 ± 3.3 years) were identified. After segmentation of bone voxels, alveolar bone and basal cortical bone regions were digitally isolated. A histogram of grey level, which is equivalent to degree of bone mineralization, was obtained from each region of the CT images. Mean, standard deviation (SD), coefficient of variation (COV), fifth percentile low (Low5) and high (High5) of alveolar bone and basal cortical bone regions were obtained. Percentage differences of grey level parameters between alveolar and basal cortical bones were computed. Results: The alveolar bone region had significantly lower Mean, Low5 and High5 values but significantly higher SD and COV than the basal cortical bone region for all CT images (p < 0.05). All parameters were significantly lower for the old cadaver group than for the young patient group (p < 0.05). Conclusions: CBCT and micro-CT provide comparable results in the assessment of relative difference in grey level distribution between alveolar and basal cortical bone regions in the human mandible. The percentage difference relative to an internal reference (basal cortical bone) can be a reliable method when assessing the degree of bone mineralization using CBCT images for both cross-sectional and longitudinal comparisons. [ABSTRACT FROM AUTHOR]

Abstract: Biomaterials based on bioinspired mineralization are expected to offer osteoconductive and osteoinductive scaffolds for bone regeneration. An important role in the mediation of in vivo biomineralization process is played by highly anionic non-collagenous phosphoproteins (NCP) bound to the collagen matrix. Inspired by this fact, synthetic analogues of the NCPs like polyvinyl phosphonic acid which provide surface nucleation sites have been employed successfully for mineralization of hard tissues. In this study, electrospun nanofibrous scaffolds of partially phosphorylated polyvinyl alcohol (PPVA) are prepared and studied for matrix mineralization and maturation of human pre-osteoblasts like MG63 cells. Partial phosphorylation was found to affect many solution properties of PVA like increase in surface tension, conductivity and semi-crystalline intermolecular hydrogen bond formations narrowing down the electrospinning window for PPVA. In vitro mineralization under SBF treatment was uniform along the length of fibers on PPVA nanofibers. Further, MG63 cells showed increased adherence and proliferation on PPVA nanofibers and the expression of alkaline phosphatase activity and cell-matrix calcium levels were about two times higher than PVA nanofibers. The study established fabrication of electrospun nanofibers of a partially phosphorylated polymer, PVA resulting in improved osteoconduction and expression of early markers of osteoinduction in MG63 cells. [Copyright &y& Elsevier]

The effect of osthol on osteoblasts was investigated in primary osteoblastic cells isolated from newborn Wistar rats. Osthol was supplemented into cultured medium at 10-7, 10-6, 10-5 and 10-4 mol/l, respectively. No stimulating effect was found on cell proliferation, but 10-5 mol/l osthol caused a significant increase in alkaline phosphatase (ALP) activity. Osteogenic differentiation markers were examined over a period of time at this concentration, and compared with control cells that were not supplemented with osthol. The results showed that the ALP activity, osteocalcin secretion and calcium deposition level in cells treated with osthol were 1.52, 2.74 and 2.0 times higher, respectively, than in the control cells. Results of ALP histochemical staining and mineralized bone nodule assays both showed that the number and area achieved in osthol-treated cells were 1.53-fold higher than in control cells. The gene expression of the growth and transcription factors basic fibroblast growth factor, insulin-like growth factor I, bone morphogenetic protein 2 (BMP-2), runt-related gene 2 (Runx-2) and osterix, which are associated with bone development, were also investigated. The increase in mRNA expression was 1.94, 1.74, 1.68, 1.83 and 2.31 times, respectively, higher compared to the control. Furthermore, osthol increased the protein expression of p38 mitogen-activated protein kinase (MAPK) and type I collagen. p38MAPK protein and collagen in osthol-treated cells were 1.42 and 1.58 times higher in osthol-treated cells compared to the control. The results of these studies support the conclusion that osthol significantly enhances the osteogenic differentiation of cultured osteoblasts. The results also indicated that osthol could stimulate the osteoblastic differentiation of rat calvarial osteoblast cultures by the BMP-2/p38MAPK/Runx-2/osterix pathway and that osthol may be used as an important compound in the development of new antiosteoporosis drugs. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: In addition to bone quantity, bone quality affects bone strength. Bone quality depends in part on the degree of mineralization of bone tissue (DMB). The relationship between the DMB distribution and the risk of osteoporotic hip fractures remains incompletely investigated. Here, our aim was to compare DMB distribution in femoral neck cortex specimens from 23 women with hip fractures (age, 65–96years) and 14 control women (age, 75–103years). Mineralization was determined using quantitative microradiography. We evaluated the following parameters of DMB frequency histograms, for both osteons and interstitial tissue: mode (oDMBAl mode and intDMBAl mode, respectively); 25th (oDMBAl q25, intDMBAl q25), 50th (oDMBAl q50, intDMBAl q50), and 75th (oDMBAl q75, intDMBAl q75) percentiles; and interquartile range (oDMBAl iqr, intDMBAl iqr). For each specimen, we also calculated the variance of pixel mineral content for osteons and interstitial tissue (oDMBAl var and intDMBAl var). We used nonparametric tests to compare frequency histogram parameters between hip-fractured women and controls and Fisher''s test to compare variances between groups. All frequency histogram parameters for osteons and interstitial tissue except the 25th percentile, and the variances of pixel mineral content in osteons and interstitial tissue, were significantly different between hip-fractured women and controls, indicating greater heterogeneity of mineralization in the hip-fracture patients than in the controls. These cross-sectional data suggest that bone fragility may be related to greater DMB heterogeneity in osteons and interstitial tissue. [Copyright &y& Elsevier]

We demonstrated the capability of in vivo synchrotron radiation CT (SRCT) in analyzing short-term changes in trabecular bone architecture (TBA) and the degree of bone mineralization (DBM) in small animals. Mice underwent unilateral sciatic neurectomy (SN) and sham operation on the contralateral side (SO) at 13 weeks of age. In vivo SRCT scans (11.7-μm cubic voxel) were made of both knees 7 and 17 days (group 1, n = 7) or only 17 days (group 2, n = 6) after surgery. In three mice in group 2, one knee was scanned twice on the same day in different orientations for reproducibility testing. Two scan data sets of the tibial proximal metaphysis acquired at different time points (group 1) or at the same time point (group 2) were registered for detecting differences in volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), connectivity density (Conn.D), and mean DBM (mDBM). The reproducibility test showed small errors of <2.5% in the TBA indexes and <3.0% in mDBM, while mismatched bone regions amounted to >25%. In group 1, Tb.Th increased but Tb.N and Conn.D decreased in both SN and SO; BV/TV and mDBM increased only in SO; accordingly, BV/TV, Tb.Th, and mDBM became lower in SN than in SO. No significant interaction between SN and irradiation was found; the SN effects on TBA and DBM were similar between groups 1 and 2, although synchrotron irradiation led to higher Tb.Th and lower Tb.N in group 1. In conclusion, in vivo SRCT has potential use for detecting short-term bone dynamics of small animals. [ABSTRACT FROM AUTHOR]

Abstract: Vitamin A (retinol) is the only molecule known to induce spontaneous fractures in laboratory animals and we have identified retinol as a risk factor for fracture in humans. Since subsequent observational studies in humans and old animal data both show that high retinol intake appears to only have small effects on bone mineral density (BMD) we undertook a mechanistic study of how excess retinol reduces bone diameter while leaving BMD essentially unaffected. We fed growing rats high doses of retinol for only 1week. Bone analysis involved antibody-based methods, histology, pQCT, biomechanics and bone compartment-specific PCR together with Fourier Transform Infrared Spectroscopy of bone mineral. Excess dietary retinol induced weakening of bones with little apparent effect on BMD. Periosteal osteoclasts increased but unexpectedly endosteal osteoclasts disappeared and there was a reduction of osteoclastic serum markers. There was also a lack of capillary erythrocytes, endothelial cells and serum retinol transport protein in the endosteal/marrow compartment. A further indication of reduced endosteal/marrow blood flow was the increased expression of hypoxia-associated genes. Also, in contrast to the inhibitory effects in vitro, the marrow of retinol-treated rats showed increased expression of osteogenic genes. Finally, we show that hypervitaminotic bones have a higher degree of mineralization, which is in line with biomechanical data of preserved stiffness in spite of thinner bones. Together these novel findings suggest that a rapid primary effect of excess retinol on bone tissue is the impairment of endosteal/marrow blood flow leading to hypoxia and pathological endosteal mineralization. [Copyright &y& Elsevier]

lthough considerable advances in our understanding of the mechanisms of phosphate homeostasis and skeleton mineralization have recently been made, little is known about the initial events involving the detection of changes in the phosphate serum concentrations and the subsequent downstream regulation cascade. Recent data has strengthened a long-established hypothesis that a phosphate-sensing mechanism may be present in various organs. Such a phosphate sensor would detect changes in serum or local phosphate concentration and would inform the body, the local environment, or the individual cell. This suggests that phosphate in itself could represent a signal regulating multiple factors necessary for diverse biological processes such as bone or vascular calcification. This review summarizes findings supporting the possibility that phosphate represents a signaling molecule, particularly in bone and cartilage, but also in other tissues. The involvement of various signaling pathways (ERK1/2), transcription factors (Fra-1, Runx2) and phosphate transporters (PiT1, PiT2) is discussed. [ABSTRACT FROM AUTHOR]

Abstract: The hypothalamic–pituitary–thyroid axis plays a key role in skeletal development, acquisition of peak bone mass and regulation of adult bone turnover. Euthyroid status is essential for maintenance of optimal bone mineralization and strength. In population studies, hypothyroidism and hyperthyroidism have both been associated with an increased risk of fracture. Furthermore, recent studies in healthy euthyroid post-menopausal women indicate that thyroid status in the upper normal range is also associated with low bone mineral density and an increased risk of non-vertebral fracture. Studies in mutant mice have demonstrated that thyroid hormone receptor α is the major mediator of T3 action in bone and that thyroid hormones exert anabolic actions during growth but have catabolic effects on the adult skeleton. Nevertheless, TSH has also been proposed to be a direct negative regulator of bone turnover, although the relative importance of T3 and TSH actions in the skeleton has yet to be clarified. [ABSTRACT FROM AUTHOR]

Abstract: In this study, bone mineral density (BMD) of normal (CON), ovariectomized (OVX), and partially nephrectomized (NFR) rats was measured by 31P NMR spectroscopy; bone matrix density was measured by 1H water- and fat-suppressed projection imaging (WASPI); and the extent of bone mineralization (EBM) was obtained by the ratio of BMD/bone matrix density. The capability of these MR methods to distinguish the bone composition of the CON, OVX, and NFR groups was evaluated against chemical analysis (gravimetry). For cortical bone specimens, BMD of the CON and OVX groups was not significantly different; BMD of the NFR group was 22.1% (by 31P NMR) and 17.5% (by gravimetry) lower than CON. For trabecular bone specimens, BMD of the OVX group was 40.5% (by 31P NMR) and 24.6% (by gravimetry) lower than CON; BMD of the NFR group was 26.8% (by 31P NMR) and 21.5% (by gravimetry) lower than CON. No significant change of cortical bone matrix density between CON and OVX was observed by WASPI or gravimetry; NFR cortical bone matrix density was 10.3% (by WASPI) and 13.9% (by gravimetry) lower than CON. OVX trabecular bone matrix density was 38.0% (by WASPI) and 30.8% (by gravimetry) lower than CON, while no significant change in NFR trabecular bone matrix density was observed by either method. The EBMs of OVX cortical and trabecular specimens were slightly higher than CON but not significantly different from CON. Importantly, EBMs of NFR cortical and trabecular specimens were 12.4% and 26.3% lower than CON by 31P NMR/WASPI, respectively, and 4.0% and 11.9% lower by gravimetry. Histopathology showed evidence of osteoporosis in the OVX group and severe secondary hyperparathyroidism (renal osteodystrophy) in the NFR group. These results demonstrate that the combined 31P NMR/WASPI method is capable of discerning the difference in EBM between animals with osteoporosis and those with impaired bone mineralization. [Copyright &y& Elsevier]

Abstract: Bone mineral is a major determinant of the mechanical resistance of bones. In bone structural units (BSUs), mineralization of osteoid tissue begins with a rapid primary mineralization followed by a secondary mineralization phase, i.e., a slow and gradual maturation of the mineral component leading to complete mineralization during an unknown period. The aim of this study was to determine the chronology of secondary bone mineralization in ewes, an animal model with a remodeling activity close to humans. Eighteen ewes received different fluorescent labels every 6 months to date the “age” of each labeled BSU. The degree of mineralization of bone (DMB) and Vickers microhardness were measured in labeled BSUs, while mineralization at the crystal level was assessed by Fourier transform infrared microspectroscopy (FTIRM). During the first 6 months of mineralization, degree of mineralization and microhardness significantly increased. They then increased more slowly until at 30 months they reach their maximal values. This progression during secondary mineralization was associated with an improvement of both the maturation and the crystal perfection of the mineral part of bone matrix. Finally, secondary mineralization in BSUs is completed after a period of 30 months. This observation should be taken into account for understanding the effects of long-term treatments of bone diseases. [Copyright &y& Elsevier]

Abstract: The intra-specimen spatial variation in mineralization of bone tissue can be changed by drug treatments that alter bone remodeling. However, the contribution of such changes to the overall biomechanical effect of a treatment on bone strength is not known. To provide insight into this issue, we used a rat model to determine the effects of ovariectomy, parathyroid hormone, and raloxifene (vs. sham) on the contribution of spatial variations in mineralization to treatment-induced changes in vertebral stiffness. Mineral density was measured from 6-µm voxel-sized quantitative micro-CT scans. Whole-vertebral and trabecular stiffness values were estimated using finite element analysis of these micro-CT scans, first including all intra-specimen variations in mineral density in the model and then excluding such variations by using a specimen-specific average density throughout each specimen. As expected, we found appreciable effects of treatment on overall bone stiffness, the effect being greater for the trabecular compartment (up to 52% reduction vs. sham, p <0.0001) than the whole vertebra (p =0.055). Intra-specimen mean mineralization was not changed with treatment but the intra-specimen variation in mineralization was, although the effect was small (4%). Intra-specimen spatial variations in mineralization accounted for 10–12% and 5–6% of overall stiffness of the trabecular compartment and whole vertebral body, respectively. However, after accounting for all treatment effects on bone geometry and trabecular microstructure, any treatment effects due to changes in mineralization were negligible (<2%), although statistically detectable (p <0.02). We conclude that, despite a role in the general biomechanical behavior of bone, the spatial variations in tissue mineralization, as measured by quantitative micro-CT, did not appreciably contribute to ovariectomy-, PTH-, or raloxifene-induced changes in stiffness of the whole bone or the trabecular compartment in these rat vertebrae. [Copyright &y& Elsevier]

The purpose of this study was to determine whether a mineral-rich extract derived from the red marine algae Lithothamnion calcareum could be used as a dietary supplement for prevention of bone mineral loss. Sixty C57BL/6 mice were divided into three groups based on diet: the first group received a high-fat Western-style diet (HFWD), the second group was fed the same HFWD along with the mineral-rich extract included as a dietary supplement, and the third group was used as a control and was fed a low-fat rodent chow diet (AIN76A). Mice were maintained on the respective diets for 15 months. Then, long bones (femora and tibiae) from both males and females were analyzed by three-dimensional micro-computed tomography (micro-CT) and (bones from female mice) concomitantly assessed in bone strength studies. Tartrate-resistant acid phosphatase (TRAP), osteocalcin, and N-terminal peptide of type I procollagen (PINP) were assessed in plasma samples obtained from female mice at the time of sacrifice. To summarize, female mice on the HFWD had reduced bone mineralization and reduced bone strength relative to female mice on the low-fat chow diet. The bone defects in female mice on the HFWD were overcome in the presence of the mineral-rich supplement. In fact, female mice receiving the mineral-rich supplement in the HFWD had better bone structure/function than did female mice on the low-fat chow diet. Female mice on the mineral-supplemented HFWD had higher plasma levels of TRAP than mice of the other groups. There were no differences in the other two markers. Male mice showed little diet-specific differences by micro-CT. [ABSTRACT FROM AUTHOR]

Broilers selected for increased body size and breast muscle have imposed stress on the skeletal system, resulting in poorer walking ability. Our objectives were to determine the relationship between bone mineralization and gait score in 4 crosses of commercial broilers and to ascertain if mineralization of the toe is correlated to the tibia. Three chickens per pen each with good (gait score of 0 or 1) or poorer (gait score of 3) walking ability were killed and weighed. The left humerus, the left middle toe, and both drumsticks were collected for determination of bone mineral density (BMD), bone mineral content, and bone size traits using dual-energy x-ray absorptiometry. The BMD and bone size traits were similar among the 4 crosses of commercial broilers at 6 wk of age. However, gait scores differed among genotypes, with cross C having better gait scores than crosses A and B, but did not differ from cross D. The bone mineral content and bone size traits did not differ between birds with good walking ability as compared with those broilers of poorer walking ability. However, birds with poorer walking ability had higher BMD (P < 0.05) and BW (P < 0.001) than males with good walking ability. Within a cross, the correlation between gait score and BMD was not significant except for cross D birds. Broilers of cross D with better walking ability had decreased bone mineralization (r = 0.19, P = 0.03). The stronger correlation between gait score and BW for all crosses of commercial broilers (r = 0.38, P < 0.0001) suggests that the low association between gait score and bone mineralization for cross D was mainly due to BW. The BMD of the left toe was correlated to the BMD of the left tibia (r = 0.91, P < 0.0001) and right tibia (r = 0.87, P < 0.0001). In conclusion, bone mineralization was similar among crosses of meat-type chickens, and it had little influence on the gait score of male broilers. [ABSTRACT FROM AUTHOR]

Abstract: The differentiation and degree of the effects of mineral content and/or morphology on bone quality remain, to a large extent, unanswered due to several microarchitectural particularities in spatial measuring fields (e.g., force transfer, trajectories, microcalli). Therefore, as the smallest basic component of cancellous bone, we focused on single trabeculae to investigate the effects of mineralization and structure, both independently and in superposition. Transiliac Bordier bone cores and T12 vertebrae were obtained from 20 females at autopsy for specimen preparation, enabling radiographical analyses, histomorphometry, Bone Mineral Density Distribution (BMDD) analyses, and trabecular singularization to be performed. Evaluated contact X-rays and histomorphometric limits from cases with osteoporotic vertebral fractures generated two subdivisions, osteoporotic (n =12, Ø 78 years) and non-osteoporotic (n =8, Ø 49 years) cases, based on fracture appearance and bone volume (BV/TV). Measurements of trabecular number (Tb.N.), trabecular separation (Tb.Sp.), trabecular thickness (Tb.Th.), trabecular bone pattern factor (TBPf) and eroded surface (ES/BS) were carried out to provide detailed structural properties of the investigated groups. The mechanical properties of 400 rod-like single vertebral trabeculae, assessed by three-point bending, were matched with mineral properties as quantified by BMDD analyses of cross-sectioned rod-like and plate-like trabeculae, both in superposition and independently. Non-osteoporotic iliac crests and vertebrae displayed linear dependency on structure parameters, whereas osteoporotic compartments proved to be non-correlated with bone structure. Independent of trabecular thickness, osteoporotic rod-like trabeculae showed decreases in Young''s modulus, fracture load, yield strength, ultimate stress, work to failure and bending stiffness, along with significantly increased mean calcium content and calcium width. Non-osteoporotic trabeculae showed biomechanically beneficial properties due to a homogeneous mineralization configuration, whereas osteoporotic trabeculae predominantly demonstrated various mineralized bone packets, eroded surfaces, highly mineralized cement lines and microcracks. The Young''s moduli of single trabeculae exhibited significantly negative linear correlations with trabecular thickness. Because of increased, but inhomogeneously distributed, calcium content, osteoporotic trabeculae may be subject to shear stresses that render bone fragile beyond structure impairment due to cracks and lacunae. [Copyright &y& Elsevier]

Abstract: The present study aimed to investigate whether genetic background in combination with the development of more intensive production regimes influences bone mineralization and the development of vertebral deformities in cultured Atlantic salmon. Triplicate salmon parr groups of wild, farmed, and hybrid origin (here-on referred to as wild, farmed and hybrid genotypes) were reared as underyearling (0+, SW transfer 8months post hatching in Jan 2006) or yearling smolt (1+, SW transfer 12.5months post hatching in May 2006), and evaluated for vertebral mineralization (mineral content, as % of bone dry weight; n =90; 15 per group) and deformities (radiology; n =540; 90 per group) in March 2007. Farmed fish (0+ 2.5kg, 1+ 2.1kg) were significantly larger than wild (0+ 0.9kg, 1+ 0.8kg) and hybrid (0+ 1.5kg, 1+ 1.3kg) fish. Bone mineral content in the vertebrae was significantly higher in farmed (57.6%) than in hybrid (57.2%) and wild (56.5%) fish, and significantly higher in hybrid than in wild fish within the 1+ smolt, whereas there were no differences in the mineral content between genotypes within the 0+ smolt, or effect of production strategy within each genotype. The prevalence of deformed fish (% individuals with one or more deformed vertebrae) ranged from 6.6% and 17.1%, but there were no significant effect of genotype or production strategy. The results of the present study indicate that neither off season smoltification, smolt quality or genetic background are important factors in the etiology of vertebral deformities in farmed Atlantic salmon. The data may serve as a base-line for further studies on vertebral deformities in Atlantic salmon. [Copyright &y& Elsevier]

Abstract: Spatial patterns of mineralization for human iliac crest cancellous bone were measured from images obtained by quantitative backscattered electron microscopy. Biopsies collected from vertebral fracture patients and healthy individuals with high or low bone formation rate (BFRs) were examined (fracture/low BFRs: N =12, fracture/high BFRs: N =10, normal/low BFRs: N =12, normal/high BFRs: N =15). 20 by 20 pixel square areas or smaller were sampled from superficial and deep remodeling packets. Mean (Z mean) and standard deviation (SD) of mineralization were measured, and coefficients of variation (CV=SD/ Z mean) were calculated. Fast Fourier transform analysis was used to quantify the distribution of the mineral in the packets. “FFT_ratio” was defined as the ratio magnitude of the principal spatial frequency to the average atomic number density. A higher FFT_ratio occurred in specimens with more pronounced alternating layers of light and dark as visible in the backscattered electron image, which was defined as lamellar patterning. Two-way ANOVA revealed that the coefficients of variation of mineralization for both superficial and deep packets were significantly lower in fracture patients than in normal individuals. However, the interaction between turnover rate and group (fracture/non-fracture) indicated that the difference in packet CV occurred among the low turnover individuals and not among those with high turnover. Mean mineralization levels and CV between deep and superficial packets were highly correlated. Regressions of packet CV of mineralization and FFT_ratio were highly significant (p <0.001) for all packets pooled and for packets divided by group (fracture/normal). However, analyses of packet CV and FFT_ratio by individual were variable (R 2 from 0.00338 to 0.700). Packet-level mineralization variability may be associated with fracture toughness, and fracture patients had less variable packet-level mineralization. The result that the packet CV varied significantly between fracture and non-fracture individuals with low turnover suggests that for low turnover subjects without fracture, high variability in mineralization may have a protective effect. In high turnover patients, the accelerated turnover may prevent the lamellar variability from developing over time. Strong correlations between CV and Z mean for both superficial and deep packets imply that newly formed bone is created similarly to older bone within an individual. Fourier transform results show that the mineralization variability found within packets is associated with lamellar patterning. Lamellar structure has been hypothesized to guide microcrack propagation in order to optimize bone strength and toughness. Osteoporotics with fracture had less pronounced lamellation than healthy normals and may be more prone to fracture. [Copyright &y& Elsevier]

The variability of bone traits was assessed in purebred lines of meat-type chickens using dual-energy x-ray absorptiometry. Experiment 1 evaluated changes in bone mineralization and size traits of the tibia and humerus in 4 purebred lines from 6 to 24 wk of age. Experiment 2 compared the same traits of the tibia, radius, and ulna of 9 purebred lines at 6 wk of age. Differences in bone traits among purebred lines were apparent in both experiments. Of the 4 purebred lines compared in experiment 1, line C demonstrated the best phenotypic traits relative to bone quality. Even though line C had the longest tibia, one of the largest bone areas, one of the heaviest BW, and one of the highest bone mineral content (BMC) at 24 wk of age, the tibia of line C did not become less dense in mineral as this line of chickens approached sexual maturity as did the tibia of the other purebred lines of chickens. Specifically, its tibial bone mineral density (BMD) showed age-related increases unlike the other purebred lines of chickens, which showed little change in tibial BMD from 6 to 24 wk of age. In experiment 2, all bone traits as well as BW were different among purebred lines (P < 0.001). The 2 purebred lines (7 and 8) with the lightest 6-wk-old BW (2,033 and 2,055 g, respectively) had diverse skeletal traits. Birds of line 7 had the lowest BMD (0.1131 g/cm²), BMC (1.05 g), shortest bone length (69.2 mm), and smallest bone area (8.0 cm²); however, the other purebred line low in BW (line 8) showed the opposite trend in that bones from these birds were the highest in BMD (0.1276 g/cm²), BMC (1.38 g), bone length (74.6 mm), and area (9.2 cm²) when compared with all of the other lines. In conclusion, several purebred lines of meat-type chickens expressed large differences in bone traits, suggesting the potential to genetically select birds for increased BMD. [ABSTRACT FROM AUTHOR]

Abstract: Background: The incidence of osteoporotic hip fractures increases with age, more sharply in women than in men, as a result of qualitative and quantitative bone alterations. Mineralization (a qualitative parameter) showed no differences with age or sex in cancellous bone in earlier studies. Few studies assessed such differences in cortical bone, a major contributor to femoral bone strength. The aim of this in vitro cross-sectional study of a large group of human femoral midshafts was to look for age- and sex-related differences in the degree and distribution of cortical mineralization that might be implicated in bone fragility. Methods: Cortical bone specimens from 193 femurs were studied using quantitative microradiography, with an aluminum step-wedge reference. The femurs were from 99 females and 94 males in a Caucasian anthropological collection covering a broad age spectrum. We determined the mean degree of mineralization of osteons (On.DMB-Al), interstitial tissue (Int.DMB-Al), and total bone (Tt.DMB-Al), and representative parameters of density histograms. Results were expressed as relative values. Age- and sex-related differences in DMB-Al values were evaluated using non-parametric tests. Results: Degree of tissue mineralization (Tt.DMB-Al) decreased significantly with age in females (r =−0.257; P =0.010) but did not change in males. Tt.DMB-Al was higher in females than males until 50 years of age (P =0.001) but was lower in elderly females than elderly males (P =0.016). DMB-Al distribution varied significantly with sex and age. The first DMB-Al quartiles in osteons and interstitial tissue were not different between males and females, but the third quartile and interquartile range differed significantly (P =0.032 and P =0.000, respectively). The mineralization difference between the two tissues indicated greater bone heterogeneity in females than males (P =0.000). Conclusions: In this in vitro cross-sectional study of anterior midfemoral cortical specimens, the degree and distribution of mineralization varied with age and sex. In females, mineralization started at a higher level than in males but was lower in the sixth decade, falling below the level in males. Mineralization was far more stable throughout life in males. In elderly females, the lower degree and greater heterogeneity of mineralization may have consequences on bone strength and the risk of fracture. [Copyright &y& Elsevier]

Abstract: Aims: Bodyweight is a significant predictor of bone mass. Hormonal factors are thought to play a role in the mechanisms controlling the association of body weight and fat mass with bone mass. Very recently, the orexigenic hormone ghrelin has also been implicated in bone metabolism. In this study we examined the associations of circulating acylated and des-acyl ghrelin concentrations with measures of bone in a group of obese children and adolescents as well as in a group of healthy control children. We also determined whether the associations were independent of body composition, chronological age, gender, Tanner stage, and leptin, glucose, insulin and insulin-like growth factor (IGF)-1 levels. Methods: We performed a prospective cross-sectional study of 100 obese children [age, 8.9 (8.3 to 9.4); BMI-Standard Deviation Score (SDS), 2.2 (2.0 to 2.3)], and 100 age-matched lean healthy subjects. Fasting insulin, leptin, IGF-1, acylated and total ghrelin were measured by radioimmunoassay. Des-acyl ghrelin values were calculated as total ghrelin minus acylated ghrelin. Whole body (WB) and lumbar spine (LS) BMD, and BMC as well as body composition were assessed by DXA (Hologic QDR-4500W). LS volumetric BMD (BMAD) was estimated using the formula of Katzman (BMC/area1.5), while WB BMC data were expressed as BMC/height. Results: Backward linear regression analysis was performed for individual groups, with age, gender, Tanner stage, weight, height, body composition (lean and fat mass), acylated ghrelin, des-acyl ghrelin, leptin, glucose, insulin, and IGF-1, entered into the model. In healthy children, acylated ghrelin was a significant and independent negative predictor of WB BMD, and WB BMC/height, while lean mass was positively associated significantly with these bone measures. In contrast, in obese children, a positive significant association was observed between des-acyl ghrelin and WB BMD as well as WB BMC/height, along with lean mass, and to a lesser degree, with fat mass. Acylated as well as des-acyl ghrelin were not significant predictors of LS BMD and LS BMAD in obese as well as control children. Conclusions: The results of this investigation indicate that the influence of the two distinct isoforms of ghrelin on BMD is mediated by specific body composition parameters in obese and control healthy children. [Copyright &y& Elsevier]

Abstract: The strength of bone depends on both bone quantity and bone quality. One determinant of bone quality is the degree of mineralization of bone tissue (DMB). To assess the role for DMB in osteoporotic hip fractures, we compared the degree of mineralization in femoral neck cortex from 23 women with hip fractures (age, 65–96 years) and 14 female controls (age, 75–103 years) using quantitative microradiography calibrated with an aluminum step wedge. Variables were DMB in osteons (oDMBAl mean) and interstitial tissue (exDMBAl mean). Wilcoxon signed-rank tests were used to compare oDMBAl mean to exDMBAl mean in each group, and Mann–Whitney tests to compare oDMBAl mean and exDMBAl mean between hip-fracture patients and controls. DMB was significantly lower in the osteons than in the interstitial tissue in both groups (hip-fracture group, P =0.000; control group, P =0.001). DMB values in osteons and interstitial tissue were significantly greater in the hip-fracture patients than in the controls (P =0.007 and P =0.005, respectively). These cross-sectional data suggest that bone fragility may be related to a higher degree of tissue mineralization. [Copyright &y& Elsevier]

Abstract: Semicarbazide (SEM) is an azodicarbonamide by-product present in glass jar packaged foods including babyfoods, in bleaching steps and flour treatment. Experimental data showed SEM acting as osteolathyrogen agent, but few toxicological data are available in susceptible life-stages. This study aimed to evaluate effects of SEM oral administration for 28 days at 0, 40, 75, 140mg/kg bw day during the juvenile period in Sprague–Dawley rats. Histopatological examinations of: epiphyseal cartilage – potential target of SEM lathyrogen action - testes, ovary, uterus, thyroid, thymus, spleen, adrenals, representative of the main developing organs relevant to juvenile toxicity, and neurobehavioural tests in males, were performed. Mortality at high and mid dose levels and significantly decreased body weight gain were observed in males even at the lowest dose. Lack of mineralization in cartilage at all dose levels was present. Marked alterations of spontaneous motor and exploratory behaviours were evident even at 40mg/kg. Histological alterations were observed in all tissues; thyroid and ovary effects were present also at 40mg/kg. The present study indicate that the NOAEL in juvenile rats is lower than 40mg/kg for SEM oral administration. SEM administration during juvenile period exerted pleiotropic effects and further studies are suggested to elucidate mechanisms. [Copyright &y& Elsevier]

Abstract: Long-lived species are particularly susceptible to bioaccumulation of lead in bone tissues. In this paper we gain insights into the sublethal effects of lead contamination on Egyptian vultures (Neophron percnopterus). Our approach was done on the comparison of two populations (Canary Islands and Iberian Peninsula) differing in exposures to the ingestion of lead ammunition. Blood lead levels were higher in the island population (Canary Islands range: 5.10–1780μg L−1 n =137; Iberian Peninsula range: 5.60–217.30μg L−1 n =32) showing clear seasonal trends, peaking during the hunting season. Moreover, males were more susceptible to lead accumulation than females. Bone lead concentration increased with age, reflecting a bioaccumulation effect. The bone composition was significatively altered by this contaminant: the mineralization degree decreased as lead concentration levels increased. These results demonstrate the existence of long-term effects of lead poisoning, which may be of importance in the declines of threatened populations of long-lived species exposed to this contaminant. [Copyright &y& Elsevier]

Abstract: We describe a procedure for quantification of vitamin K1 in human plasma by HPLC. Samples, enriched with a vitamin K derivative as internal standard, were deproteinized, purified on polymeric RP-SPE cartridges and injected into HPLC equipped with a post-column on-line zinc metal reactor and a fluorometric detector. Median level in blood donors (n =87) was 1.967nmol/L (0.93–4.01, 5th–95th percentiles), with a significant correlation between plasma levels and age (r =0.276, p =0.00958) and a lower (not significant) value in women than in men. This method, easy-to-handle and with a high throughput, can be used to identify covert states of vitamin K intake deficiency in patients thus at risk of alterations in blood clotting or bone mineralization. [Copyright &y& Elsevier]

Abstract: Physical activity in adolescence is beneficial for increasing bone mineral accrual; however, it''s unclear whether these benefits persist into adulthood. This prospective study investigated whether physically active adolescents maintained their higher bone mineral content (BMC) into the third decade of life when compared to their less active peers. Data were from 154 subjects (82 females and 72 males) who participated in the University of Saskatchewan''s Pediatric Bone Mineral Accrual Study (1991–1997), entry age 8 to 15 years. Participants returned for follow-up as young adults (2002–2006), follow-up age 23 to 30 years. Dual energy X-ray absorptiometry was used to measure BMC of total body (TB), lumbar spine (LS), total hip (TH) and femoral neck (FN) annually from 1991 to 1997 and from 2002 to 2006. Peak height velocity (PHV) was determined for each child as a measure of maturity. Age and gender-specific activity Z-scores were calculated for each participant based on the mean physical activity scores obtained from bi-annual questionnaire data during childhood and adolescence. Subjects were ranked into three adolescent activity groups: active, average and inactive (top, middle two, and bottom quartiles, respectively). Analysis of covariance (ANCOVA) was used to compare adjusted TB, LS, TH and FN BMC across the three adolescent activity groups at 1 year post PHV and in young adulthood. When compared to the inactive group, active males had 8% greater adjusted BMC at the TB, 13% at the LS and 11% at the TH (p <0.05) in adolescence. Active females also had 8% and 15% more adjusted BMC (p <0.05) at the TB and LS, respectively, during adolescence. In young adulthood the male and female adolescent active groups were still significantly more active than their peers (p >0.05). It was found that active adolescent males had 8–10% more adjusted BMC at the TB, TH and FN (p <0.05) in young adulthood and that active adolescent females had 9% and 10% more adjusted BMC at the TH and FN. These results suggest that the skeletal benefits of physically activity in adolescents are maintained into young adulthood. [Copyright &y& Elsevier]