Noncollagenous Bone Proteins Research Articles

Osteocalcin: Promoter or Inhibitor of Hydroxyapatite Growth?

Osteocalcin is the most abundant noncollagenous bone protein and the functions in bone remineralization as well as in inhibition of bone growth have remained unclear. In this contribution, we explain the dual role of osteocalcin in the nucleation of new calcium phosphate during bone remodeling and in the inhibition of hydroxyapatite crystal growth at the molecular scale. The mechanism was derived using pH-resolved all-atom models for the protein, phosphate species, and hydroxyapatite, along with molecular dynamics simulations and experimental and clinical observations. Osteocalcin binds to (hkl) hydroxyapatite surfaces through multiple residues, identified in this work, and the fingerprint of binding residues varies as a function of the (hkl) crystal facet and pH value. On balance, the affinity of osteocalcin to hydroxyapatite slows down crystal growth. The unique tricalcium γ-carboxylglutamic acid (Gla) domain hereby rarely adsorbs to hydroxyapatite surfaces and faces instead toward the solution. The Gla domain enables prenucleation of calcium phosphate for new bone formation at a slightly acidic pH of 5. The growth of prenucleation clusters of calcium phosphate continues upon increase in pH value from 5 to 7 and is much less favorable, or not observed, on the native osteocalcin structure at and above neutral pH values of 7. The results provide mechanistic insight into the early stages of bone remodeling from the molecular scale, help inform mutations of osteocalcin to modify binding to apatites, support drug design, and guide toward potential cures for osteoporosis and hyperosteogeny.

Porous titanium scaffolds modified with Zeolitic Imidazolate Framework (ZIF-8) with enhanced osteogenic activity for the prevention of implant-associated infections.

In this study, zeolitic imidazolate framework 8 (ZIF-8) was coated on porous Ti6Al4V scaffolds, either bare or previously modified using hydroxyapatite (HA) or HA and gelatin (HAgel), via a growing single-step method in aqueous media using two contact times at 6h and 24h. The coated scaffolds termed ZIF-8@Ti, ZIF-8@HA/Ti, and ZIF-8@HAgel/Ti were characterized via scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), attenuated total reflectance-Fourier transform infrared (ATR-FTIR), and molecular plasma-atomic emission spectroscopy (MP-AES). In order to assess the cell proliferation rate, the cytocompatibility of the scaffolds was evaluated in primary osteoblasts (hOBs) using alamarBlue assay, while the osteoconductivity was analyzed in hOBs using a real-time approach, evaluating the expression of secreted phosphoprotein 1 (SPP1). Osteopontin, which is the protein encoded by this gene, represents the major non-collagenous bone protein that binds tightly to HA. The scaffolds were shown to be non-cytotoxic based on hOB proliferation at all time points of analysis (24h and 72h). In hOB cultures, the scaffolds induced the upregulation of SPP1 with different fold changes. Some selected scaffolds were assayed in vitro for their antibacterial potential against Staphylococcus epidermidis; the scaffolds coated with ZIF-8 crystals, regardless of the presence of HA and gelatin, strongly inhibited bacterial adhesion to the materials and reduced bacterial proliferation in the culture medium, demonstrating the suitable release of ZIF-8 in a bioactive form. These experiments suggest that the innovative scaffolds, tested herein, provide a good microenvironment for hOB adhesion, viability, and osteoconduction with effective prevention of S. epidermidis adhesion.

Selective excitation with recoupling pulse schemes uncover properties of disordered mineral phases in bone-like apatite grown with bone proteins

Bone construction has been under intensive scrutiny for many years using numerous techniques. Solid-state NMR spectroscopy helped unravel key characteristics of the mineral structure in bone owing to its capability of analyzing crystalline and disordered phases at high-resolution. This has invoked new questions regarding the roles of persistent disordered phases in structural integrity and mechanical function of mature bone as well as regarding regulation of early events in formation of apatite by bone proteins which interact intimately with the different mineral phases to exert biological control.Here, spectral editing tethered to standard NMR techniques is employed to analyze bone-like apatite minerals prepared synthetically in the presence and absence of two non-collagenous bone proteins, osteocalcin and osteonectin. A 1H spectral editing block allows excitation of species from the crystalline and disordered phases selectively, facilitating analysis of phosphate or carbon species in each phase by magnetization transfer via cross polarization. Further characterization of phosphate proximities using SEDRA dipolar recoupling, cross-phase magnetization transfer using DARR and T1/T2 relaxation times demonstrate that the mineral phases formed in the presence of bone proteins are more complex than bimodal. They reveal disparities in the physical properties of the mineral layers, indicate the layers in which the proteins reside and highlight the effect that each protein imparts across the mineral layers.

Proteins and Our Past: An Exploration of Human Bone Protein from the Eighteenth-Century Fortress of Louisbourg, Nova Scotia, and Its Potential Applications in Bioarchaeological Research

Bioarchaeologists can further investigate human bone metabolism at the biomolecular level by incorporating biochemical methods into their research. Recently, there has been a focus on osteocalcin, an abundant non-collagenous bone protein, because of its clinically identified relationship with biological factors (i.e., age and sex), activity, and pathological conditions (i.e., disease). For this study, osteocalcin was extracted and quantified from the femora of 27 individuals from the Fortress of Louisbourg (1713–1758) skeletal collection to explore if the clinical relationship between osteocalcin and sex, age, activity, and pathological conditions can also be established in archaeological bone. However, no significant relationships between osteocalcin concentrations and biological factors (i.e., age and sex), activity, or pathological conditions were identified. This is the first study to quantify osteocalcin from archaeological human skeletal remains from a Canadian context and provides another example of how this method may be used to study stress in bioarchaeological populations.

The role of osteocalcin in regulation of glycolipid metabolism and muscle function in children with osteogenesis imperfecta.

ObjectiveOsteoblasts are discovered to secrete hormones with endocrine effects on metabolism, and osteocalcin (OC) is the most abundant non-collagenous protein in bone. We investigate the relationship between serum OC levels and glycolipid metabolism and muscle function in children with osteogenesis imperfecta (OI).MethodsA total of 225 children with OI and 80 healthy controls matched in age and gender were included in this single center study. Serum levels of fasting blood glucose (FBG), triglyceride (TG), total cholesterol (TC), low- and high-density lipoprotein cholesterol (LDL-C, HDL-C) were measured by automated analyzers. Serum levels of fasting insulin (FINS) were measured using an automated electrochemiluminescence system. Serum levels of OC and undercarboxylated osteocalcin (ucOC) were measured by enzyme-linked immunosorbent assay. Grip strength and timed-up-and-go (TUG) test were measured. Bone mineral density (BMD) and body composition were measured using dual-energy X-ray absorptiometry.ResultsOI patients had significantly higher body mass index (BMI), FBG, and HOMA-IR, but lower HDL-C levels, lower grip strength and longer TUG than control group (all P<0.05). Serum OC, ucOC levels, and ucOC/OC in OI type III patients were significantly lower than those in OI patients with type I and IV. Serum levels of OC, ucOC, and ucOC/OC were negatively correlated to BMI, FBG, insulin levels, and HOMA-IR (all P<0.05). The ratio of ucOC/OC was positively correlated to grip strength (r=0.512, P=0.036), lean mass percentage (%LM) of the total body and limbs, and negatively correlated to fat mass percentage (%FM) of the total body, %FM and fat mass index (FMI) of the trunk (all P<0.05).ConclusionsObesity, glucolipid metabolic abnormalities, and reduced grip strength were common in children with OI. Circulating osteocalcin and ucOC may play an important role in the regulation of glucose metabolism, as well as the muscle function of children with OI.

Poisoning by Nierembergia veitchii: Effects on vascular smooth muscle cells in the pathogenesis of enzootic calcinosis.

Vascular mineralization is a hallmark of enzootic calcinosis. Histopathological, ultrastructural, and immunohistochemical investigations were performed on the external carotid arteries of seven sheep naturally poisoned by Nierembergia veitchii. Histologically, moderate to marked hyperplasia of the tunica intima was observed without mineralization. The tunica media exhibited mild to severe mineralization and osteochondroid metaplasia. Sheep with enzootic calcinosis showed arterial overexpression of osteopontin and tissue-nonspecific alkaline phosphatase and immunolabeling for osteonectin and osteocalcin in both intima and media layers of the tested arteries. The main ultrastructural finding in the tunica media was a marked phenotypic change of vascular smooth muscle cells from a contractile phenotype (VSMC-C) into a synthetic phenotype (VSMC-S). In the tunica media, VSMC-S produced matrix and extracellular vesicles, forming mineralizable granules associated with arterial mineralization. VSMC-S were also present in the tunica intima, but matrix and extracellular vesicles and mineralization were not observed. The absence of matrix and extracellular vesicles in the intimal hyperplasia, even in the presence of noncollagenous bone proteins, tissue-nonspecific alkaline phosphatase, and vitamin D receptors, reinforces the hypothesis that the presence of matrix and extracellular vesicles are crucial for the development of vascular mineralization in enzootic calcinosis. It is proposed that the two different VSMC-S phenotypes in calcinosis are due to the expression of at least two genetically different types of these cells induced by the action of 1,25(OH)2D3.

Intrinsically disordered peptides enhance regenerative capacities of bone composite xenografts

Biomaterial scientists design organic bone substitutes based on the biochemical properties of the mimicked tissue to achieve near native functionality. Several non-collagenous proteins in bone are known as intrinsically disordered proteins (IDPs), as they lack detectible ordered domains and a fixed 3D structure under physiological conditions. Many IDPs perform regulatory roles in a range of cellular functions, which motivated us to design two proline-rich disordered peptides (P2 and P6) and augmented them into the SmartBone® (SBN) biohybrid substitute. Recently we reported an improved proliferation and osteogensis of human osteoblasts and mesenchymal stem cells in the composite groups containing peptides (named here as SBN + P2 and SBN + P6) in vitro. To address the effects of these composites on bone formation and biomineralization, this in vivo study investigated their functions in critical size craniotomy defects in 16 domestic pigs after 8 and 16 weeks of healing. For this purpose, we used cone beam computed tomography (CBCT), microCT (µCT), histology, immunohistochemistry, fluorescent labeling of abundant reactive entities (FLARE), synchrotron SAXS/XRD, optical photothermal IR (O-PTIR) microscopy and nanoscale atomic force microscopy-infrared (AFM-IR) analyses. Our results represent new synthetic IDPs as potential candidates for directing bone formation and biomineralization. The SBN + P6 stimulated significantly higher bone formation and biomineralization after 8 weeks of healing compared to other groups indicating its potential in stimulating early biomineralization. After 16 weeks of healing, the SBN + P2 induced significantly higher bone formation and biomineralization compared to other groups indicating its effects on later bone formation and biomineralization processes. The vigorous stretching of amide primary and secondary IR absorbance peaks at 1660 and 1546 cm−1 in the SBN + P2 group verified that this peptide experienced more conformational changes after 16 weeks of implantation with a higher phosphate intensity at 1037 cm−1 compared to peptide 6. Overall, P2 and P6 are promising candidates for bone augmentation strategies in critical clinical applications. We concluded that FLARE and O-PTIR are promising tools in evaluating and diagnosing the biochemical structure of bone tissue and the bone-biomaterial interface.

Bone matrix quality in paired iliac bone biopsies from postmenopausal women treated for 12months with strontium ranelate or alendronate.

Bone quality is altered mainly by osteoporosis, which is treated with modulators of bone quality. Knowledge of their mechanisms of action is crucial to understand their effects on bone quality. The goal of our study was to compare the action of alendronate (ALN) and strontium ranelate (SrRan) on the determinants of bone quality. The investigation was performed on over 60 paired human iliac biopsies. Paired samples correspond to biopsies obtained from the same patient, one before treatment (baseline) and one after 12months of treatment, in postmenopausal women with osteoporosis. Vibrational spectroscopy (Raman and FTIRM) and nanoindentation were used to evaluate the effect of both drugs on bone quality at the ultrastructural level. Outcomes measured by vibrational spectroscopy and nanoindentation are sensitive to bone age. New bone packets are distinguished from old bone packets. Thus, the effect of bone age is distinguished from the treatment effect. Both drugs modify the mineral and organic composition in new and old bone in different fashions after 12months of administration. The new bone formed during ALN administration is characterized by an increased mineral content, carbonation and apatite crystal size/perfection compared to baseline. Post-translational modifications of collagen are observed through an increase in the hydroxyproline/proline ratio in new bone. The proteoglycan content is also increased in new bone. SrRan directly modulates bone quality through its physicochemical actions, independent of an effect on bone remodeling. Strontium cations are captured by the hydrated layer of the mineral matrix. The mineral matrix formed during SrRan administration has a lower carbonate content and crystallinity after 12months than at baseline. Strontium might create bonds (crosslinks) with collagen and noncollagenous proteins in new and old bone. The nanomechanical properties of bone were not modified with either ALN or SrRan, probably due to the short duration of administration. Our results show that ALN and SrRan have differential effects on bone quality in relation to their mechanism of action.

Vitamin K-Dependent Proteins in Skeletal Development and Disease.

Vitamin K and Vitamin K-dependent proteins (VKDPs) are best known for their pivotal role in blood coagulation. Of the 14 VKPDs identified in humans to date, 6 play also important roles in skeletal biology and disease. Thus, osteocalcin, also termed bone Gla-protein, is the most abundant non-collagenous protein in bone. Matrix Gla protein and Ucma/GRP on the other hand are highly abundant in cartilage. Furthermore, periostin, protein S, and growth arrest specific 6 protein (GAS 6) are expressed in skeletal tissues. The roles for these VKDPs are diverse but include the control of calcification and turnover of bone and cartilage. Vitamin K plays an important role in osteoporosis and serum osteocalcin levels are recognized as a promising marker for osteoporosis. On the other hand, matrix Gla protein and Ucma/GRP are associated with osteoarthritis. This review focuses on the roles of these three VKDPs, osteocalcin, matrix Gla protein and Ucma/GRP, in skeletal development and disease but will also summarize the roles the other skeletal VKDPs (periostin, protein S and GAS6) in skeletal biology.

Single-Molecule Force Spectroscopy Reveals Adhesion-by-Demand in Statherin at the Protein-Hydroxyapatite Interface.

Achieving strong adhesion in wet environments remains a technological challenge in biomedical applications demanding biocompatibility. Attention for adhesive motifs meeting such demands has largely been focused on marine organisms. However, bioadhesion to inorganic surfaces is also present in the human body, in the hard tissues of teeth and bones, and is mediated through serines (S). The specific amino acid sequence DpSpSEEKC has been previously suggested to be responsible for the strong binding abilities of the protein statherin to hydroxyapatite, where pS denotes phosphorylated serine. Notably, similar sequences are present in the non-collagenous bone protein osteopontin (OPN) and the mussel foot protein 5 (Mefp5). OPN has previously been shown to promote fracture toughness and physiological damage formation. Here, we investigated the adhesion strength of the motif D(pS)(pS)EEKC on substrates of hydroxyapatite, TiO2, and mica using atomic force microscopy (AFM) single-molecule force spectroscopy (SMFS). Specifically, we investigated the dependence of adhesion force on phosphorylation of serines by comparing findings with the unphosphorylated variant DSSEEKC. Our results show that high adhesion forces of over 1 nN on hydroxyapatite and on TiO2 are only present for the phosphorylated variant D(pS)(pS)EEKC. This warrants further exploitation of this motif or similar residues in technological applications. Further, the dependence of adhesion force on phosphorylation suggests that biological systems potentially employ an adhesion-by-demand mechanism via expression of enzymes that up- or down-regulate phosphorylation, to increase or decrease adhesion forces, respectively.

Functions of Osteocalcin in Bone, Pancreas, Testis, and Muscle.

Osteocalcin (Ocn), which is specifically produced by osteoblasts, and is the most abundant non-collagenous protein in bone, was demonstrated to inhibit bone formation and function as a hormone, which regulates glucose metabolism in the pancreas, testosterone synthesis in the testis, and muscle mass, based on the phenotype of Ocn−/− mice by Karsenty’s group. Recently, Ocn−/− mice were newly generated by two groups independently. Bone strength is determined by bone quantity and quality. The new Ocn−/− mice revealed that Ocn is not involved in the regulation of bone formation and bone quantity, but that Ocn regulates bone quality by aligning biological apatite (BAp) parallel to the collagen fibrils. Moreover, glucose metabolism, testosterone synthesis and spermatogenesis, and muscle mass were normal in the new Ocn−/− mice. Thus, the function of Ocn is the adjustment of growth orientation of BAp parallel to the collagen fibrils, which is important for bone strength to the loading direction of the long bone. However, Ocn does not play a role as a hormone in the pancreas, testis, and muscle. Clinically, serum Ocn is a marker for bone formation, and exercise increases bone formation and improves glucose metabolism, making a connection between Ocn and glucose metabolism.

Spider Silk Fusion Proteins for Controlled Collagen Binding and Biomineralization.

The development of biomaterials for the interface between tendon and bone is important for realizing functional tendon replacements. Toward the development of new materials for such applications, engineered recombinant spider silk proteins were modified with peptide tag sequences derived from noncollagenous proteins in bone, so-called SIBLING proteins, such as osteopontin and sialoprotein, which are known to interact with collagen and to initiate mineralization. Materials made of these spider silk-SIBLING hybrids were analyzed concerning mineralization and interaction with cells. They showed enhanced calcium phosphate formation upon incubation in mineralization agents. In gradient films, MC3T3-E1 mouse preosteoblasts adhered preferentially along the gradient toward the variant with a collagen binding motif.

Osteocalcin is necessary for the alignment of apatite crystallites, but not glucose metabolism, testosterone synthesis, or muscle mass

The strength of bone depends on bone quantity and quality. Osteocalcin (Ocn) is the most abundant noncollagenous protein in bone and is produced by osteoblasts. It has been previously claimed that Ocn inhibits bone formation and also functions as a hormone to regulate insulin secretion in the pancreas, testosterone synthesis in the testes, and muscle mass. We generated Ocn-deficient (Ocn-/-) mice by deleting Bglap and Bglap2. Analysis of Ocn-/-mice revealed that Ocn is not involved in the regulation of bone quantity, glucose metabolism, testosterone synthesis, or muscle mass. The orientation degree of collagen fibrils and size of biological apatite (BAp) crystallites in the c-axis were normal in the Ocn-/-bone. However, the crystallographic orientation of the BAp c-axis, which is normally parallel to collagen fibrils, was severely disrupted, resulting in reduced bone strength. These results demonstrate that Ocn is required for bone quality and strength by adjusting the alignment of BAp crystallites parallel to collagen fibrils; but it does not function as a hormone.

Study of bone formation marker levels in rheumatoid arthritis

Background: Rheumatoid arthritis (RA) is an autoimmune disease of unknown cause that affects the joints principally. The disease affects between 0.5% and 1% of the adult population worldwide. Two to three times as many women as men suffer from the disease. Osteocalcin (OC) is a small protein of 49 amino acids long. OC is the most abundant non-collagenous protein in bone. OC originates from osteoblasts and is deposited into bones or released into circulation, where it correlates with histological measures of bone formation. Bone alkaline phosphatase (ALP) is a glycoprotein that is found on the surface of osteoblasts. This enzyme reflects the biosynthetic activity of these bone-forming cells. The presence of OC and ALP in the circulation may, therefore, provide a specific chemical index of osteoblastic activity. Objectives: This study was undertaken to estimate the values of serum OC and ALP among patients with RA and healthy control groups and to compare and find out any changes in levels of serum OC and ALP between the study and control groups. Materials and Methods: It was a case–control study done on 76 RA patients and 76 age- and sex-matched healthy individuals. Serum OC and serum ALP values were evaluated among all 76 cases and 76 controls. Serum OC was measured using immunoenzymatric assay and ALP was measured by colorimetric method. Statistical analysis was performed and results were tabulated and analyzed. Results: Mean ± standard deviation of serum OC level is significantly higher (P < 0.001) among cases (18.50 ± 8.72 ng/ml) than controls (9.98 ± 7.68 ng/ml). Similarly, the values of ALP are higher (P < 0.001) among cases (216.22 ± 59.96 IU/L) than controls (164.17 ± 50.70 IU/L). A significantly positive correlation was found between serum OC and serum ALP levels. Patient with the highest mean value of serum OC also has the highest values of ALP. The values of serum ALP and OC levels increase significantly in both early and late stages when compared with control values. Conclusions: A significant difference between the values of serum OC and ALP among cases and controls was seen in the study. Levels of both these parameters are elevated in subjects with RA compared to controls. Furthermore, the levels of serum OC correlated with the levels of serum ALP. This study demonstrates that increased bone formation is associated with RA together with bone resorption.

Osteocalcin as a Skeletal Maturity Indicator:A Review

Bone mineral metabolism helps in understanding the sequence of boneloss and bone remodeling and in identifying those persons at risk from osteoporosis. One of the approaches that has yielded useful information is the examination of the role of collagenous and noncollagenous bone proteins in bone mineral metabolism. Osteoblasts are the cells responsible for new bone formation, and osteoclasts the cells for resorption. Osteocytes are derived from osteoblasts but their function although not precisely established probably involves calcium homeostasis and bone cell destruction comparable to osteoclastosis. The term remodeling or bone turnover refers to the sequential processes of resorption and subsequent formation for maintenance of mineral homeostasis. In normal man this remodeling cycle occurs at a particular site about every 3 months. The osteoclasts and osteoblasts which are responsible for remodeling appear to be anatomically linked or coupled in equilibrium. Osteocalcin (OC) acts as a regulator of bone mineralization. It also regulates osteoblast and osteoclast activity. This review article lays emphasis on the structure and interaction of OC with various other molecules; its skeletal effects and therapeutic applications.

Osteocalcin affects bone mineral and mechanical properties in female mice.

Osteocalcin is one of the most abundant noncollagenous proteins in bone. Phenotypes of osteocalcin knock-out mice (OC-/-) may vary on different backgrounds and with sex. Previous studies using adult female (OC-/-) mice on a mixed genetic background (129/B6) showed osteocalcin inhibited bone formation leading to weaker bone in wild-type (OC+/+). Yet on a pure (B6) genetic background male mice revealed osteocalcin improved fracture resistance and OC-/- bones were more prone to fracture. Osteocalcin is decreased with age and in some diseases (diabetes) where bone weakness is observed. The effect of osteocalcin in adult female bone from mice on a pure B6 background is unknown. We investigated differences in bone mineral properties and bone strength in female adult (6 months) (OC+/+) and (OC-/-) mice on a pure C57BL/6J background using Fourier Transform Infrared Imaging (FTIRI), micro-computed tomography (uCT), biomechanical measurements, histomorphometry and serum turnover markers (P1NP, CTX). Similar to female age matched mice on the (129/C57) background we found B6 OC-/- mice had a higher bone formation rate, no change in bone resorption, more immature mineral, decreased crystallinity and increased trabecular bone as compared to OC+/+. In contrast, the OC-/- mice on a pure B6 background had a lower bone mineral density, lower mineral to matrix ratio resulting in reduced stiffness and weaker bone strength. Our results demonstrate some properties of the OC-/- phenotype are dependent on genetic background. This may suggest that reduced osteocalcin may contribute to fracture and weaker bone in some groups of elderly and adults with diseases where osteocalcin is low.

Tuning mechanical reinforcement and bioactivity of 3D printed ternary nanocomposites by interfacial peptide-polymer conjugates

We present a study on ternary nanocomposites consisting of medical grade poly(ε-caprolactone) (mPCL) matrix, hydroxyapatite nanopowder (nHA) and compatibilized magnesium fluoride nanoparticle (cMgF2) fillers. MgF2 nanoparticles were compatibilized by following a design approach based on the material interfaces of natural bone. MgF2-specific peptide-poly(ethylene glycol) conjugates were synthesized and used as surface modifiers for MgF2 nanoparticles similarly to the non-collagenous proteins (NPC) of bone which compatibilize hydroxyapatite nanocrystallites. Different compositions of mPCL/nHA/cMgF2 composites were blended together and processed into three dimensional (3D) scaffolds using solvent-free techniques including cryomilling and melt extrusion-based additive manufacturing. The use of two different inorganic fillers in mPCL resulted in nanocomposite materials with enhanced mechanical and biological properties. In particular, cMgF2 nanoparticles were found to be the primary constitent leading to the significant improvements in the mechanical properties of these composites. The scaffolds of the ternary nanocomposites provided the best in vitro performance in terms of osteogenic differentiation and stimulated mineralization. In summary, we demonstrated that the concept of bioinspired interface engineering facilitates the development of homogeneous ternary nanocomposites with increased processability in additive biomanufacturing. Additionally, the concept leads to scaffolds exhibiting enhanced mechanical and biological properties. Overall, these multicomponent nano-interfaced building blocks add a new group of advanced functional materials with tunable mechanical properties, degradation and bioactivity.

Acute calcium kinetics in haemodialysis patients.

To avoid excessive calcium loading in haemodialysis (HD) patients, current guidelines suggest a dialysate calcium concentration (dCa) of 2·5 mEq/L based on relatively stable intradialytic serum calcium levels. However, the latter do not account for possible calcium storage in acutely accessible pools. A rapidly exchangeable calcium pool located at the bone level has been previously proposed to be involved in acute (minute-to-minute) extracellular calcium regulation. To evaluate the contribution of this pool in the maintenance of serum calcium levels, acute calcium buffer capacity was assessed by measuring intradialytic dialysate-sided ionized calcium mass balance (iCaMB ) and change in extracellular fluid calcium mass in chronic HD patients using a dCa of 3·5 (n = 28) and 2·5 (n = 10) mEq/L. Serum osteocalcin, the most abundant noncollagenous bone protein, was measured before the HD session. iCaMB was invariably positive for both 2·5 and 3·5 mEq/L dCa, with a mean of 434 (±125) and 725 (±162) mg/HD, respectively (P < 0·001). Buffered intradialytic calcium load was 410 (±116) and 565 (±130) mg/HD, and acute calcium buffer capacity was 95 (±8)% and 78 (±7)% (mean values at 2·5 and 3·5 mEq/L dCa, respectively) (P < 0·001). Using 3·5 mEq/L dCa, an independent association of acute calcium buffer capacity with undercarboxylated osteocalcin (β = 0·512, P = 0·002) was demonstrated. Our data strongly suggest the existence of a rapidly exchangeable calcium pool that counteracts acute serum calcium deviations in HD patients. This study provides, for the first time, experimental evidence for the involvement of bone in acute extracellular calcium regulation in vivo.

Different osteocalcin forms, markers of metabolic syndrome and anthropometric measures in children within the IDEFICS cohort

ObjectiveOsteocalcin (OC), an aboundant non-collagenous bone protein, is inversely associated with parameters of glucose metabolism. Interactions between bone tissue and energy metabolism have not been thoroughly investigated during childhood. This study investigated OC, metabolic parameters and anthropometric characteristics in normal weight and overweight/obese children. MethodsThis study comprised 108 (46 normal weight/62 overweight/obese) Swedish 2–9year old children. Anthropometric data, insulin, glucose, glycosylated haemoglobin (HbA1c), HOMA index, vitamin D, adiponectin, total OC, carboxylated OC (cOC) and undercarboxylated OC (ucOC) were analysed. ResultsNo difference was found for total OC between the normal and overweight/obese groups, with a mean (±SD) value of 82.6 (±2.8) ng/mL and 77.0 (±2.4) ng/mL, (P=0.11), respectively. Overweight children had lower cOC levels, mean 69.1 (±2.2) ng/mL, vs. normal weight children, mean 75.6 (±2.5) ng/mL (P=0.03). The mean ucOC levels of 7.9 (±0.4) ng/mL in overweight children did not differ vs. normal weight children, mean level 7.0 (±0.4) ng/mL, (P=0.067). None of the three OC forms correlated with any of the measured parameters. ConclusionsThe cOC levels were lower in overweight children. There was no correlation between the three OC forms and any of the measured anthropometric or metabolic parameters. OC has been suggested to have a possible metabolic role, but in general the current study in prepubertal children does not support the hypothesis of an association between OC and a positive metabolic profile.

Family with sequence similarity member 20C is the primary but not the only kinase for the small-integrin-binding ligand N-linked glycoproteins in bone.

Recent studies have identified family with sequence similarity member 20C (FAM20C) as a kinase that phosphorylates the Ser in Ser-X-Glu/phospho-Ser (pSer) motifs in the small-integrin-binding ligand N-linked glycoproteins (SIBLINGs). There is no in vivo evidence that validates this finding, and it is unclear whether FAM20C is the only kinase for SIBLINGs. We extracted bone noncollagenous proteins (NCPs) from Fam20C-knockout (KO) mice and analyzed the phosphorylation levels. The total NCPs were separated into osteopontin-, bone sialoprotein-, and dentin matrix protein-1-enriched fractions by anion-exchange chromatography and analyzed by SDS-PAGE, native PAGE, and Western immunoblot analysis. The NCP phosphorylation level in the KO mice was lower than that in the wild-type (WT). On the native gel, the SIBLINGs from KO mice showed a lower migration rate (Mr) than those from the WT. Calf intestine phosphatase treatment shifted SIBLINGs from the WT mice to the level adjacent to the KO, but failed to shift the latter, suggesting a phosphorylation loss of SIBLINGs in the KO mice. Mass spectrometry identified less pSers in the SIBLINGs from the KO mice [including the region of the acidic Ser- and aspartate-rich motif (ASARM) peptides]. In an intriguing finding, several pSers in the Ser-X-Glu motifs in the KO mice maintained their phosphorylation, whereas several others in non-Ser-X-Glu motifs did not. Phospho-Tyrs and phospho-Thrs in the SIBLINGs did not appear to be associated with FAM20C. Our results indicate that FAM20C is the primary, but not the only, kinase for the SIBLINGs.-Yang, X., Yan, W., Tian, Y., Ma, P., Opperman, L. A., Wang, X. Family with sequence similarity member 20C is the primary but not the only kinase for the small-integrin-binding ligand N-linked glycoproteins in bone.