Mud in the blood: the role of protein-mineral complexes and extracellular vesicles in biomineralisation and calcification.

Protein-mineral interaction is known to regulate biomineral stability and morphology. We hypothesise that fluid phases produce highly dynamic protein-mineral complexes involved in physiology and pathology of biomineralisation. Here, we specifically focus on calciprotein particles, complexes of vertebrate mineral-binding proteins and calcium phosphate present in the systemic circulation and abundant in extracellular fluids - hence the designation of the ensuing protein-mineral complexes as "mud in the blood". These complexes exist amongst other extracellular particles that we collectively refer to as "the particle zoo".

Correction: Li, P. et al. Mechanical Characteristics, In Vitro Degradation, Cytotoxicity, and Antibacterial Evaluation of Zn-4.0Ag Alloy as a Biodegradable Material. Int. J. Mol. Sci. 2018, 19, 755.

The authors wish to make the following corrections to this paper [...].

Prothrombin Loading of Vascular Smooth Muscle Cell-Derived Exosomes Regulates Coagulation and Calcification.

OBJECTIVE: The drug warfarin blocks carboxylation of vitamin K-dependent proteins and acts as an anticoagulant and an accelerant of vascular calcification. The calcification inhibitor MGP (matrix Gla [carboxyglutamic acid] protein), produced by vascular smooth muscle cells (VSMCs), is a key target of warfarin action in promoting calcification; however, it remains unclear whether proteins in the coagulation cascade also play a role in calcification. APPROACH AND RESULTS: Vascular calcification is initiated by exosomes, and proteomic analysis revealed that VSMC exosomes are loaded with Gla-containing coagulation factors: IX and X, PT (prothrombin), and proteins C and S. Tracing of Alexa488-labeled PT showed that exosome loading occurs by direct binding to externalized phosphatidylserine (PS) on the exosomal surface and by endocytosis and recycling via late endosomes/multivesicular bodies. Notably, the PT Gla domain and a synthetic Gla domain peptide inhibited exosome-mediated VSMC calcification by preventing nucleation site formation on the exosomal surface. PT was deposited in the calcified vasculature, and there was a negative correlation between vascular calcification and the levels of circulating PT. In addition, we found that VSMC exosomes induced thrombogenesis in a tissue factor-dependent and PS-dependent manner. CONCLUSIONS: Gamma-carboxylated coagulation proteins are potent inhibitors of vascular calcification suggesting warfarin action on these factors also contributes to accelerated calcification in patients receiving this drug. VSMC exosomes link calcification and coagulation acting as novel activators of the extrinsic coagulation pathway and inducers of calcification in the absence of Gla-containing inhibitors.

The Calamistrum of the Feather-Legged Spider Uloborus plumipes Investigated by Focused Ion Beam and Scanning Electron Microscopy (FIB-SEM) Tomography.

Spiders are natural specialists in fiber processing. In particular, cribellate spiders manifest this ability as they produce a wool of nanofibers to capture prey. During its production they deploy a sophisticated movement of their spinnerets to darn in the fibers as well as a comb-like row of setae, termed calamistrum, on the metatarsus which plays a key role in nanofiber processing. In comparison to the elaborate nanofiber extraction and handling process by the spider's calamistrum, the human endeavors of spinning and handling of artificial nanofibers is still a primitive technical process. An implementation of biomimetics in spinning technology could lead to new materials and applications. Despite the general progress in related fields of nanoscience, the expected leap forward in spinning technology depends on a better understanding of the specific shapes and surfaces that control the forces at the nanoscale and that are involved in the mechanical processing of the nanofibers, respectively. In this study, the authors investigated the morphology of the calamistrum of the cribellate spider Uloborus plumipes. Focused ion beam and scanning electron microscopy tomography provided a good image contrast and the best trade-off between investigation volume and spatial resolution. A comprehensive three-dimensional model is presented and the putative role of the calamistrum in nanofiber processing is discussed.

Mechanical Characteristics, In Vitro Degradation, Cytotoxicity, and Antibacterial Evaluation of Zn-4.0Ag Alloy as a Biodegradable Material.

Zn-based biodegradable metallic materials have been regarded as new potential biomaterials for use as biodegradable implants, mainly because of the ideal degradation rate compared with those of Mg-based alloys and Fe-based alloys. In this study, we developed and investigated a novel Zn-4 wt % Ag alloy as a potential biodegradable metal. A thermomechanical treatment was applied to refine the microstructure and, consequently, to improve the mechanical properties, compared to pure Zn. The yield strength (YS), ultimate tensile strength (UTS) and elongation of the Zn-4Ag alloy are 157 MPa, 261 MPa, and 37%, respectively. The corrosion rate of Zn-4Ag calculated from released Zn ions in DMEM extracts is approximately 10.75 ± 0.16 µg cm-2 day-1, which is higher than that of pure Zn [corrected]. In vitro cytotoxicity tests showed that the Zn-4Ag alloy exhibits acceptable toxicity to L929 and Saos-2 cells, and could effectively inhibit initial bacteria adhesion. This study shows that the Zn-4Ag exhibits excellent mechanical properties, predictable degradation behavior, acceptable biocompatibility, and effective antibacterial properties, which make it a candidate biodegradable material.

Clearance of fetuin-A--containing calciprotein particles is mediated by scavenger receptor-A.

RATIONALE: Fetuin-A is a liver-derived plasma protein involved in the regulation of calcified matrix metabolism. Biochemical studies showed that fetuin-A is essential for the formation of protein-mineral complexes, called calciprotein particles (CPPs). CPPs must be cleared from circulation to prevent local deposition and pathological calcification. OBJECTIVE: We studied CPP clearance in mice and in cell culture to identify the tissues, cells, and receptors involved in the clearance. METHODS AND RESULTS: In mice, fetuin-A-containing CPPs were rapidly cleared by the reticuloendothelial system, namely Kupffer cells of the liver and marginal zone macrophages of the spleen. Macrophages from scavenger receptor-AI/II (SR-A)-deficient mice cleared CPPs less efficiently than macrophages from wild-type mice, suggesting that SR-AI/II is involved in CPP binding and endocytosis. Accordingly, we found reduced clearance of CPPs in SR-A/MARCO-deficient mice. CONCLUSIONS: We could demonstrate that fetuin-A-containing CPPs facilitate the clearance of mineral debris by macrophages via SR-A. Since the same receptor also contributes to the uptake of modified low-density lipoprotein particles in atherosclerosis, defective endocytosis of both types of particle may impinge on lipid as well as mineral debris clearance in calcifying atherosclerosis.

Fetuin-A regulation of calcified matrix metabolism.

The final step of biomineralization is a chemical precipitation reaction that occurs spontaneously in supersaturated or metastable salt solutions. Genetic programs direct precursor cells into a mineralization-competent state in physiological bone formation (osteogenesis) and in pathological mineralization (ectopic mineralization or calcification). Therefore, all tissues not meant to mineralize must be actively protected against chance precipitation of mineral. Fetuin-A is a liver-derived blood protein that acts as a potent inhibitor of ectopic mineralization. Monomeric fetuin-A protein binds small clusters of calcium and phosphate. This interaction results in the formation of prenucleation cluster-laden fetuin-A monomers, calciprotein monomers, and considerably larger aggregates of protein and mineral calciprotein particles. Both monomeric and aggregate forms of fetuin-A mineral accrue acidic plasma protein including albumin, thus stabilizing supersaturated and metastable mineral ion solutions as colloids. Hence, fetuin-A is a mineral carrier protein and a systemic inhibitor of pathological mineralization complementing local inhibitors that act in a cell-restricted or tissue-restricted fashion. Fetuin-A deficiency is associated with soft tissue calcification in mice and humans.

Development, Processing and Aging of Novel Zn-Ag-Cu Based Biodegradable Alloys.

The use of biodegradable materials for implants is a promising strategy to overcome known long-term clinical complications related to permanent implants. Ideally, biodegradable implants support the damaged tissue for a certain period and then degrade, while the physiological function of the surrounding tissue is restored. Although Mg-based alloys nearly ideally lend themselves to biodegradable implants, a few critical shortcomings promoted the development of alternative alloy systems. Due to their reasonably good biocompatibility, moderate corrosion rate without hydrogen evolution and adequate mechanical properties, increasing attention has been paid to Zn alloys. In this work, precipitation-hardening alloys in the system Zn-Ag-Cu were developed relying on thermodynamic calculations. After casting the alloys, their microstructures were refined by thermomechanical treatment. The processing was tracked and directed, respectively, by routine investigations of the microstructure, associated with hardness assessments. Although microstructure refinement increased the hardness, the material proved to be susceptible to aging as the homologous temperature of zinc is at 0.43 Tm. Besides mechanical performance and corrosion rate, long-term mechanical stability is another crucial factor that must be taken into consideration to ensure the safety of the implant and thus requires a profound understanding of the aging process.

A red herring in vascular calcification: 'nanobacteria' are protein-mineral complexes involved in biomineralization.

Biomineralization at pathological extraosseous sites (i.e. vasculature and soft tissues) is associated with increased morbidity and mortality. So-called 'nanobacteria' have been described as pathogenic agents causing many diseases including calcification. Initially, their appearance, and having a content consisting of nucleic acids plus proteins and properties of growing structures, suggested that they were living organisms. However, it could be demonstrated that the so-called nanobacteria were in fact mineralizing nanoparticles that contain mineral and non-mineral compounds, that these particles bind to charged molecules and that supersaturation enables in vitro growth of these nanoparticles. Recent data indicate that nanoparticles consisting of protein-mineral complexes can be seen both in vitro and in vivo as precursors of matrix calcification.

Fetuin-A is a mineral carrier protein: small angle neutron scattering provides new insight on Fetuin-A controlled calcification inhibition.

Clinical studies and animal experiments have shown that the serum protein fetuin-A is a highly effective inhibitor of soft tissue calcification. This inhibition mechanism was elucidated on the basis of an in vitro fetuin-A-mineral model system. In a previous study, we found that in a two-stage process ∼100-nm sized calciprotein particles (CPPs) were formed whose final stage was stabilized by a compact outer fetuin-A monolayer against further growth. Quantitative small-angle neutron scattering data analysis revealed that even at a fetuin-A concentration close to the stability limit, only approximately one-half of the mineral ions and only 5% of the fetuin-A were contained in the CPPs. To uncover the interplay of the remaining supersaturated mineral ion fraction and of the 95% non-CPP fetuin-A, we explored the fetuin-A monomer fraction in solution by contrast variation small-angle neutron scattering. Our results suggest that the mineral ions coalesce to subnanometer-sized clusters, reminiscent of Posner clusters, which are stabilized by fetuin-A monomers. Hence, our experiments revealed a second mechanism of long-term mineral ion stabilization by the fetuin-A that is complementary to the formation of CPPs.

Response of human periosteal cells to degradation products of zinc and its alloy.

Zinc (Zn) and its alloys are proposed as promising resorbable materials for osteosynthesis implants. Detailed studies should be undertaken to clarify their properties in terms of degradability, biocompatibility and osteoinductivity. Degradation products of Zn alloys might affect directly adjacent cellular and tissue responses. Periosteal stem cells are responsible for participating in intramembranous ossification during fracture healing. The present study aims at examining possible effects emanating from Zn or Zn-4Ag (wt%) alloy degradation products on cell viability and osteogenic differentiation of a human immortalized cranial periosteal cell line (TAg cells). Therefore, a modified extraction method was used to investigate the degradation behavior of Zn and Zn-4Ag alloys under cell culture conditions. Compared with pure Zn, Zn-4Ag alloy showed almost fourfold higher degradation rates under cell culture conditions, while the associated degradation products had no adverse effects on cell viability. Osteogenic induction of TAg cells revealed that high concentration extracts significantly reduced calcium deposition of TAg cells, while low concentration extracts enhanced calcium deposition, indicating a dose-dependent effect of Zn ions. Our results give evidence that the observed cytotoxicity effects were determined by the released degradation products of Zn and Zn-4Ag alloys, rather than by degradation rates calculated by weight loss. Extracellular Zn ion concentration was found to modulate osteogenic differentiation of TAg cells. These findings provide significant implications and guidance for the development of Zn-based alloys with an optimized degradation behavior for Zn-based osteosynthesis implants.

A shielding topology stabilizes the early stage protein-mineral complexes of fetuin-A and calcium phosphate: a time-resolved small-angle X-ray study.

We report on the earliest stages of the formation of complexes of calcium phosphate in the presence of the serum protein alpha(2)-HS glycoprotein/fetuin-A termed calciprotein particles (CPPs). Time-resolved small-angle X-ray scattering (TR-SAXS) and stopped-flow analysis were used to monitor the growth of protein mineral particles nucleating from supersaturated salt solutions. It was found that fetuin-A did not influence the formation of mineral nuclei. However, fetuin-A did prevent the aggregation of nuclei and thus mineral precipitation. Hence, fetuin-A shielded spontaneously formed mineral nuclei, leading to stable calciprotein particles in the first stage of mineralization. Fetuin-A is therefore critically required during the earliest stages of the formation of protein-mineral complexes in order to prevent uncontrolled mineralization.

Selection of extraction medium influences cytotoxicity of zinc and its alloys.

Zinc (Zn) alloys have been considered as promising absorbable metals, mainly due to their moderate degradation rates ranging between magnesium alloys and iron alloys. The degradation behavior depends on the specific physiological environment. Released metallic ions and corrosion products directly influence biocompatibility. The initial contact of orthopedic implants or vascular stents after implantation will be with blood. In this study, fetal bovine serum (FBS) was used as a model system of blood components. We investigated the influence of FBS on in vitro degradation behavior and cytotoxicity of pure Zn, and Zn-4Ag and Zn-2Ag-1.8Au-0.2 V (wt%) alloys. The initial degradation rates in FBS were assessed and compared with the degradation and toxicity in four other common physiological model systems: DMEM cell culture medium ±â€¯FBS and McCoy's 5A medium ±â€¯FBS. Test samples in pure FBS showed the highest initial degradation rates, and accordingly, FBS supplemented media accelerated the degradation process as well. Moreover, an extract test according to ISO 10993-5 and -12 with L929 and Saos-2 cells was performed to investigate the role of FBS in the extraction medium. The cytotoxic effects observed in the tests were correlated with FBS-mediated Zn2+ release. These findings have significant implications regarding the selection of appropriate media for in vitro degradation and cytotoxicity evaluation of Zn and its alloys. STATEMENT OF SIGNIFICANCE: Metallic zinc and its alloys have been considered as promising biodegradable metals, mainly due to their moderate degradation rates. However, in vitro cytotoxicity tests according to the current ISO 10993 standard series are not suitable to predict biocompatibility of Zn alloys due to the inconsistent correlation between in vitro and in vitro biocompatibility. In this study, we show that the outcomes of standardized in vitro cytotoxicity tests of Zn and Zn alloys are influenced by fetal bovine serum in the extraction vehicle because FBS promotes Zn2+ release during the extraction process. The results of the study provide significant information for selection of appropriate model systems to evaluate in vitro degradation behavior and cytotoxicity.

Evaluation of a Zn-2Ag-1.8Au-0.2V Alloy for Absorbable Biocompatible Materials.

Zinc (Zn) and Zn-based alloys have been proposed as a new generation of absorbable metals mainly owing to the moderate degradation behavior of zinc between magnesium and iron. Nonetheless, mechanical strength of pure Zn is relatively poor, making it insufficient for the majority of clinical applications. In this study, a novel Zn-2Ag-1.8Au-0.2V (wt.%) alloy (Zn-Ag-Au-V) was fabricated and investigated for use as a potential absorbable biocompatible material. Microstructural characterization indicated an effective grain-refining effect on the Zn alloy after a thermomechanical treatment. Compared to pure Zn, the Zn-Ag-Au-V alloy showed significantly enhanced mechanical properties, with a yield strength of 168 MPa, an ultimate tensile strength of 233 MPa, and an elongation of 17%. Immersion test indicated that the degradation rate of the Zn-Ag-Au-V alloy in Dulbecco's phosphate buffered saline was approximately 7.34 ± 0.64 µm/year, thus being slightly lower than that of pure Zn. Biocompatibility tests with L929 and Saos-2 cells showed a moderate cytotoxicity, alloy extracts at 16.7%, and 10% concentration did not affect metabolic activity and cell proliferation. Plaque formation in vitro was reduced, the Zn-Ag-Au-V surface inhibited adhesion and biofilm formation by the early oral colonizer Streptococcus gordonii, indicating antibacterial properties of the alloy.

The serum protein alpha 2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification.

Ectopic calcification is a frequent complication of many degenerative diseases. Here we identify the serum protein alpha2-Heremans-Schmid glycoprotein (Ahsg, also known as fetuin-A) as an important inhibitor of ectopic calcification acting on the systemic level. Ahsg-deficient mice are phenotypically normal, but develop severe calcification of various organs on a mineral and vitamin D-rich diet and on a normal diet when the deficiency is combined with a DBA/2 genetic background. This phenotype is not associated with apparent changes in calcium and phosphate homeostasis, but with a decreased inhibitory activity of the Ahsg-deficient extracellular fluid on mineral formation. The same underlying principle may contribute to many calcifying disorders including calciphylaxis, a syndrome of severe systemic calcification in patients with chronic renal failure. Taken together, our data demonstrate a critical role of Ahsg as an inhibitor of unwanted mineralization and provide a novel therapeutic concept to prevent ectopic calcification accompanying various diseases.

Enhanced antibacterial activity of silver-ruthenium coated hollow microparticles.

The oxidation based antimicrobial activity of silver is long known. Microparticles with a particular silver-ruthenium coating and specific physical properties were developed. The coating showed a considerably increased silver ion release rate in comparison to a plain silver coating. Accordingly, an exposure of Escherichia coli and Staphylococcus aureus to these silver-ruthenium coated microparticles resulted in a time and concentration dependent cell killing. Even though contact killing may contribute to this efficacy, rather a release associated diffusion gradient dependent killing was observed. Moreover, cell killing did not involve lysis. The coated microparticles manifested no reduction in antibacterial activity for months. Due to their specific size and density, they sedimented slowly in aqueous solution, showed a low aggregation tendency, and could be recycled easily. Hence, these silver-ruthenium coated microparticles lend themselves to a wide range of antibacterial applications as they combine long-term stability and high efficacy with ease of use.

Tissue distribution and activity testing suggest a similar but not identical function of fetuin-B and fetuin-A.

Fetuins are serum proteins with diverse functions including the regulation of osteogenesis and inhibition of unwanted mineralization. Besides the alpha2-Heremans and Schmid glycoprotein/fetuin-A, the recently identified fetuin-B is a second member of the fetuin family [Olivier, Soury, Risler, Smih, Schneider, Lochner, Jouzeau, Fey and Salier (1999) Genomics 57, 352-364; Olivier, Soury, Ruminy, Husson, Parmentier, Daveau and Salier (2000) Biochem. J. 350, 589-597], which belongs to the cystatin superfamily. We compared the expressions of fetuin-B and fetuin-A at the RNA level and established that both genes are most highly expressed in liver tissue. Like fetuin-A, fetuin-B mRNA is also highly expressed in tongue and placenta tissues. We demonstrated for the first time that fetuin-B is also expressed at the protein level in sera and several organs of mouse, rat and human. We isolated contiguous genomic clones containing both fetuin-B and fetuin-A genes, indicating that these genes are closely linked at the genome level. The close proximity of both these genes may explain our observation that fetuin-B expression was decreased in fetuin-A-deficient mice. Unlike fetuin-A, the amount of fetuin-B protein in human serum varied with gender and was higher in females than in males. Functional analysis revealed that fetuin-B, similarly to fetuin-A, is an inhibitor of basic calcium phosphate precipitation, albeit less active when compared with fetuin-A. Therefore fetuin-B may have a function that is partly overlapping, if not identical, with the function of fetuin-A.

Crystallization of calcium oxalates is controlled by molecular hydrophilicity and specific polyanion-crystal interactions.

To gain more insight into protein structure-function relationships that govern ectopic biomineralization processes in kidney stone formation, we have studied the ability of urinary proteins (Tamm-Horsfall protein, osteopontin (OPN), prothrombin fragment 1 (PTF1), bikunin, lysozyme, albumin, fetuin-A), and model compounds (a bikunin fragment, recombinant-, milk-, bone osteopontin, poly-L-aspartic acid (poly asp), poly-L-glutamic acid (poly glu)) in modulating precipitation reactions of kidney stone-related calcium oxalate mono- and dihydrates (COM, COD). Combining scanning confocal microscopy and fluorescence imaging, we determined the crystal faces of COM with which these polypeptides interact; using scanning electron microscopy, we characterized their effects on crystal habits and precipitated volumes. Our findings demonstrate that polypeptide adsorption to COM crystals is dictated first by the polypeptide's affinity for the crystal followed by its preference for a crystal face: basic and relatively hydrophobic macromolecules show no adsorption, while acidic and more hydrophilic polypeptides adsorb either nonspecifically to all faces of COM or preferentially to {100}/{121} edges and {100} faces. However, investigating calcium oxalates grown in the presence of these polypeptides showed that some acidic proteins that adsorb to crystals do not affect crystallization, even if present in excess of physiological concentrations. These proteins (albumin, bikunin, PTF1, recombinant OPN) have estimated total hydrophilicities from 200 to 850 kJ/mol and net negative charges from -9 to -35, perhaps representing a "window" in which proteins adsorb and coat urinary crystals (support of excretion) without affecting crystallization. Strongest effects on crystallization were observed for polypeptides that are either highly hydrophilic (>950 kJ/mol) and highly carboxylated (poly asp, poly glu), or else highly hydrophilic and highly phosphorylated (native OPN isoforms), suggesting that highly hydrophilic proteins strongly affect precipitation processes in the urinary tract. Therefore, the level of hydrophilicity and net charge is a critical factor in the ability of polypeptides to affect crystallization and to regulate biomineralization processes.

Hierarchical role of fetuin-A and acidic serum proteins in the formation and stabilization of calcium phosphate particles.

The serum protein fetuin-A is a potent systemic inhibitor of soft tissue calcification. Fetuin-A is highly effective in the formation and stabilization of protein-mineral colloids, referred to as calciprotein particles (CPPs). These particles ripen in vitro in a two-step process, indicated by a morphological conversion from spheres to larger prolate ellipsoids. Using a combined light scattering and electron microscopic imaging approach we determined that the second-stage particles resulted from a highly anisotropic outgrowth of the first-stage particles. Electron microscopy of ascites fluid from a patient with calcifying peritonitis revealed particles reminiscent of secondary CPPs. Thus, CPPs form in the body and undergo the two-step ripening at least in pathological conditions. Unlike in vitro generated CPPs, ascites-derived CPPs contained little fetuin-A but large amounts of albumin. This prompted us to study the role of fetuin-A combined with other serum proteins in CPP formation. Fetuin-A was indispensable for primary CPP formation. Albumin and acidic proteins in general greatly enhanced the fetuin-A triggered formation of secondary CPPs and, thus, substituted substantial amounts of fetuin-A without loss of inhibition of calcium phosphate precipitation. Thus, direct mineral deposition from solute in the body is unlikely even at low fetuin-A serum levels as long as sufficient bulk acidic protein is available. Collectively fetuin-A and other acidic bulk plasma proteins may be considered as mineral chaperones mediating the stabilization, safe transport, and clearance in the body of calcium and phosphate as colloidal complexes, thus, preventing ectopic calcification.

Structural basis of calcification inhibition by alpha 2-HS glycoprotein/fetuin-A. Formation of colloidal calciprotein particles.

Genetic evidence from mutant mice suggests that alpha(2)-HS glycoprotein/fetuin-A (Ahsg) is a systemic inhibitor of precipitation of basic calcium phosphate preventing unwanted calcification. Using electron microscopy and dynamic light scattering, we demonstrate that precipitation inhibition by Ahsg is caused by the transient formation of soluble, colloidal spheres, containing Ahsg, calcium, and phosphate. These "calciprotein particles" of 30-150 nm in diameter are initially amorphous and soluble but turn progressively more crystalline and insoluble in a time- and temperature-dependent fashion. Solubilization in Ahsg-containing calciprotein particles provides a novel conceptual framework to explain how insoluble calcium precipitates may be transported and removed in the bodies of mammals. Mutational analysis showed that the basic calcium phosphate precipitation inhibition activity resides in the amino-terminal cystatin-like domain D1 of Ahsg. A structure-function analysis of wild type and mutant forms of cystatin-like domains from Ahsg, full-length fetuin-B, histidine-rich glycoprotein, and kininogen demonstrated that Ahsg domain D1 is most efficient in inhibiting basic calcium phosphate precipitation. The computer-modeled domain structures suggest that a dense array of acidic residues on an extended beta-sheet of the cystatin-like domain Ahsg-D1 mediates efficient inhibition.