CNTs-CaP/chitosan-coated AZ91D magnesium alloy extract promoted rat dorsal root ganglia neuron growth via activating ERK signalling pathway.

Increasing attention has been paid on the application of biodegradable materials such as magnesium and its alloys in neuron repair. AZ91D magnesium alloy coated with carbon nanotubes (CNTs) and/or calcium phosphate (CaP)/chitosan (CS) was fabricated in this study. To evaluate the bioactivity of these AZ91D-based composites, the extracts were prepared by immersing samples in modified simulated body fluid (m-SBF) for 0, 2, 8, 16, 24, 34, 44, 60, or 90 days. Immunofluorescence staining for neuronal class III ß-tubulin (TUJ1) revealed that both CNTs-CaP/CS-AZ91D and CaP/CS-AZ91D extracts promoted axon outgrowth of dorsal root ganglia (DRG) neurons, accompanied with increased expression of phosphorylated focal adhesion kinase (p-FAK) and growth associated protein-43 (GAP-43). Besides, the extracts increased the expression and the release of neurotrophic factors including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). ERK signalling was activated in DRG neurons after treating with either CNTs-CaP/CS-AZ91D or CaP/CS-AZ91D extracts, and its inhibition with U0126 counteracted the beneficial effects of these extracts on DRG neuron. Overall, the extracts from these AZ91D-based composites might promote DRG neuron growth via activating ERK signalling pathway. Notably, CNTs-CaP/CS-AZ91D extracts showed a better promoting effect on neuron growth than CaP/CS-AZ91D. Assessment of ion elements showed that the addition of CNTs coating enhanced magnesium corrosion resistance and reduced the deposition of calcium and phosphorus on the surface of CaP/CS-AZ91D alloy. These findings demonstrate that CNTs-CaP/CS-AZ91D likely provide a more suitable environment for neuron growth, which suggests a potential implantable biomaterial for the treatment of nerve injury. SIGNIFICANCE: AZ91D magnesium alloy coated with carbon nanotubes (CNTs) and/or calcium phosphate (CaP)/chitosan (CS) was fabricated and their immersion extracts were prepared using modified simulated body fluid in this study. Both extracts from CNTs-CaP/CS and CaP/CS-coated AZ91D magnesium alloy promotes rat dorsal root ganglia (DRG) neuron growth via activating ERK signalling pathway. Notably, the addition of CNTs improves the performance of CaP/CS-AZ91D. For the first time, our research demonstrates that CNTs-CaP/CS-AZ91D likely provide a suitable environment for neuron growth, suggesting these AZ91D-based composites as potential implantable biomaterials for the treatment of nerve injury.

A new approach to removing and recovering phosphorus from livestock wastewater using dolomite.

Recovering phosphorus from livestock wastewater could partly mitigate the global phosphorus resource crisis. Crystallization is a promising method for removing phosphorus from wastewater, but the costs of calcium- and magnesium-containing reagents are increasing. Cheap, available, efficient materials are required to replace conventional calcium and magnesium reagents. Here, we describe a new approach to removing and recovering phosphorus from livestock wastewater of a large pig farm, containing a high phosphorus concentration. The effects of the pH, stirring speed, stirring time, and extract dose (containing calcium and magnesium) on phosphorus removal from livestock wastewater were investigated. The product was characterized by X-ray diffractometry, Fourier-transform infrared spectroscopy, and scanning electron microscopy. Under optimized conditions (pH 9.0, stirring speed 200 r/m, stirring time 600 s, Ca 207.62 mg/L, Mg 122.86 mg/L), 92% of the phosphorus was removed from livestock wastewater. The product was mainly the hydroxyapatite (Ca5(PO4)3OH) precursor amorphous calcium phosphate but also contained 1.65% (by mass) magnesium ammonium phosphate (MgNH4PO4·6H2O) crystals. The cost of dolomite to treat 1 m3 of high-phosphorus wastewater was 0.20 yuan (45.9%, 25.9%, and 75.9% lower than for pure MgCl2, MgSO4, and CaCl2, respectively) in 2019. Using dolomite to provide calcium and magnesium effectively decreases the crystallization process cost and should encourage the use of crystallization to remove phosphorus from wastewater.

Comprehensive organic profiling of biological particles derived from blood.

Mineral nanoparticles form in physiological and pathological processes occurring in the human body. The calcium phosphate mineral phase of the particles has affinity for proteins and lipids, but the complete profiling of the organic molecules that bind to the particles has not been described in detail. We report here a comprehensive analysis of organic components found in mineralo-organic particles derived from body fluids. Based on biological staining, fluorescent tagging, proteomics and metabolomics, our results indicate that the mineral particles bind to proteins, amino acids, carbohydrates, polysaccharides, phospholipids, fatty acids, DNA and low molecular weight metabolites. These results can be used to study the formation and effects of mineralo-organic particles in biological fluids.

Phosphate-water interplay tunes amorphous calcium carbonate metastability: spontaneous phase separation and crystallization vs stabilization viewed by solid state NMR.

Organisms tune the metastability of amorphous calcium carbonates (ACC), often by incorporation of additives such as phosphate ions and water molecules, to serve diverse functions, such as modulating the availability of calcium reserves or constructing complex skeletal scaffolds. Although the effect of additive distribution on ACC was noted for several biogenic and synthetic systems, the molecular mechanisms by which additives govern ACC stability are not well understood. By precipitating ACC in the presence of different PO4(3-) concentrations and regulating the initial water content, we identify conditions yielding either kinetically locked or spontaneously transforming coprecipitates. Solid state NMR, supported by FTIR, XRD, and electron microscopy, define the interactions of phosphate and water within the initial amorphous matrix, showing that initially the coprecipitates are homogeneous molecular dispersions of structural water and phosphate in ACC, and a small fraction of P-rich phases. Monitoring the transformations of the homogeneous phase shows that PO4(3-) and waters are extracted first, and they phase separate, leading to solid-solid transformation of ACC to calcite; small part of ACC forms vaterite that subsequently converts to calcite. The simultaneous water-PO4(3-) extraction is the key for the subsequent water-mediated accumulation and crystallization of hydroxyapatite (HAp) and carbonated hydroxyapatite. The thermodynamic driving force for the transformations is calcite crystallization, yet it is gated by specific combinations of water-phosphate levels in the initial amorphous coprecipitates. The molecular details of the spontaneously transforming ACC and of the stabilized ACC modulated by phosphate and water at ambient conditions, provide insight into biogenic and biomimetic pathways.

Obtaining Ca(H2PO4)(2)·H2O, monocalcium phosphate monohydrate, via monetite from brushite by using sonication.

Brushite was synthesized by precipitation of calcium chloride (CaCl(2)) and sodium phosphate monobasic (Na(2)HPO(4)) dried in vacuum and monetite was obtained from this brushite by sonication with a frequency of 90kHz at 500W for 90min. Monetite itself was also transformed in Ca(H(2)PO(4))(2)·H(2)O, monocalcium phosphate monohydrate (MCPM), by sonication with a frequency of 90kHz at 500W for 60min followed by lyophilization. The MCPM was sonicated and lyophilized by three times more until reach over 240min, but any other phase transformation was observed. All these phase transformations were analyzed by X-ray diffraction (XRD) and infrared spectroscopy (FTIR). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) indicated a grain size of about 200nm in all the samples. The morphology observed was a corn-flake-like grain for brushite, a pseudo-needle-like grains for monetite, and lamellar-like grains for MCPM.

Removal of phosphate from solution by adsorption and precipitation of calcium phosphate onto monohydrocalcite.

The sorption behavior and mechanism of phosphate on monohydrocalcite (CaCO(3)·H(2)O: MHC) were examined using batch sorption experiments as a function of phosphate concentrations, ionic strengths, temperatures, and reaction times. The mode of PO(4) sorption is divisible into three processes depending on the phosphate loading. At low phosphate concentrations, phosphate is removed by coprecipitation of phosphate during the transformation of MHC to calcite. The sorption mode at the low-to-moderate phosphate concentrations is most likely an adsorption process because the sorption isotherm at the conditions can be fitted reasonably with the Langmuir equation. The rapid sorption kinetics at the conditions is also consistent with the adsorption reaction. The adsorption of phosphate on MHC depends strongly on ionic strength, but slightly on temperature. The maximum adsorption capacities of MHC obtained from the regression of the experimental data to the Langmuir equation are higher than those reported for stable calcium carbonate (calcite or aragonite) in any conditions. At high phosphate concentrations, the amount of sorption deviates from the Langmuir isotherm, which can fit the low-to-moderate phosphate concentrations. Speciation-saturation analyses of the reacted solutions at the conditions indicated that the solution compositions which deviate from the Langmuir equation are supersaturated with respect to a certain calcium phosphate. The obtained calcium phosphate is most likely amorphous calcium phosphate (Ca(3)(PO(4))(2)·xH(2)O). The formation of the calcium phosphate depends strongly on ionic strength, temperature, and reaction times. The solubility of MHC is higher than calcite and aragonite because of its metastability. Therefore, the higher solubility of MHC facilitates the formation of the calcium phosphates more than with calcite and aragonite.

Proteomic identification of a novel hsp90-containing protein-mineral complex which can be induced in cells in response to massive calcium influx.

Fetuin-A is known for limiting the expansion and formation of hydroxyapatite crystals from calcium phosphate aggregates in circulation by forming a soluble fetuin-mineral complex. This study was aimed to uncover potential proteins involved in the regulation of calcium phosphate precipitation within cells. We found that a novel protein-mineral complex (PMC) can be generated after introduction of calcium chloride and sodium phosphate into the porcine brain protein extract prepared in Tris-HCl buffer. Selectively enriched proteins in the pellet were confirmed by immunoblotting, including heat shock protein 90 (Hsp90), annexin A5, calreticulin, nucleolin, and other proteins. In addition, purified native Hsp90 directly bound both amorphous calcium phosphate and hydroxyapatite and underwent conformational changes and oligomerization in the presence of excess calcium and phosphate. The morphology of the PMC prepared from Hsp90, calcium, and phosphate was distinctly different from that of hydroxyapatite under transmission electron microscope observation. When cultured SiHa cells were treated with a calcium ionophore or damaged by scratch to induce the massive calcium influx, a complex was formed and observed at discrete sites near the plasma membrane as revealed by antibodies against Hsp90, annexin A5, calreticulin, nucleolin, and other proteins. This complex could also be probed in situ with fetuin-A suggesting the existence of calcium phosphate aggregates in this complex. Inhibition of the complex formation by bisphosphonates hindered cell recovery from A23187 assault. Our results show that following membrane damage amorphous calcium phosphate develops at sites near membrane rupture where saturated calcium phosphate concentration is achieved. As a result, Hsp90 and other proteins are recruited, and the cytosolic PMC is formed. Inhibition of the cytosolic PMC formation may in part contribute to the cellular toxicity and in vivo side effects of bisphosphonates, particularly in cells prone to membrane damage under physiological conditions such as gastrointestinal epithelial and oral cavity epithelial cells.

Highly sinterable calcium phosphate fabricated by using starfish bone.

Nano-sized calcium phosphate powders were simply synthesized by using dried starfish bone. The calcined bone was mixed with phosphoric acid and the dried mixtures were heated for synthesis. The powder compacts were fully sintered at 1100 degrees C for 1 h. The densified samples showed CaO-free calcium phosphate phase and non-uniform, over sized grains.

Detection of basic calcium phosphate crystals in osteoarthritis.

Osteoarthritis (OA) is the most common human joint disorder. Its complex pathogenesis remains poorly understood but appears multifactorial. To date, no specific pharmacological agent has been identified to alter the disease course of OA. Calcification of articular cartilage is a hallmark of OA and evidence suggests it contributes directly to joint degeneration. Calcium crystals are frequently found in OA synovial fluid but their exact role in the disease process is unclear. Basic calcium phosphate (BCP) crystals are the predominant crystal type found in OA and recent data indicate a pathogenic role for these crystals in OA. However, information on the exact frequency and distribution of BCP crystals vary considerably, mainly due to the lack of simple and reliable methods of detection. The purpose of this review is to describe the current available methods for detecting BCP crystals and to highlight their obvious advantages and limitations. Recent developments in the field are also discussed with particular reference to their potential clinical applicability. Improved methods of detection for BCP crystals could potentially aid the diagnosis of OA and the development of novel therapies.

Thermal preparation of highly porous calcium phosphate bone filler derived from marine algae.

A sustainable marine-derived bioceramic with a unique porous structure has been developed for hard tissue repair. The conversion of alga was achieved through a novel technique, involving well controlled thermal processing followed by low pressure-temperature hydrothermal synthesis. In its preparation, a heat treatment step was required to remove the organic compounds from the algae, which reinforces the mineralised matrices. Its removal is necessary to prevent issue such as immune biocompatibility and ensure phase purity of the resultant biomaterial. This paper investigates the hydrothermal technique used for the transformation of mineralised red algae to hydroxyapatite that preserves the algae's unique structure. It specifically focuses on the effects of heat treatment on the morphology of the algae, TGA, SEM and hot stage XRD to quantity the changes.

Impact of anti-scalant on fouling of reverse osmosis membranes in reclamation of secondary effluent.

The objective of the study was to evaluate the impact of anti-scalant on fouling of reverse osmosis (RO) membranes in reclamation of secondary effluent which was produced by a conventional activated sludge process at Kranji Water Reclamation Plant with the capacity of 151,000 m3/d. The study was carried out using a RO pilot plant with the capacity of 2.4 m3/h. The RO plant was in 2:1 configuration and was operated at 75% recovery and at membrane flux of 17 l m(-2) h(-1). Pilot trials were conducted with and without anti-scalant. Compositions of feed and concentrate streams were analyzed and the pilot data were normalized. The results of the study showed that the plant operation was stable during the first few days after stopping dosage of anti-scalant but after 3-6 days of operation the membranes were fouled. The time lag effect of anti-scalant without dosage was not reported previously and could be potentially beneficial to save chemicals. The membrane fouling was more serious at the second stage due to the formation of calcium phosphate scale when the pilot plant was operated without anti-scalant. The flux of fouled membranes could be completely recovered after clean-in-place (CIP) with citric acid, indicating that scaling dominated the fouling of the RO membranes. These findings in the study could be applied to select an appropriate anti-scalant for prevention from formation of calcium phosphate scale in the RO operation.

The preparation, cytocompatibility, and in vitro biodegradation study of pure beta-TCP on magnesium.

Biodegradable and bioactive beta-tricalcium phosphate (beta-TCP) coatings were prepared on magnesium (Mg) in order to improve its biocompatibility by a chemical method. The tensile bonding strength of beta-TCP coating and Mg substrate was measured by the standard adhesion test (ISO 13779-4). And the cytocompatibility of beta-TCP coated Mg was studied by using human osteoblast-like MG63 cells. It was found that the MG63 cells could grow well on the surface of beta-TCP coated Mg and the cell viability on beta-TCP coated Mg was above 80% during the cocultivation of MG63 cells and beta-TCP coated Mg for 10 days, indicating no cytotoxicity. It was concluded that the beta-TCP coated Mg had good cytocompatibility. The degradation of Mg substrate with beta-TCP coating in vitro was studied in detail by XRD, EDX, SEM, and ICP. The results showed that a bone-like apatite continually formed on the surface of the sample with the degradation of both Mg substrate and beta-TCP coating in Hank's solution (a simulated body fluid). The biodegradation mechanism was preliminarily analyzed in the paper.

Isolation of calcium phosphate crystals from complex biological fluids using bisphosphonate-modified superparamagnetic beads.

The surface of superparamagnetic magnetic beads was modified with bisphosphonates to selectively capture calcium phosphate crystals from complex biological fluids (i.e. synovial fluid).

Calcinosis cutis universalis in a patient with systemic lupus erythematosus.

Deposition of calcium salts in the skin and subcutaneous tissue occurs in a variety of rheumatic diseases, being most commonly associated with scleroderma, CREST (calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly, and telangiectasia), dermatomyositis, and overlap syndromes but is a rare complication of systemic lupus erythematosus (SLE). Calcinosis is classified into four subsets: dystrophic, metastatic, idiopathic, or calciphylaxis/iatrogenic. The pathophysiology of calcinosis cutis remains unclear. Our patient developed extensive areas of calcifications in the trunk and extremities (calcinosis universalis) 8 years after SLE diagnosis, which would correspond to a form of dystrophic calcification. No response was observed after treatment with oral diltiazem for 3 months. We review the literature on the pathogenesis and prevalence of calcinosis universalis in SLE.

Histological assessment in grafts of highly purified beta-tricalcium phosphate (OSferion) in human bones.

Prominent osteoconductive activity and the biodegradable nature of commercially available beta-tricalcium phosphate (beta-TCP, OSferion) have been documented in animal experiments. We analyzed four cases of involving grafted OSferion in human bone with respect to histological features by routine hematoxylin and eosin staining, silver impregnation, immunohistochemistry and in situ hybridization. OSferion affords early bioresorption by osteoclasts, vascular invasion of macropores and osteoblastic cell attachment on the surface on the ceramic surface 14 days after grafting. Prominent bone formation and direct bone connection between preexisting bone and OSferion were evident 28 days after grafting. Nearly the entire TCP surface was covered by lamellar bone; additionally, active osteoblastic lining and attachment of the osteoclast-like giant cells were not observed 72 weeks after grafting. Silver impregnation revealed the presence of collagen fibrils within probable micropores of OSferion.

Free-standing nanogold membranes as supports for the growth of calcium phosphate crystals.

Current strategies for bone tissue regeneration focus on the development of implantable matrices that mimic biological tissues. Inorganic composites are of special interest for bone substitute applications. It is necessary to create an artificial three-dimensional scaffold-like porous material with certain geometrical structure to induce bone growth. We report here the growth of calcium phosphate crystals on free-standing carboxylic acid functionalized gold nanoparticle membranes. The gold nanoparticle membrane is synthesized by the spontaneous reduction of aqueous chloroaurate ions by a diamine molecule at a liquid-liquid interface. This membrane is robust and malleable, and most importantly, the gold nanoparticles in the membrane may be functionalized with suitable ligands. In this study, the amino acids aspartic acid and cysteine together with an aromatic bifunctional molecule, anthranilic acid, were used to modify the surface of the gold nanoparticles in the membrane. The free carboxylic acid groups on the gold nanoparticles further to functionalization with these molecules were then used to bind Ca(2+) ions and reacted with phosphate ions to yield calcium phosphate. The nature of the nanogold surface modifier directed the formation of either crystalline hydroxyapatite or amorphous calcium phosphate. The nanogold membrane thus suggests potential biomedical application as biocompatible implants and grafts.

Bone formation and resorption of highly purified beta-tricalcium phosphate in the rat femoral condyle.

The aim of this study was to examine the chronological histology associated with highly purified beta-tricalcium phosphate (beta-TCP) implanted in the rat femoral condyle. Specimens were harvested on days 4, 7, 14, 28 and 56 after implantation, and were analyzed by tartrate-resistant acid phosphatase (TRAP) staining, immunohistochemistry of the ED1 protein as a marker of the phagocyte system, and in situ hybridization with digoxigenin-labeled alpha1 chain of type I procollagen (COL1A1), osteopontin and osteocalcin. beta-TCP was resorbed in a chronological manner. Although new bone was not observed on day 4, fibroblast-like cells around beta-TCP were positive for COL1A1 and osteopontin mRNA. New bone formation presented after day 7. In the double-staining for OPN and ED1 on day 7, most cells around beta-TCP were positive for either osteopontin mRNA or ED1 protein. However, there were some doubly positive multinucleated cells, suggesting that they belonged to the mononuclear phagocyte system. After day 28, the implanted region was replaced with bone marrow. Multinucleated TRAP-positive and ED1-positive cells which adhered to beta-TCP at all stages seemed to be osteoclasts and they continuously resorbed beta-TCP. beta-TCP has a good biocompatibility since both bioresorption and bone formation started at an early stage after implantation.

Apatite formation in the reaction-setting mixture of Ca(OH)2--KH2PO4 system.

Development of a new calcium phosphate cement (CPC) system as an alternative to that commonly used, basically consisting of tetracalcium phosphate and dicalcium phosphate self-setting mixtures, could be of interest in achieving special properties of the product. Powder mixtures of Ca(OH)(2) and KH(2)PO(4) were studied to assess their potential for the precipitation of apatite-like phase with the use of potassium phosphate salt solution as the cement liquid. X-ray diffraction and infrared (IR) spectroscopy studies and pH and setting time measurements were performed. The set cement was revealed to consist of a low crystalline carbonate-substituted apatite-like phase. The setting time of the cement was about 5 min. Its dissolution in distilled water led to an increase in solution pH to about 11.5, the pH slowly decreasing to 10.2 at day 10. The results showed the cement to be of an increased carbonate substitution ability compared to the tetracalcium phosphate-dicalcium phosphate anhydrous cement.

An equilibrium thermodynamic model of the sequestration of calcium phosphate by casein phosphopeptides.

Sequestration of calcium phosphate by caseins occurs in the Golgi region of mammary secretory cells during lactation, where it helps to prevent calcification of the gland and to deliver high concentrations of calcium and phosphate to the neonate in the form of milk. Calcium phosphate nanoclusters are formed when a core of amorphous calcium phosphate is sequestered within a shell of casein or casein phosphopeptides. The nanoclusters can form spontaneously from a supersaturated solution or by dispersion of a precipitate of calcium phosphate, demonstrating that they are thermodynamically stable complexes. The average size and chemical composition of the complexes are largely independent of the solution conditions (pH, temperature, peptide concentration, salt composition and rate of reaction) under which they form. Larger, metastable, colloidal particles can form if there is not enough of the phosphopeptide to sequester all the calcium phosphate, or, transiently, if the salt and peptide solutions are mixed together without sufficient care. A thermodynamic model of the sequestration process is presented which makes use of an invariant ion activity product observed in nanocluster-containing solutions. In any given solution that has thermodynamic stability, the extent of the sequestration reaction can be calculated from the empirical formula of the nanoclusters using the criterion that the solution should have the equilibrium value of the invariant ion activity product. Other members of the paralogous group of secretory calcium-binding phosphoproteins to which caseins belong may also be able to sequester calcium phosphate in biological fluids such as saliva and in the extracellular matrix of mineralizing tissues.

An equilibrium thermodynamic model of the sequestration of calcium phosphate by casein micelles and its application to the calculation of the partition of salts in milk.

An equilibrium thermodynamic model of the interaction of calcium, phosphate and casein in milk is described in which the micellar calcium phosphate is assumed to be in the form of calcium phosphate nanoclusters. A generalized empirical formula for the nanocluster is used to define the molar ratios of small ions (Ca, Mg, P(i) and citrate) to a casein phosphorylated sequence (phosphate centre, PC). From this model, a method of calculating the partition of milk salts into diffusible and non-diffusible fractions is obtained. No arbitrary assumptions are made, no fitting of adjustable parameters is done and the PCs in the caseins are defined by inspection of their primary structures. In addition to the salt partition, the mole fractions of the individual caseins not complexed to the calcium phosphate through one or more of their PCs are computed and a generic stability rule for milks is derived. The use of the model is illustrated by calculations of the partition of salts in a standard milk and by comparison with experimental data on the partition of salts in the milk of individual cows. The generic stability rule is applied to the individual milks to determine whether the micellar calcium phosphate is thermodynamically stable. According to the calculations, compositions that might lead to pathological calcification in the lumen of the mammary gland were seldom found in primiparous healthy cows in early or mid lactation but occurred more often in multiparous animals, in late lactation and during mastitic infection.