Calcium Phosphate Precipitation Research Articles

From sewage sludge ash to a recycled feed phosphate – digestibility of precipitated calcium phosphate in broiler chickens and growing pigs

Today, EU is largely (∼92%) dependent on the import of phosphates as most mines are located outside Europe. Because of the limited availability, phosphorus (P) is included on the list of Critical Raw Materials. Precipitated calcium phosphate (PCP) recovered from sewage sludge ash is a novel and sustainable option to replace mined P as raw material in feed phosphates, e.g. monocalcium phosphate (MCP) or dicalcium phosphate, but the digestibility has not yet been tested in vivo. The aim was therefore to determine PCP and MCP apparent ileal digestibility (AID) of P in broiler chickens and apparent (ATTD) and true (TTTD) total tract digestibility of P in growing pigs. A chicken study comprised 240 Ross 308 chickens that were housed in groups of eight from day 21 to day 28. Five diets were used, a basal diet and two test diets, which contributed either 0.075% (low) or 0.150% (high) additional P for each of the test sources (MCP and PCP). The basal and test diets were composed to achieve increasing levels of P and AID was calculated with regression analysis. In the pig study, eight individually housed pigs were used in a change-over study with two experimental periods. The pigs were fed a basal P-free diet in a preperiod to be able to estimate endogenous P losses and then two different diets in two periods using a change-over design, where MCP and PCP were the only P source, providing in total 0.33 (basal diet), 4.42 (MCP) and 3.53 (PCP) g kg-1P, respectively. The AID of P in PCP and MCP for chickens was 58.4 and 75.1% (P = 0.166). The ATTD and TTTD of P in PCP for pigs were 58.4 and 67.2%, respectively, which was lower (P < 0.001) than the corresponding values for MCP (82.1 and 89.1%), respectively. The digestibility of calcium (Ca) did not differ in the chicken diets with high inclusion levels of PCP and MCP (54.7 and 55.3%, respectively, P = 0.535), but was lower for PCP than MCP in the pig study (57.8 and 70.8% respectively, P = 0.001). In conclusion, the digestibility of P in PCP for chickens did not differ from conventional MCP, whereas for pigs, it was lower, but could be a viable alternative to other common sources of P.

Impact of borosilicate bioactive glass scaffold processing and reactivity on in-vitro dissolution properties

In this study, bulk borosilicate glasses and 3D scaffolds (processed by the burn-off technique and by robocasting) were synthesized to investigate the impact of the manufacturing method, glass composition and preincubation time on in vitro dissolution and cell response. The studied compositions are based on commercial bioactive glass S53P4 (BonAlive) where 12.5% SiO2 has been replaced by B2O (labelled B12.5), and part of the CaO is replaced with MgO and SrO (labelled B12.5-Mg-Sr). First, the impact of the processing and glass composition, on the dissolution rate, was assessed. As expected, scaffolds were found to exhibit faster dissolution, due to the increased surface area, when compared to the bulk glass. Furthermore, the 3D printed scaffolds were found to dissolve faster than the burn-off scaffolds. Moreover, scaffolds made from B12.5-Mg-Sr glass composition exhibited slower ion release and precipitation of calcium phosphate (CaP) layer, when compared to B12.5, due to the stabilizing effect of Mg and Sr. Finally, dynamic condition produces lower ion releases that static condition and could be more optimal for in vitro cell growth. Secondly, in culture with murine MC3T3-E1 cells, it was shown that 3 days preincubation would be optimal to decrease the burst of ions that is known to lead to cell death. However, it was found that MC3T3-E1 survived and proliferated only in presence of B12.5-Mg-Sr scaffolds. Finally, it was shown that despite scaffolds having different porosities, they had no significant difference on human adipose-derived stem cells (hADSCs) survival. This manuscript brings new information on 1) the impact of material design (porosity) and composition on dissolution kinetic sand reactivity, 2) the impact of static vs dynamic testing on in-vitro dissolution and 3) the impact of materials’ pre-incubation on cell behavior.

New strategy to remove phosphate from low concentration solution by MOFs-modified resin: High affinity and thermal desorption

Adsorption process was usually torn between higher affinity and easier regeneration. Herein, for removing phosphate from low concentration solution, a new strategy was developed to break this tradeoff by metal organic frameworks (MOFs) confined in resins. For comparison, a series of metal oxide modified resins were prepared as adsorbents together with MOFs modified resins (transformation from corresponding oxides). MIL-101(Fe) presents higher adsorption capacity (41.79 mg/g) than Oxide(Fe)@201(36.07 mg/g), as well as high anti-interference ability with removal efficiency of 99.8% (2 mg/L phosphate), even if the loading amounts of iron in Oxide(Fe)@201 is almost twice of that in MIL-101(Fe)@201. Instrumental analysis and molecular dynamics simulations show that the higher adsorption affinity is mainly due to direct interaction between Fe3+ and phosphate via the formation of Fe-O-P, leading to much lower adsorption binding energy (Eads) of phosphate on MIL-101(Fe)@201 (-42.48 kcal/mol) than Oxide(Fe)@201; In desorption process, the MOFs structure can be stably restored with push of organic salt and high temperature (60 °C) around neutral pH due to the reversible structural transformation of MOFs. Moreover, MIL-101(Fe)@201 can continuously purify wastewater containing low concentration phosphate in fixed bed system after multiple regeneration, the water treatment capacity remained above ∼3000 BV and the regenerants can be intermittently recycled with precipitation of calcium phosphate from the solutions.

From Balloon to Crystalline Structure in the Calcium Phosphate Flow-Driven Chemical Garden.

We have studied the calcium phosphate precipitation reaction by producing chemical gardens in a controlled manner using a three-dimensional flow-driven technique. The injection of the phosphate containing solution into the calcium ion reservoir has resulted in structures varying from membranes to crystals. Dynamical phase diagrams are constructed by varying chemical composition and flow rates from which three different growth mechanisms have been revealed. The microstructural analysis by scanning electron microscopy and powder X-ray diffraction confirmed the morphological transition from membrane tubes to crystalline branches upon decreasing pH.

Avoiding and allowing apatite precipitation in oxygenic photolithotrophs.

The essential elements Ca and P, taken up and used metabolically as Ca2+ and H2 PO4 - /HPO4 2- respectively, could precipitate as one or more of the insoluble forms calcium phosphate (mainly apatite) if the free ion concentrations and pH are high enough. In the cytosol, chloroplast stroma, and mitochondrial matrix, the very low free Ca2+ concentration avoids calcium phosphate precipitation, apart from occasionally in the mitochondrial matrix. The low free Ca2+ concentration in these compartments is commonly thought of in terms of the role of Ca2+ in signalling. However, it also helps avoids calcium phosphate precipitation, and this could be its earliest function in evolution. In vacuoles, cell walls, and xylem conduits, there can be relatively high concentrations of Ca2+ and inorganic orthophosphate, but pH and/or other ligands for Ca2+ , suggests that calcium phosphate precipitates are rare. However, apatite is precipitated under metabolic control in shoot trichomes, and by evaporative water loss in hydathodes, in some terrestrial flowering plants. In aquatic macrophytes that deposit CaCO3 on their cell walls or in their environment as a result of pH increase or removal of inhibitors of nucleation or crystal growth, phosphate is sometimes incorporated in the CaCO3 . Calcium phosphate precipitation also occurs in some stromatolites.

Main clinical findings of oral and maxillofacial osteonecrosis, bisphosphonates and alternative treatment with photobiomodulation and magnetotherapy: a systematic review

Introduction: Bisphosphonates are drugs of a class widely used in several medical specialties. Its main property is to inhibit calcium phosphate precipitation, decrease bone calcification and reabsorption, and reduce osteoclastic action by inducing apoptosis in these cells. Bisphosphonates have anti-angiogenic effects. Low Power Laser is widely used for surgical purposes and it acts through various effects, such as photothermal, photomechanical-acoustic, photoionizing, and photoablation. Laser with low power can be divided into 2 groups, according to the respective types of photoreceptors: Low-Intensity Laser (LIL), with endogenous action, and Photodynamic Therapy (PDT), acting in the exogenous environment, as well as magnetotherapy. Objective: It was to highlight the main clinical findings of oral and maxillofacial osteonecrosis through the use of bisphosphonates, as well as an alternative treatment with photobiomodulation and magnetotherapy. Methods: The systematic review rules of the PRISMA Platform were followed. The search was carried out from December 2022 to February 2023 in the Scopus, PubMed, Science Direct, Scielo, and Google Scholar databases, using articles from 1985 to 2022. The quality of the studies was based on the GRADE instrument and the risk of bias was analyzed according to the Cochrane instrument. Results and Conclusion: A total of 320 articles were found, 59 articles were evaluated in full and 39 were included and developed in this systematic review study. Considering the Cochrane tool for risk of bias, the overall assessment resulted in 101 studies with a high risk of bias and 42 studies that did not meet GRADE. Patients being treated with bisphosphonates should be informed about the potential risk of bisphosphonates-related osteonecrosis of the jaws. In addition, providers responsible for breastfeeding therapy would be advised to refer patients for dental check-ups before starting treatment, allowing for patient follow-up by a multidisciplinary team. Although the morbidity of this pathology is not high, prevention must be mandatory, thus avoiding mutilating and painful processes. However, if the surgical procedure is required, the use of these new adjuvant therapies such as low-intensity laser and magnetotherapy.

Water-Mediated attraction between Like-charged species involved in calcium phosphate nucleation

There is overwhelming evidence that the biomineralization pathway involves prenucleation clusters (PNCs). Studies of calcium phosphate precipitation have shown that the thermodynamically favorable pathway to form the PNC identified as calcium triphosphate (Ca(HPO4)34-) includes two stable ion species (CaHPO4, Ca(HPO4)22–). However, the underlying mechanism remains unclear. Here, we reveal that the formation of these three species exhibits extremely distinct thermodynamic mechanisms. In sharp contrast to an entropy-driven Ca2+-HPO42- (CaP1) pairing compensated by unfavorable energy variation, free energy calculations display an energy-dominated association for CaHPO4-HPO42- (CaP2) and Ca(HPO4)22–-HPO42- (CaP3) with a slightly favorable entropy change. Further free energy decompositions demonstrate that the ion-induced favorable entropic and energetic variations are responsible for CaP1 and CaP2 associations, while the solvent-induced energy change accounts for the CaP3 pairing between two species carrying the same charges. In the complete prenucleation process starting from aqueous solution, the solvent-induced energy change initially hinders the CaP1 pairing but eventually makes a favorable contribution to the CaP3 association. Specifically, the enhanced stability and alignment of the hydrated water molecules around the ion species with charges of the same sign lead to the substantial favorable water-induced energy change and thus promote the CaP3 association even without any counterions.

Nutritional content of pastures with phosphate fertilization in 2 calcareous soils

ABSTRACT Alkaline soils present large amounts of calcium carbonates, with precipitation of insoluble calcium phosphate. The objective of the research was to determine the effects of the P application on the nutrient levels of the foliar tissue of Pennisetum pastures in calcareous soils in Córdoba-Colombia. Soil samples were collected from two locations in the Department of Córdoba. A completely randomized design with a 2 x 3 x 6 factorial arrangement (Vista Hermosa and Carolina), there Pennisetum species (king grass, Cuba OM-22, and Pennisetum purpureum), and six P doses (0, 80, 150, 250, 400, and 650 kg ha-1), applied as P2O5, was used. The addition of P did not increase the contents of N, K, Ca, and Mg in the king grass, Pennisetum purpureum, and Cuba OM-22 pastures. However, in the calcareous soils of Carolina, king grass, Pennisetum purpureum, and Cuba, OM-22 absorbed higher amounts of P.

Can Differently Stabilized Silver Nanoparticles Modify Calcium Phosphate Precipitation?

Calcium phosphates (CaPs) composites with silver nanoparticles (AgNPs) attract attention as a possible alternative to conventional approaches to combating orthopedic implant-associated infections. Although precipitation of calcium phosphates at room temperatures was pointed out as an advantageous method for the preparation of various CaP-based biomaterials, to the best of our knowledge, no such study exists for the preparation of CaPs/AgNP composites. Motivated by this lack of data in this study we investigated the influence of AgNPs stabilized with citrate (cit-AgNPs), poly(vinylpyrrolidone) (PVP-AgNPs), and sodium bis(2-ethylhexyl) sulfosuccinate (AOT-AgNPs) in the concentration range 5-25 mg dm-3 on the precipitation of CaPs. The first solid phase to precipitate in the investigated precipitation system was amorphous calcium phosphate (ACP). The effect of AgNPs on ACP stability was significant only in the presence of the highest concentration of AOT-AgNPs. However, in all precipitation systems containing AgNPs, the morphology of ACP was affected, as gel-like precipitates formed in addition to the typical chain-like aggregates of spherical particles. The exact effect depended on the type of AgNPs. After 60 min of reaction time, a mixture of calcium-deficient hydroxyapatite (CaDHA) and a smaller amount of octacalcium phosphate (OCP) formed. PXRD and EPR data point out that the amount of formed OCP decreases with increasing AgNPs concentration. The obtained results showed that AgNPs can modify the precipitation of CaPs and that CaPs properties can be fine-tuned by the choice of stabilizing agent. Furthermore, it was shown that precipitation can be used as a simple and fast method for CaP/AgNPs composites preparation which is of special interest for biomaterials preparation.

Synergistic effect of sulfonation followed by precipitation of amorphous calcium phosphate on the bone-bonding strength of carbon fiber reinforced polyetheretherketone

Sulfonation and applications of amorphous calcium phosphate are known to make polyetheretherketone (PEEK) bioactive. Sulfonation followed by precipitation of amorphous calcium phosphate (AN-treatment) may provide PEEK with further bone-bonding strength. Herein, we prepared a carbon-fiber-reinforced PEEK (CPEEK) with similar tensile strength to cortical bone and a CPEEK subjected to AN-treatment (CPEEK-AN). The effect of AN-treatment on the bone-bonding strength generated at the interface between the rabbit’s tibia and a base material was investigated using a detaching test at two time-points (4 and 8 weeks). At 4 weeks, the strength of CPEEK-AN was significantly higher than that of CPEEK due to the direct bonding between the interfaces. Between 4 and 8 weeks, the different bone forming processes showed that, with CPEEK-AN, bone consolidation was achieved, thus improving bone-bonding strength. In contrast, with CPEEK, a new bone was absorbed mainly on the interface, leading to poor strength. These observations were supported by an in vitro study, which showed that pre-osteoblast on CPEEK-AN caused earlier maturation and mineralization of the extracellular matrix than on CPEEK. Consequently, AN-treatment, comprising a combination of two efficient treatments, generated a synergetic effect on the bonding strength of CPEEK.

Comparison of bovine serum albumin and chitosan effects on calcium phosphate formation in the presence of silver nanoparticles.

The precipitation of calcium phosphates (CaPs) in the presence of more than one type of additive is of interest both from a fundamental point of view and as a possible biomimetic route for the preparation of multicomponent composites in which the activity of the components is preserved. In this study, the effect of bovine serum albumin (BSA) and chitosan (Chi) on the precipitation of CaPs in the presence of silver nanoparticles (AgNPs) stabilized with sodium bis(2-ethylhexyl)sulfosuccinate (AOT-AgNPs), poly(vinylpyrrolidone) (PVP-AgNPs), and citrate (cit-AgNPs) was investigated. In the control system, the precipitation of CaPs occurred in two steps. Amorphous calcium phosphate (ACP) was the first precipitated solid, which transformed into a mixture of calcium-deficient hydroxyapatite (CaDHA) and a smaller amount of octacalcium phosphate (OCP) after 60 min of ageing. Both biomacromolecules inhibited ACP transformation, with Chi being a stronger inhibitor due to its flexible molecular structure. As the concentration of the biomacromolecules increased, the amount of OCP decreased both in the absence and presence of AgNPs. In the presence of cit-AgNPs and two highest BSA concentrations, a change in the composition of the crystalline phase was observed. Calcium hydrogen phosphate dihydrate was formed in the mixture with CaDHA. An effect on the morphology of both the amorphous and crystalline phases was observed. The effect depended on the specific combination of biomacromolecules and differently stabilized AgNP. The results obtained suggest a simple method for fine-tuning the properties of precipitates using different classes of additives. This could be of interest for the biomimetic preparation of multifunctional composites for bone tissue engineering.

A microfluidic double emulsion platform for spatiotemporal control of pH and particle synthesis.

The temporal control of pH in microreactors such as emulsion droplets plays a vital role in applications including biomineralisation and microparticle synthesis. Typically, pH changes are achieved either by passive diffusion of species into a droplet or by acid/base producing reactions. Here, we exploit an enzyme reaction combined with the properties of a water-oil-water (W/O/W) double emulsion to control the pH-time profile in the droplets. A microfluidic platform was used for production of ∼100-200 μm urease-encapsulated double emulsions with a tuneable mineral oil shell thickness of 10-40 μm. The reaction was initiated on-demand by addition of urea and a pulse in base (ammonia) up to pH 8 was observed in the droplets after a time lag of the order of minutes. The pH-time profile can be manipulated by the diffusion timescale of urea and ammonia through the oil layer, resulting in a steady state pH not observed in bulk reactive solutions. This approach may be used to regulate the formation of pH sensitive materials under mild conditions and, as a proof of concept, the reaction was coupled to calcium phosphate precipitation in the droplets. The oil shell thickness was varied to select for either brushite microplatelets or hydroxyapatite particles, compared to the mixture of different precipitates obtained in bulk.

Enhancing the activity of biocatalysts supported on calcium phosphate by inducing mesoporosity with phosphopeptides

Preventing enzyme deactivation while increasing their accessibility by adsorption in mesopores remains a key challenge in the design of biocompatible supported enzymatic catalysts. Herein, it is expected that food-grade monomeric or polymeric glycopeptides containing organic phosphate groups (as casein phosphopeptide derivatives) enhances the entrapment of the enzyme over the one pot method of wet precipitation of calcium phosphate (CaP) leading to a biocompatible hybrid organic/inorganic catalyst. The supported catalyst was prepared by wet precipitation of calcium and phosphorous in the presence of either monomeric glycopeptide or polymeric glycopeptide. These organic glycopeptides had an influence on the electrostatic stabilization during the nucleation of calcium phosphate particles, as tracked by dynamic light scattering and by scanning electron microscopy.The phosphopeptide derivatives, were able to decrease the crystallization degree in calcium phosphate particles, as demonstrated by XRD and solid-state NMR analysis. Furthermore, according to N2 sorption data, the glycopeptide derivatives induced interparticle mesopores, which might be responsible of the enhanced enzymatic activity of the supported biocatalyst that improved from 52 (+/−19) NLU·g−1 without organic compound to 607 (+/−34) NLU·g−1.

The role of protein contents in promoting wastewater phosphorus and bioenergy recovery during anaerobic digestion

Proteins present in domestic and industrial wastewater, yet their role in nutrient and energy recovery during anaerobic digestion (AD) has not been well understood. This study aimed to examine the impact of feedwater protein content on the calcium phosphate (CaP) precipitation under a low supersaturation condition and the methane production in an AD process. Four 1.0 L upflow anaerobic sludge blanket (UASB) reactors were fed with the synthetic feeds with different protein contents. The results showed that the amino acid degradation caused the pH elevation, triggering the CaP precipitation. A 25% bovine serum albumin (BSA) content in the feedwater helped shape a superior microbial community that contributed to a methanisation rate of 82.7%. The high prevalence of the phylum Synergistetes and the methanogen Methanosaeta suggested that methane was mainly produced through the acetate utilization.

Assessment of Physical/Mechanical Performance of Dental Resin Sealants Containing Sr-Bioactive Glass Nanoparticles and Calcium Phosphate.

The aim of this study was to assess the chemical/mechanical properties of ion-releasing dental sealants containing strontium-bioactive glass nanoparticles (Sr-BGNPs) and monocalcium phosphate monohydrate (MCPM). Two experimental sealants, TS1 (10 wt% Sr-BGNPs and 2 wt% MCPM) and TS2 (5 wt% Sr-BGNPs and 4 wt% MCPM), were prepared. Commercial controls were ClinproXT (CP) and BeautiSealant (BT). The monomer conversion (DC) was tested using ATR−FTIR (n = 5). The biaxial flexural strength (BFS) and modulus (BFM) were determined (n = 5) following 24 h and 7 days of immersion in water. The Vickers surface microhardness (SH) after 1 day in acetic acid (conc) versus water was tested (n = 5). The bulk and surface calcium phosphate precipitation in simulated body fluid was examined under SEM-EDX. The ion release at 4 weeks was analyzed using ICP-MS (n = 5). The DC after 40 s of light exposure of TS1 (43%) and TS2 (46%) was significantly lower than that of CP (58%) and BT (61%) (p < 0.05). The average BFS of TS1 (103 MPa), TS2 (123 MPa), and BT (94 MPa) were lower than that of CP (173 MPa). The average BFM and SH of TS1 (2.2 GPa, 19 VHN) and TS2 (2.0 GPa, 16 VHN) were higher than that of CP (1.6 GPa, 11 VHN) and BT (1.3 GPa, 12 VHN). TS1 showed higher Ca, P, and Sr release than TS2. Bulk calcium phosphate precipitation was detected on TS1 and TS2 suggesting some ion exchange. In conclusion, the DC of experimental sealants was lower than that of commercial materials, but their mechanical properties were within the acceptable ranges. The released ions may support remineralizing actions.

Structure, stability, and mechanism of dextran–CPP–Ca2+ conjugates: A novel high-efficiency calcium ion delivery system

Calcium has limited bioavailability because of the formation of calcium phosphate deposits in the gastrointestinal tract. In this study, we prepared a dextran-casein phosphopeptide (CPP)–Ca2+ delivery system and evaluated for Ca2+ binding mechanism, structure, stability, and sustained release of Ca2+ and assessed inhibition of calcium phosphate precipitation. The results revealed that Ca2+ binds to dextran–CPP through the phosphate, carboxyl, and amino groups and forms crystal clusters. Furthermore, compared with single polymer CPP–Ca2+ conjugates, copolymer dextran–CPP–Ca2+ conjugates exhibited improved stability at various conditions (pH, temperature, and coexisting food), efficiently reduced the calcium phosphate precipitation, and improved sustained-release of Ca2+. Collectively, dextran–CPP–Ca2+ conjugates can be an efficient Ca2+ delivery system.

Measuring pH of skim milk and milk permeate at ultra-high temperatures at laboratory and pilot scale

Changes in the pH of skim milk and skim milk ultrafiltration permeate on heating from 25 to 140 °C were examined. Results showed that the decrease in pH with increase in temperature up to 140 °C was not linear. Hydrogen ion release due to changes in the milk mineral balance were responsible for the reduction in pH with increase in temperature. The presence of milk proteins offered little buffering against the drop in pH. The precipitation of calcium phosphate resulted in sediment in milk permeate heated above ∼70 °C, but this did not occur in skim milk, with the pH remaining lower in milk permeate after heat treatment when measured at 25 °C. This study has shown that in-line pH measurements of milk at ultra-high temperatures is feasible, and could prove useful at laboratory and pilot-scale for studying interactions within, and stability of, more complex formulations with added minerals.

Influence of Biomacromolecules on Calcium Phosphate Formation on TiO2 Nanomaterials

Calcium phosphates (CaP) composites with biomacromolecules and/or nanomaterials have recently emerged as a potential solution to improve the poor mechanical properties and biological response of CaP. Among the methods available for preparation of such composites, precipitation at low temperatures attracts special interest as it allows preservation of the activity of biomacromolecules. However, precipitation of CaP in the presence of two additives is a complex process that needs to be studied in detail to rationalize composite preparation. This study aimed to investigate co-precipitation of CaP on different TiO2 nanomaterials (TiNMs), including nanoparticles (TiNPs), nanoplates (TiNPls), nanotubes (TiNTs), and nanowires (TiNWs), in the presence of bovine serum albumin (BSA) and chitosan (Chi). The obtained results have shown that both BSA and Chi inhibited transformation of amorphous to crystalline CaP, even in the presence of TiNMs at concentrations that promoted transformation. Chi proved to be a stronger inhibitor due to its more flexible structure. The presence of BSA and Chi did not influence the composition of the CaP formed as calcium-deficient hydroxyapatite (CaDHA) was formed in all the systems. However, both macromolecules influenced the morphology of the formed CaDHA in different ways depending on the type of TiNM used. BSA and Chi adsorbed on all the TiNMs, as confirmed by zeta potential measurements, but this adsorption reduced the amount of CaP formed on TiNMs only in the case of TiNWs. The obtained results contribute to the understanding of the influence of BSA and Chi on CaP precipitation in the presence of nanomaterials and thus to the rational design of CaP-based multi-composite materials.

Shedding Light on the Role of Na,K-ATPase as a Phosphatase during Matrix-Vesicle-Mediated Mineralization

Matrix vesicles (MVs) contain the whole machinery necessary to initiate apatite formation in their lumen. We suspected that, in addition to tissue-nonspecific alkaline phosphatase (TNAP), Na,K,-ATPase (NKA) could be involved in supplying phopshate (Pi) in the early stages of MV-mediated mineralization. MVs were extracted from the growth plate cartilage of chicken embryos. Their average mean diameters were determined by Dynamic Light Scattering (DLS) (212 ± 19 nm) and by Atomic Force Microcopy (AFM) (180 ± 85 nm). The MVs had a specific activity for TNAP of 9.2 ± 4.6 U·mg-1 confirming that the MVs were mineralization competent. The ability to hydrolyze ATP was assayed by a colorimetric method and by 31P NMR with and without Levamisole and SBI-425 (two TNAP inhibitors), ouabain (an NKA inhibitor), and ARL-67156 (an NTPDase1, NTPDase3 and Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) competitive inhibitor). The mineralization profile served to monitor the formation of precipitated calcium phosphate complexes, while IR spectroscopy allowed the identification of apatite. Proteoliposomes containing NKA with either dipalmitoylphosphatidylcholine (DPPC) or a mixture of 1:1 of DPPC and dipalmitoylphosphatidylethanolamine (DPPE) served to verify if the proteoliposomes were able to initiate mineral formation. Around 69-72% of the total ATP hydrolysis by MVs was inhibited by 5 mM Levamisole, which indicated that TNAP was the main enzyme hydrolyzing ATP. The addition of 0.1 mM of ARL-67156 inhibited 8-13.7% of the total ATP hydrolysis in MVs, suggesting that NTPDase1, NTPDase3, and/or NPP1 could also participate in ATP hydrolysis. Ouabain (3 mM) inhibited 3-8% of the total ATP hydrolysis by MVs, suggesting that NKA contributed only a small percentage of the total ATP hydrolysis. MVs induced mineralization via ATP hydrolysis that was significantly inhibited by Levamisole and also by cleaving TNAP from MVs, confirming that TNAP is the main enzyme hydrolyzing this substrate, while the addition of either ARL-6715 or ouabain had a lesser effect on mineralization. DPPC:DPPE (1:1)-NKA liposome in the presence of a nucleator (PS-CPLX) was more efficient in mineralizing compared with a DPPC-NKA liposome due to a better orientation of the NKA active site. Both types of proteoliposomes were able to induce apatite formation, as evidenced by the presence of the 1040 cm-1 band. Taken together, the findings indicated that the hydrolysis of ATP was dominated by TNAP and other phosphatases present in MVs, while only 3-8% of the total hydrolysis of ATP could be attributed to NKA. It was hypothesized that the loss of Na/K asymmetry in MVs could be caused by a complete depletion of ATP inside MVs, impairing the maintenance of symmetry by NKA. Our study carried out on NKA-liposomes confirmed that NKA could contribute to mineral formation inside MVs, which might complement the known action of PHOSPHO1 in the MV lumen.

Development of a cyclic-inverso AHSG/Fetuin A-based peptide for inhibition of calcification in osteoarthritis

Ectopic calcification is an important contributor to chronic diseases, such as osteoarthritis. Currently, no effective therapies exist to counteract calcification. We developed peptides derived from the calcium binding domain of human Alpha-2-HS-Glycoprotein (AHSG/Fetuin A) to counteract calcification. A library of seven 30 amino acid (AA) long peptides, spanning the 118 AA Cystatin 1 domain of AHSG, were synthesized and evaluated in an in vitro calcium phosphate precipitation assay. The best performing peptide was modified (cyclic, retro-inverso and combinations thereof) and evaluated in cellular calcification models and the rat Medial Collateral Ligament Transection + Medial Meniscal Tear (MCLT+MMT) osteoarthritis model. A cyclic peptide spanning AA 1-30 of mature AHSG showed clear inhibition of calcium phosphate precipitation in the nM-pM range that far exceeded the biological activity of the linear peptide variant or bovine Fetuin. Biochemical and electron microscopy analyses of calcium phosphate particles revealed a similar, but distinct, mode of action in comparison with bFetuin. A cyclic-inverso variant of the AHSG 1-30 peptide inhibited calcification of human articular chondrocytes, vascular smooth muscle cells and during osteogenic differentiation of bone marrow derived stromal cells. Lastly, we evaluated the effect of intra-articular injection of the cyclic-inverso AHSG 1-30 peptide in a rat osteoarthritis model. A significant improvement was found in histopathological osteoarthritis score and animal mobility. Serum levels of IFNγ were found to be lower in AHSG 1-30 peptide treated animals. The cyclic-inverso AHSG 1-30 peptide directly inhibits the calcification process and holds the potential for future application in osteoarthritis.