Humidity-Responsive Amorphous Calcium-Magnesium Pyrophosphate/Cassava Starch Scaffold for Enhanced Neurovascular Bone Regeneration.

Developing a neurovascular bone repair scaffold with an appropriate mechanical strength remains a challenge. Calcium phosphate (CaP) is similar to human bone, but its scaffolds are inherently brittle and inactive, which require recombination with active ions and polymers for bioactivity and suitable strength. This work discussed the synthesis of amorphous magnesium-calcium pyrophosphate (AMCP) and the subsequent development of a humidity-responsive AMCP/cassava starch (CS) scaffold. The scaffold demonstrated enhanced mechanical properties by strengthening the intermolecular hydrogen bonds and ionic bonds between AMCP and CS during the gelatinization and freeze-thawing processes. The release of active ions was rapid initially and stabilized into a long-term stable release after 3 days, which is well-matched with new bone growth. The release of pyrophosphate ions endowed the scaffold with antibacterial properties. At the cellular level, the released active ions simultaneously promoted the proliferation and mineralization of osteoblasts, the proliferation and migration of endothelial cells, and the proliferation of Schwann cells. At the animal level, the scaffold was demonstrated to promote vascular growth and peripheral nerve regeneration in a rat skull defect experiment, ultimately resulting in the significant and rapid repair of bone defects. The construction of the AMCP/CS scaffold offers practical suggestions and references for neurovascular bone repair.

17O solid state NMR as a valuable tool for deciphering reaction mechanisms in mechanochemistry: the case study on the 17O-enrichment of hydrated Ca-pyrophosphate biominerals.

The possibility of enriching in 17O the water molecules within hydrated biominerals belonging to the Ca-pyrophosphate family was investigated, using liquid assisted grinding (LAG) in the presence of 17O-labelled water. Two phases with different hydration levels, namely triclinic calcium pyrophosphate dihydrate (Ca2P2O7·2H2O, denoted t-CPPD) and monoclinic calcium pyrophosphate tetrahydrate (Ca2P2O7·4H2O, denoted m-CPPT ß) were enriched in 17O using a "post-enrichment" strategy, in which the non-labelled precursors were ground under gentle milling conditions in the presence of stoichiometric quantities of 17O-enriched water (introduced here in very small volumes ∼10 µL). Using high-resolution 17O solid-state NMR (ssNMR) analyses at multiple magnetic fields, and dynamic nuclear polarisation (DNP)-enhanced 17O NMR, it was possible to show that the labelled water molecules are mainly located at the core of the crystal structures, but that they can enter the lattice in different ways, namely by dissolution/recrystallisation or by diffusion. Overall, this work sheds light on the importance of high-resolution 17O NMR to help decipher the different roles that water can play as a liquid-assisted grinding agent and as a reagent for 17O-isotopic enrichment.

Tunable Behavior in Solution of Amorphous Calcium Ortho/Pyrophosphate Materials: An Acellular In Vitro Study.

Amorphous calcium phosphate-based materials are of major interest in the field of bone substitution. Very recently, the low-temperature synthesis of a new family of amorphous calcium phosphate containing both orthophosphate and pyrophosphate ions in controlled proportions has been reported. Despite their interest, especially due to the biochemical role and the hydrolysis of pyrophosphate occurring in vivo, the behavior of such materials when interacting with aqueous media has never been described. Consequently, we herein report the in vitro acellular evolution of three compositions of mixed calcium ortho- and pyrophosphate amorphous materials including a different orthophosphate proportion. As a first step to assess the physicochemical reactivity of these amorphous materials, they were tested in two different media at 37 °C, acidified water and simulated body fluid solution, from 1 h up to 15 days. The results demonstrated that they were quite stable and that they progressively released part of their constitutive ions, highlighting their potential for controlled delivery of bioactive ions (calcium, orthophosphate, and pyrophosphate ions). In addition to these properties, we showed that the material with the highest ortho/(ortho + pyro) phosphate ratio started to crystallize into nanocrystalline apatite analogous to bone mineral within 2 days or 2 weeks depending on the medium. For the other material compositions, no layer of apatite was detected at their surface with SBF testing despite the favorable supersaturation indexes, crystallization being probably inhibited by pyrophosphate ions released in the medium. This varying apatite-forming ability emphasizes the key role of the ortho/(ortho + pyro) phosphate ratio of these materials in their in vitro reactivity and bioactivity, which paves the way for the development of this promising family of amorphous calcium phosphate materials with tunable physicochemical and biological properties.

A Phage Display-Identified Short Peptide Capable of Hydrolyzing Calcium Pyrophosphate Crystals-The Etiological Factor of Chondrocalcinosis.

Chondrocalcinosis is a metabolic disease caused by the presence of calcium pyrophosphate dihydrate crystals in the synovial fluid. The goal of our endeavor was to find out whether short peptides could be used as a dissolving factor for such crystals. In order to identify peptides able to dissolve crystals of calcium pyrophosphate, we screened through a random library of peptides using a phage display. The first screening was designed to select phages able to bind the acidic part of alendronic acid (pyrophosphate analog). The second was a catalytic assay in the presence of crystals. The best-performing peptides were subsequently chemically synthesized and rechecked for catalytic properties. One peptide, named R25, turned out to possess some hydrolytic activity toward crystals. Its catalysis is Mg2+-dependent and also works against soluble species of pyrophosphate.

Effect of pathogenic crystals on the production of pro- and anti-inflammatory cytokines by different leukocyte populations.

PURPOSE: To evaluate cytokine production in vitro by different types of leukocytes stimulated with monosodium urate (MSU), calcium pyrophosphate (CPP) and basic calcium phosphate (BCP) crystals. MATERIAL AND METHODS: Polymorphonuclear cells (PMN), monocytes and lymphocytes, isolated from healthy volunteer blood, were stimulated for different time periods with increasing MSU, CPP or BCP crystal concentrations. IL-1ß, IL-8, IL-6, CCL2, IL-1Ra and TGFß1 were determined by ELISA. RESULTS: Exposure of PMN to different crystals resulted in a moderate IL-8 and IL-1Ra release. Stimulation of monocytes induced a significant production of all the cytokines evaluated. The highest levels of IL-1ß, IL-6, CCL2 and IL-8 were observed with MSU at 0.5 mg/ml, CPP at 0.01-0.05 mg/ml and BCP at 1 mg/ml after 18-48 h and then decreased. At the same crystal concentrations, IL-1Ra and TGFß1 increased until the end of the experiment. Treatment of lymphocytes with different crystals did not induce cytokine release. CONCLUSION: This study demonstrates that PMN, monocytes and lymphocytes from the same donor respond differently after stimulation with MSU, CPP or BCP crystals, depending on the dose and the time of exposure. Crystals induce a rapid increase of pro-inflammatory cytokines, whereas longer time is required to release high levels of anti-inflammatory cytokines.

Analysis of the osteogenic and mechanical characteristics of iron (Fe2+/Fe3+)-doped ß­calcium pyrophosphate.

The calcium phosphate is the main mineral constituent of bone. Although there has been significant amount of research on finding ideal synthetic bone, no suitable scaffold material has yet been found. In this investigation, the iron doped brushite (CaHPO4·2H2O) has been investigated for osteogenic potential and mechanical properties. The synthesis of iron-oxide doping in the form of Fe2+,3+-ions were carried out using the solution based method in which the ammonium hydrogen phosphate and calcium nitrate solutions were used in stoichiometric ratio for synthesizing CaHPO4·2H2O, with doping concentrations of Fe2+,3+-ions between 5 mol% and 30 mol%. The synthesized powders were analysed using X-ray powder diffraction, FTIR, SEM and Raman spectroscopic techniques. The heat treatment of synthesized powder was carried out at 1000 °C in air for 5 h, and it was found that the dominant crystalline phase in samples with <20 mol% was ß-CPP, which also formed an iron-rich solid solution phase. Increasing the concentrations of Fe2+,3+-ions enhances the phase fraction of FePO4 and amorphous phase. Amongst the Fe2+,3+-doped ß-CPP minerals, it was found that the 10 mol% Fe2+,3+-doped ß-CPP offers the best combination of bio-mechanical and osteogenic properties as a scaffold for bone tissue regenerative engineering.

Mechanochemical immobilization of lead contaminated soil by ball milling with the additive of Ca(H2PO4)2.

Lead (Pb) pollution in the soil is becoming more and more serious, and lead poisoning incidents also constantly occur. Therefore, the remediation of lead pollution in the soil has attracted widespread attention. In this study, heavy metal lead in soil was remediated by mechanochemical methods. The effects of different ball milling conditions on the toxic leaching concentration and morphological distribution (BCR sequential extraction procedure) of lead in contaminated soil were analyzed, including the addition of calcium dihydrogen phosphate (Ca(H2PO4)2), ball milling time, and ball milling speed. The reaction mechanism was analyzed by X-ray diffractometry (XRD), scanning electron microscopy (SEM), and a laser particle size analyzer. The results show that the optimal conditions for mechanochemical immobilization were 10% additive (Ca(H2PO4)2), milling speed of 550 rpm, and ball milling time for 2 h. Under this condition, the toxic leaching concentration of lead from contaminated soil was 4.36 mg L-1, and in the BCR sequential extraction procedure, Pb was mainly present in the residual fraction (54.96%). The mechanism of mechanochemical solidification of heavy metal lead in soil is that, during the ball milling process, the lead precipitates with Ca(H2PO4)2 to produce dense agglomerates (Pb3(PO4)2 and PbxCa10-x(PO4)6(OH)2), which fixes the lead in the soil and hampers its leaching.

Calcium Pyrophosphate Dihydrate Crystals Increase the Granulocyte/Monocyte Progenitor (GMP) and Enhance Granulocyte and Monocyte Differentiation In Vivo.

Calcium pyrophosphate dihydrate (CPPD) crystals are formed locally within the joints, leading to pseudogout. Although the mobilization of local granulocytes can be observed in joints where pseudogout has manifested, the mechanism of this activity remains poorly understood. In this study, CPPD crystals were administered to mice, and the dynamics of splenic and peripheral blood myeloid cells were analyzed. As a result, levels of both granulocytes and monocytes were found to increase following CPPD crystal administration in a concentration-dependent manner, with a concomitant decrease in lymphocytes in the peripheral blood. In contrast, the levels of other cells, such as dendritic cell subsets, T-cells, and B-cells, remained unchanged in the spleen, following CPPD crystal administration. Furthermore, an increase in granulocytes/monocyte progenitors (GMPs) and a decrease in megakaryocyte/erythrocyte progenitors (MEPs) were also observed in the bone marrow. In addition, CPPD administration induced production of IL-1ß, which acts on hematopoietic stem cells and hematopoietic progenitors and promotes myeloid cell differentiation and expansion. These results suggest that CPPD crystals act as a "danger signal" to induce IL-1ß production, resulting in changes in course of hematopoietic progenitor cell differentiation and in increased granulocyte/monocyte levels, and contributing to the development of gout.

Prevalence and incidence of non-gout crystal arthropathy in southern Sweden.

OBJECTIVE: To estimate the prevalence and incidence of non-gout crystal arthropathy in relation to socioeconomic factors in southern Sweden. METHODS: All patients (age ≥ 18 years) with at least one visit to a physician with the diagnosis of interest in the Skåne region (population of 1.3 million) in 1998-2014 were identified. Non-gout crystal arthropathy (ICD-10 codes M11.0-M11.9) was subclassified in four different groups: calcium pyrophosphate crystal deposition related arthropathy (CPPD), unspecified non-gout arthropathies, chondrocalcinosis, and hydroxyapatite crystal deposition disease. The crude and age-adjusted point prevalence on December 31, 2014, and the cumulative incidence during 2014 were calculated for all non-gout crystal arthropathies, CPPD, and other unspecified non-gout arthropathies overall and in relation to occupation, income, and level of education. RESULTS: The crude 2014 point prevalence (95% CI) and 2014 cumulative incidence (95% CI) of all non-gout crystal arthropathies were 0.23% (0.23-0.24) and 21.5 (19-25) cases/100,000 persons. Mean age (range) among all prevalent cases in 2014 was 71 (20-102) years and 56% were males. The point prevalence and cumulative incidence of CPPD were 0.09% (0.08-0.09) and 8 (7-10)/100,000 persons, respectively. The corresponding data for unspecified non-gout crystal deposition disease was 0.16% (0.16-0.17) and 15.6 (13-18)/100,000 persons, respectively. The prevalence and incidence of CPPD and unspecified non-gout crystal arthropathies were slightly higher in men and increased with age irrespective of gender. Unspecified non-gout crystal arthropathy but not CPPD was less prevalent in persons with ≥ 15 years of education, whereas there were no clear associations with occupation and income. CONCLUSION: The prevalence of all diagnosed non-gout crystal arthropathies was 0.23%, thus considerably less prevalent than gout in southern Sweden. CPPD and other unspecified non-gout crystal arthropathies are the predominant diagnoses, increasing with age and in men. With the exception for unspecified non-gout crystal arthropathies being inversely correlated to a higher level of education, no convincing association with the socioeconomic factors was found.

Polydopamine-coated chitosan/calcium pyrophosphate hybrid microflowers as an effective hemostatic agent.

In this study, polydopamine (PDA) coated chitosan/calcium pyrophosphate hybrid microflowers (PDA@CS-CaP) were prepared using a facile approach and evaluated as a hemostatic agent. The surface morphology and elements, chemical groups, porous structure, thermostability, zeta potential, as well as surface wettability were systematically characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), N2 adsorption-desorption, thermogravimetry (TGA) and contact angle apparatus. Due to the synergistic effects of the high hydrophilicity (water contact angel nearly 0), chemical activation (the amino and phenol group of PDA), and the flower-like porous hierarchical structure, the prepared PDA@CS-CaP could induce hemocytes and platelets aggregation, promote the blood clotting and achieve bleeding control in vitro and in vivo. Furthermore, the PDA@CS-CaP had no exothermic adverse effects and exhibited better biocompatibility (no cytotoxicity against fibroblasts cells). Thus, the PDA@CS-CaP were expected to be a promising candidate for a safe and promising hemostatic agent because of its porous and hierarchical structures as well as the active PDA coating.

Microstructure evolution, mechanical properties, and enhanced bioactivity of Ti-13Nb-13Zr based calcium pyrophosphate composites for biomedical applications.

The present study reports the results of the microstructure, mechanical properties and in vitro bioactivity of the Ti-13Nb-13Zr based composite with 10 wt% CPP (calcium pyrophosphate), densified using spark plasma sintering process (SPS) at different sintering temperatures (900-1200 °C). The results show that the sintered composites mainly consist of ß-Ti, α-Ti, and ceramic interphases (Ti2O, CaTiO3, CaZrO3, CaO, TixPy). With the sintering temperature increasing, α-Ti and ceramic interphases gradually increase, and relative density, elastic modulus, compressive strength and yield strength of the composites also reveal an increasing tendency. However, Ti-13Nb-13Zr-10CPP composite sintered at 1000 °C exhibits high matching elastic modulus (46 GPa) and compressive strength (1617 MPa) due to uniform structure and high density. In addition, in vitro mineralization assays demonstrate the apatite-forming ability of the composite (1000 °C) and its higher surface bioactivity as compared to the Ti-13Nb-13Zr alloy. Furthermore, ROS1728 osteoblast culture evidences that the composite (1000 °C) stimulates cell adhesion and growth due to the pore characteristics and ceramic interphases. Therefore, the prepared Ti-13Nb-13Zr-10CPP composite at 1000 °C exhibits immense potential as a biomedical material.

Inflammatory Potential of Four Different Phases of Calcium Pyrophosphate Relies on NF-κB Activation and MAPK Pathways.

Background: Calcium pyrophosphate (CPP) microcrystal deposition is associated with wide clinical phenotypes, including acute and chronic arthritis, that are interleukin 1ß (IL-1ß)-driven. Two CPP microcrystals, namely monoclinic and triclinic CPP dihydrates (m- and t-CPPD), have been identified in human tissues in different proportions according to clinical features. m-CPP tetrahydrate beta (m-CPPTß) and amorphous CPP (a-CPP) phases are considered as m- and t-CPPD crystal precursors in vitro. Objectives: We aimed to decipher the inflammatory properties of the three crystalline phases and one amorphous CPP phase and the intracellular pathways involved. Methods: The four synthesized CPP phases and monosodium urate crystals (MSU, as a control) were used in vitro to stimulate the human monocytic leukemia THP-1 cell line or bone marrow-derived macrophages (BMDM) isolated from WT or NLRP3 KO mice. The gene expression of pro- and anti-inflammatory cytokines was evaluated by quantitative PCR; IL-1ß, IL-6 and IL-8 production by ELISA; and mitogen-activated protein kinase (MAPK) activation by immunoblot analysis. NF-κB activation was determined in THP-1 cells containing a reporter plasmid. In vivo, the inflammatory potential of CPP phases was assessed with the murine air pouch model via cell analysis and production of IL-1ß and CXCL1 in the exudate. The role of NF-κB was determined by a pharmacological approach, both in vivo and in vitro. Results:In vitro, IL-1ß production induced by m- and t-CPPD and m-CPPTß crystals was NLRP3 inflammasome dependent. m-CPPD crystals were the most inflammatory by inducing a faster and higher production and gene expression of IL-1ß, IL-6, and IL-8 than t-CPPD, m-CPPTß and MSU crystals. The a-CPP phase did not show an inflammatory property. Accordingly, m-CPPD crystals led to stronger activation of NF-κB, p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) MAPKs. Inhibition of NF-κB completely abrogated IL-1ß and IL-8 synthesis and secretion induced by all CPP crystals. Also, inhibition of JNK and ERK1/2 MAPKs decreased both IL-1ß secretion and NF-κB activation induced by CPP crystals. In vivo, IL-1ß and CXCL1 production and neutrophil infiltration induced by m-CPPD crystals were greatly decreased by NF-κB inhibitor treatment. Conclusion: Our results suggest that the inflammatory potential of different CPP crystals relies on their ability to activate the MAPK-dependent NF-κB pathway. Studies are ongoing to investigate the underlying mechanisms.

Effect of calcium pyrophosphate on microstructural evolution and in vitro biocompatibility of Ti-35Nb-7Zr composite by spark plasma sintering.

ß-type Ti-35Nb-7Zr alloy has attracted considerable attentions as a bone implant material. The alloy, however, has poor bioactivity, which difficult to form a strong osseointegration between the bone tissues. Combining Ti alloy with a bioactive and biodegradable ceramic has been of interest to researchers. But the large difference in physicochemical property of high-melting metal and ceramic elements would bring the manufacturing restriction. In this work, Ti-35Nb-7Zr-CPP composites were fabricated with mechanical alloy of Ti, Nb, Zr and Nano calcium pyrophosphate (CPP) powders mixture followed by spark plasma sintering (SPS) routes. The effect of CPP ceramic on microstructural evolution and in vitro biocompatibility were investigated. As the addition of CPP (10-30 wt%), ceramic elements spreading towards the matrix, the generated metal-ceramic bioactive phases CaTiO3 are observed well consolidated with ß-Ti matrix. With the CPP increasing, Ca and P atoms rapidly migrated to the ß-Ti matrix to form granulated Ti5P3, which leads to the increasing porosity (10%-18%) in the composites. The results demonstrated that the favorable cell viability (the cell proliferation rates were higher than 100%) and growth inside the pores of the composites arise from the rough micro-porous surface and the release of bioactive metal-ceramic phase ions into the biological environment. The enhanced bioactivity and microstructural evolution behaviors of the Ti-35Nb-7Zr-CPP composites may provide a strategy for designing and fabricating multifunctional implants.

Biodegradable collagen sponge reinforced with chitosan/calcium pyrophosphate nanoflowers for rapid hemostasis.

Efficient and biodegradable hemostatic materials become increasingly important in civilian and military clinical. However, traditional hemostatic materials are difficult to achieve expected effects especially in parenchymal organs with rich vascularity. In facing these challenges, we designed a biodegradable collagen sponge reinforced with chitosan/calcium pyrophosphate nanoflowers (CPNFs-Col sponge) for rapid hemostasis. With specific performances, such as rapid water absorption ability, the positive surface rich in amino groups and high specific surface area (952.5m2g-1), the obtained CNPFs-Col sponge with optimized composition could activate the intrinsic pathway of coagulation cascade, induce haemocytes and platelets adherence, promote the blood clotting and achieve hemorrhage control in vitro and in vivo. In addition, the CNPFs-Col sponge can be completely biodegraded in 3 weeks, which is suitable for post-operative treatment and peritoneal adhesion prevention. It can be concluded that the CPNFs-Col sponge would become a promising candidate for clinical hemostatic applications.

Neutrophil Extracellular Traps and Microcrystals.

Neutrophil extracellular traps represent a fascinating mechanism by which PMNs entrap extracellular microbes. The primary purpose of this innate immune mechanism is thought to localize the infection at an early stage. Interestingly, the ability of different microcrystals to induce NET formation has been recently described. Microcrystals are insoluble crystals with a size of 1-100 micrometers that have different composition and shape. Microcrystals have it in common that they irritate phagocytes including PMNs and typically trigger an inflammatory response. This review is the first to summarize observations with regard to PMN activation and NET release induced by microcrystals. Gout-causing monosodium urate crystals, pseudogout-causing calcium pyrophosphate dehydrate crystals, cholesterol crystals associated with atherosclerosis, silicosis-causing silica crystals, and adjuvant alum crystals are discussed.

Eight years of follow-up after laminectomy of calcium pyrophosphate crystal deposition in the cervical yellow ligament of patient with Coffin-Lowry syndrome: A case report.

BACKGROUND: We report 8 years of follow-up after decompression to treat cervical myelopathy in a patient with Coffin-Lowry syndrome (CLS). CLS is a rare X-linked semidominant syndrome associated with growth and psychomotor retardation, general hypotonia, and skeletal abnormalities. In this patient, the spinal cord was compressed by calcium pyrophosphate crystal deposition in the cervical yellow ligament (YL). To date, only 1 report has described clinical features after surgery for calcified cervical YL in CLS. METHODS: A 15-year-old male with tetraplegia secondary to compression of the cervical spinal cord induced by a hypoplastic posterior arch of C1 and calcification of the YL from C2 to C7 was treated surgically with laminectomy from C1 to C7. The patient's history, clinical examination, imaging findings, and treatment are reported. The patient was incapable of speech because of mental retardation, so he could not describe his symptoms. Gait disturbance worsened over the 2 months before admission to our hospital. At admission, the patient could not move his extremities, and tendon reflexes of the upper and lower extremities were significantly increased. Computed tomography of the cervical spine showed YL calcification from C2 to C7. Magnetic resonance imaging showed consecutive compression of the cervical spinal cord. We diagnosed quadriplegia secondary to cervical cord damage and performed emergency surgery. RESULTS: During C1-C7 laminectomy, YL calcification in C2-C7 was observed. The calcification was confirmed as calcium pyrophosphate by crystal analysis. Quadriplegia gradually resolved, and almost disappeared by 2 weeks after the operation. Cervical hyperlordosis was observed in radiographs starting from 1 month after the operation, but it has not progressed and is not associated with any symptoms. CONCLUSIONS: The efficacy of decompression continued, and no postoperative complications have occurred during at least 8 years of follow-up.

Reinforcement of freeze-dried chitosan scaffolds with multiphasic calcium phosphate short fibers.

The composite scaffolds of the chitosan and multiphasic calcium phosphate (HW) short fibers were prepared by freeze drying and characterized by X-ray diffractometry (XRD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM and FE-SEM). The mechanical properties of the scaffolds were assessed by compression test. The incorporation of HW fibers consisting three phases of hydroxyapatite (HA), beta-tricalcium phosphate (ß-TCP) and calcium pyrophosphate (CPP) into the chitosan matrices was associated with an increase in pore size, density and compressive strength and modulus, and a decrease in porosity and swelling ratio of the scaffolds. The strongest composite scaffolds in this study with a chitosan: HW fibers weight ratio of 1:1 showed a mean porosity of 69% and a mean strength and modulus of 420kPa and 3.87MPa, respectively. The in vitro bioactivity of the composites was confirmed by the formation of a calcium phosphate rich layer on the surface of soaked scaffolds in simulated body fluid. The findings of this initial work indicate that the chitosan-multiphasic calcium phosphate short fibers may be a suitable material for bone scaffolding.

Development of a new family of monolithic calcium (pyro)phosphate glasses by soft chemistry.

UNLABELLED: The development of bioactive phosphate-based glasses is essential in biomaterials science, and especially for bone substitution applications. In this context, the preparation of amorphous calcium-phosphorus hydroxide/oxide monoliths at low temperature is a key challenge for being able to develop novel hybrid materials for these applications. We herein report for the first time the synthesis and physical chemical characterisation of a novel family of pyrophosphate-based glasses (with the formula: {[(Ca(2+))1-x(H(+)/K(+))2x]2[(P2O7(4-))1-y(PO4(3-))4y/3]} n(H2O)), which were prepared by soft chemistry using low temperatures (T<70°C) and water as a solvent. The effect of the initial Ca/Pyrophosphate ratio on the structure and morphology of these pyrophosphate glasses was investigated in detail. Depending on this ratio, a glass (mixed calcium pyro- and orthophosphate) or a glass-ceramic (Ca10K4(P2O7)6·9H2O crystals embedded in the amorphous phase) was obtained. The proportion of the crystalline phase increased with an increase in the Ca/Pyrophosphate ratio in the batch solution. As expected for a glass, the formation of the glassy material was demonstrated not to be thermodynamically but rather kinetically driven, and the washing step was found to be crucial to prevent crystallisation. The stability of the amorphous phase was discussed considering the structural degrees of freedom of pyrophosphate entities, ionic strength of the initial solution and the inhibitory effect of orthophosphate ions. Overall, this new strategy of preparation of monolithic calcium-(pyro)phosphate based glasses using soft chemistry in water is highly promising in view of preparing new functional organic-inorganic hybrids for bone substitution applications. STATEMENT OF SIGNIFICANCE: Phosphate-based glasses have gradually emerged as a potential alternative to silicate bioactive glasses in order to induce different biological mechanisms of degradation. The synthesis of such monolithic glasses at low temperature is a key step to allow new inorganic glass compositions to be reached and hybrid materials to be prepared. Although sol-gel and coacervate methods (respectively orthophosphate and metaphosphate precursors) have already been described to prepare such glasses, the use of toxic solvents and/or the final temperature treatment associated to these processes could limit the use of these materials for biomedical applications and/or the further development of hybrids. It is shown here that pyrophosphate precursors are an alternative strategy to obtain monolithic calcium (pyro)phosphate glasses under soft conditions (water solvent, 70°C).

Crystal structure of monoclinic calcium pyrophosphate dihydrate (m-CPPD) involved in inflammatory reactions and osteoarthritis.

Pure monoclinic calcium pyrophosphate dihydrate (m-CPPD) has been synthesized and characterized by synchrotron powder X-ray diffraction and neutron diffraction. Rietveld refinement of complementary diffraction data has, for the first time, allowed the crystal structure of m-CPPD to be solved. The monoclinic system P2(1)/n was confirmed and unit-cell parameters determined: a = 12.60842 (4), b = 9.24278 (4), c = 6.74885 (2) Å and ß = 104.9916 (3)°. Neutron diffraction data especially have allowed the precise determination of the position of H atoms in the structure. The relationship between the m-CPPD crystal structure and that of the triclinic calcium pyrophosphate dihydrate (t-CPPD) phase as well as other pyrophosphate phases involving other divalent cations are discussed by considering the inflammatory potential of these phases and/or their involvement in different diseases. These original structural data represent a key step in the understanding of the mechanisms of crystal formation involved in different types of arthritis and to improve early detection of calcium pyrophosphate (CPP) phases in vivo.

Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis.

Crystals cause injury in numerous disorders, and induce inflammation via the NLRP3 inflammasome, however, it remains unclear how crystals induce cell death. Here we report that crystals of calcium oxalate, monosodium urate, calcium pyrophosphate dihydrate and cystine trigger caspase-independent cell death in five different cell types, which is blocked by necrostatin-1. RNA interference for receptor-interacting protein kinase 3 (RIPK3) or mixed lineage kinase domain like (MLKL), two core proteins of the necroptosis pathway, blocks crystal cytotoxicity. Consistent with this, deficiency of RIPK3 or MLKL prevents oxalate crystal-induced acute kidney injury. The related tissue inflammation drives TNF-α-related necroptosis. Also in human oxalate crystal-related acute kidney injury, dying tubular cells stain positive for phosphorylated MLKL. Furthermore, necrostatin-1 and necrosulfonamide, an inhibitor for human MLKL suppress crystal-induced cell death in human renal progenitor cells. Together, TNF-α/TNFR1, RIPK1, RIPK3 and MLKL are molecular targets to limit crystal-induced cytotoxicity, tissue injury and organ failure.