Pure carbonate and iodate co-doped hydroxyapatites were successfully synthesized (up 60 g/batch) by an aqueous precipitation route. CO 3 2− and IO 3 − influence each other's incorporation, indicating a competitive interaction during Hap precipitation.

ABSTRACT This study investigates the structure and microstructure of hydroxyapatite substituted with iodate (IO 3 − ) and/or carbonate (CO 3 2− ) ions, within the context of long‐lived radionuclide waste conditioning. The role of the synthesis protocol, “reverse” or “direct” precipitation from aqueous solution, as well as the nature and amount of substituted anions on the structure and microstructure of hydroxyapatite samples, was evaluated using XRD, SEM, and HRTEM. The anisotropic crystallite shape assessed by HRTEM analyses was taken into account for the Rietveld refinements of the XRD patterns. Considering the reverse synthesis protocol, these refinements revealed the formation of a single hydroxyapatite phase. Despite the use of long counting times, no crystalline secondary phases were detected by XRD, regardless of the nature and amount of the substituting anions. Iodate ions substituting for A sites tend to expand the lattice, while carbonate ions located in B sites induce lattice contraction. Clearly, the influence of iodate substitution on the structural modifications is lower than that of carbonate substitution. Moreover, co‐substitution seems to modify the incorporation mode of iodate ions within the structure. The intended application of this work requires studying high substitution rates, thereby addressing gaps in the literature regarding the structural evolution of hydroxyapatite with increasing substitution rates. In this study, substitution levels reached up to 15.4 wt.% for iodate mono‐substitutions, 11.9 wt.% for carbonate mono‐substitutions, and approximately 20 wt.% for combined iodate and carbonate ions co‐substitutions.

Freeze-casting enables the fabrication of porous scaffolds for bone reconstruction, but the homogeneous dispersion of bioactive glass (BG) in polymer solutions remains challenging. Here, we report, for the first time, the synthesis of core-shell microparticles combining Cu-doped BG with poly(D,L-lactide) (PDLLA) grafted via a surface-initiated "grafting from" approach. Covalent grafting was confirmed by FT-IR spectroscopy and TGA analyses, with grafted chain lengths close to theoretical values. In simulated body fluid (SBF), the PDLLA corona temporarily delayed glass degradation and ion release, mitigating the initial "burst effect", particularly for Cu2+. In vitro, all samples displayed dose-dependent antibacterial and cytotoxic responses, but PDLLA-grafted particles improved cell viability while preserving antibacterial activity. Notably, PDLLA-BG5 achieved the best balance between bacterial inhibition and cytocompatibility. These polymer-grafted, Cu-doped BG microspheres represent promising candidates as building blocks for future PDLLA-based scaffold fabrication via freeze-casting approaches, with scalable processing and tuneable ion-release-driven biological responses.

Pyrophosphate-stabilized amorphous calcium carbonates (PyACC) are promising compounds for bone repair due to their ability to release calcium, carbonate, and phosphate ions following pyrophosphate hydrolysis. However, shaping these metastable and brittle materials using conventional methods remains a challenge, especially in the form of macroporous scaffolds, yet essential to promote cell colonization. To overcome these limitations, this article describes for the first time the design and multiscale characterization of freeze-cast alginate (Alg)-PyACC nanocomposite scaffolds. The study initially focused on the synthesis of Alg-PyACC powder through in situ coprecipitation. The presence of alginate chains in the vicinity of the PyACC was shown to affect both the powder reactivity and the release of calcium ions when placed in water (XRD, chemical titrations). In vitro cellular assays confirmed the biocompatibility of Alg-PyACC powder, supporting its use as a filler in scaffolds for bone substitutes. In a second step, the freeze-casting process was carried out using these precursor powders with varying rates of inorganic fillers. The resulting scaffolds were compared in terms of pore size and gradient (via SEM, X-ray microtomography, and mercury intrusion porosimetry). All scaffolds exhibited a pore size gradient oriented along the solidification axis, featuring unidirectional, lamellar, and interconnected pores. Interestingly, we found that the pore size and wall thickness could be controlled by the filler rate. This effect was attributed to the in situ cross-linking of alginate chains by released Ca2+ ions from the fillers, which increased viscosity, affecting temperature-driven segregation during the freezing step. Different multiscale organizations of the porosity and spatial distribution of fillers (FEG-SEM) were correlated with changes in the scaffold mechanical properties (tested via uniaxial compression). With such tunable porous and mechanical properties, Alg-PyACC composite scaffolds present attractive opportunities for specific bone substitute applications.

OBJECTIVE:Gout caused by the formation of monosodium urate (MSU) crystals and calcium pyrophosphate (CPP) deposition disease are two major types of microcrystalline pathologies in adults. They are responsible for recurrent flares that rely on interleukin (IL) 1β via activation of the NLRP3 inflammasome. Intermittent fasting (IF) is a nonpharmacologic intervention that improves age-related diseases and reduces inflammation. METHODS:In an air pouch model, crystal-induced inflammation was compared between mice fed ad libitum and mice under IF. Systemic (liver and serum) and local (air pouch cavity) modifications were evaluated by metabolomics analysis. The anti-inflammatory potential of metabolites was tested in vitro and in vivo. RESULTS:We observe that two nonconsecutive days of fasting during one week significantly prevents the inflammation provoked by MSU and CPP crystal injection into the air pouch cavity in a mouse model. This short-term fasting is associated with increased serum abundances of numerous anti-inflammatory metabolites, including β-hydroxybutyrate and spermidine (SPD), a polyamine able to reproduce biologic effects of IF. Conversely, crystal stimulation in mice fed ad libitum decreases the local production of these metabolites in the air pouch membrane. Supplementation of SPD reproduces the anti-inflammatory effect of IF and prevents crystal-induced inflammation through inhibition of NF-κB and NLRP3 inflammasome. Finally, down-regulation of SPD/spermine acetyltransferase 1, which encodes the enzyme that degrades SPD, reduces IL-1β production induced by crystal stimulation. CONCLUSION:In summary, we have identified several metabolites that recapitulate the anti-inflammatory effects of IF and could be used as IF mimetics to prevent microcrystal inflammation.

Introduction Les cristaux d’urate monosodique (UMS) et de pyrophosphate de calcium (PPC) sont responsables de poussées inflammatoires récurrentes dépendantes de l’interleukine (IL)-1β produite par les macrophages. La coexistence des cristaux d’UMS et de PPC est fréquente dans les liquides articulaires et dans les tophus. Les dépôts de cristaux d’UMS et de PPC sont organisés en lobules entourés de cellules géantes multinucléées (CGM) qui proviennent de la fusion des macrophages. Les cristaux activent les macrophages soit par interactions directes avec la membrane cellulaire soit après leur phagocytose. Nos objectifs sont de décrire la phagocytose des cristaux et leurs devenirs intracellulaires dans les macrophages et les CGM. Matériels et méthodes Des cristaux synthétiques d’UMS et de PPC ont été utilisés pour stimuler différents types de macrophages (lignées murine RAW264.7 et humaine THP-1, macrophages isolés de la moelle osseuse (BMDM) et cellules mononucléées du sang périphérique (PBMC)). La phagocytose des cristaux a été évaluée in vitro en utilisant des cristaux greffés de nanoparticules organiques fluorescentes (FON). Le devenir intracellulaire a été évalué par enregistrement continu de 18 à l’aide d’un microscope apotome. Le rôle des lysosomes et de la pompe V-ATPase a été évalué en utilisant une sonde à pH (Lysosensor Dnd dextran) et un inhibiteur pharmacologique (bafilomycine A1). Résultats Les macrophages (lignées et cellules primaires murines et humaines) et les CGM ont phagocyté rapidement les cristaux d’UMS et de PPC. Tous les cristaux ont été phagocytés par les macrophages après une heure de stimulation. En histologie, les cristaux d’UMS ont été identifiés dans les CGM des tophus prélevés des patients. In vitro, les cristaux d’UMS et de PPC ont induit la formation des CGM à partir des RAW264.7. Ces CGM ont phagocyté une plus grande quantité de cristaux que les macrophages d’origine. Après leur phagocytose, les cristaux pouvaient être expulsés vers le milieu extérieur ou vers une autre cellule, ou être dissouts. La dissolution n’a été observée que pour les cristaux d’UMS et seulement après 48heures de stimulation (Figure 1A). Après 5jours de stimulation, tous les cristaux d’UMS ont été dissouts. Aucune dissolution des cristaux de CPP n’a été observée, y compris après 7jours de stimulation. À 48h de stimulation, la capacité de dissolution des cristaux d’UMS variait en fonction des cellules : les RAW264.7 ont dissout 17,5 % des cristaux d’UMS présents dans le champ d’enregistrement, les BMDM et THP-1 ont respectivement dissout 24,3 % et 56,0 % des cristaux (Figure 1B). Les CGM on dissout une grande partie des cristaux qu’elles ont phagocytés. La dissolution des cristaux d’UMS était accompagnée d’une mort de la cellule dans une grande majorité des cellules RAW264.7 et des PBMC. En revanche, elle n’était pas associée à une mort des THP-1. Enfin, nous avons observé que la phagocytose et la dissolution des cristaux d’UMS étaient associées à une augmentation du nombre de lysosomes et de son acidification. L’inhibition de l’acidification des lysosomes par la bafilomycine A1 a empêché complètement la dissolution des cristaux d’UMS (Figure 1C) Conclusion Après leur phagocytose, les cristaux d’UMS peuvent être expulsés ou dissouts. La dissolution des cristaux est tardive et semble dépendre de l’acidification des lysosomes. L’identification des facteurs impliqués dans ces phénomènes permettra de proposer des nouvelles approches thérapeutiques visant à accélérer la dissolution des cristaux.

In the biomedical field, nanocrystalline hydroxyapatite is still one of the most attractive candidates as a bone substitute material due to its analogies with native bone mineral features regarding chemical composition, bioactivity and osteoconductivity. Ion substitution and low crystallinity are also fundamental characteristics of bone apatite, making it metastable, bioresorbable and reactive. In the present work, biomimetic apatite and apatite/chitosan composites were produced by dissolution–precipitation synthesis, using mussel shells as a calcium biogenic source. With an eye on possible bone reconstruction and drug delivery applications, apatite/chitosan composites were loaded with strontium ranelate, an antiosteoporotic drug. Due to the metastability and temperature sensitivity of the produced composites, sintering could be carried out by conventional methods, and therefore, cold sintering was selected for the densification of the materials. The composites were consolidated up to ~90% relative density by applying a uniaxial pressure up to 1.5 GPa at room temperature for 10 min. Both the synthesised powders and cold-sintered samples were characterised from a physical and chemical point of view to demonstrate the effective production of biomimetic apatite/chitosan composites from mussel shells and exclude possible structural changes after sintering. Preliminary in vitro tests were also performed, which revealed a sustained release of strontium ranelate for about 19 days and no cytotoxicity towards human osteoblastic-like cells (MG63) exposed up to 72 h to the drug-containing composite extract.

Introduction L’inflammation induite par les cristaux d’urate de sodium (UMS) et de pyrophosphate de calcium (PPC) dépend de l’interleukine (IL)-1β dont la production est régulée par l’inflammasome NLRP3. NLRP3 peut être activé par des variations de l’osmolarité. Lors d’un stress hypotonique extracellulaire, il se produit un gonflement cellulaire par entrée d’eau osmo-induite, suivi d’un mécanisme de régulation, appelé regulatory volume decrease (RVD), permettant un retour au volume initial. Ce mécanisme RVD dépend du canal anionique LRRC8. Nos objectifs sont de déterminer comment le canal osmo-sensible LRRC8 régule l’inflammation induite par les cristaux. Matériels et méthodes In vitro, les monocytes de la lignée humaine THP1, les macrophages murins isolés de la moelle osseuse (BMDM) sont stimulés par des cristaux synthétiques dans des milieux de culture iso-, hypo- ou hypertoniques. Le rôle du canal LRRC8A est évalué en utilisant un inhibiteur pharmacologique (DCPIB) et des cellules dont l’expression de LRRC8A est délétée par shRNA ou knock-down (KO). La production des cytokines inflammatoires et de l’ATP est dosée par ELISA. Les courants chlorure et ATP sont enregistrés par patch-clamp. La taille des cellules est évaluée par méthodes de fluorescence. In vivo, l’inflammation a été évaluée dans le modèle de poche à air sur des souris sauvages (notées Lrrc8Acont) et des souris dont LRRC8A est invalidé dans la lignée macrophagique (notées Lrrc8AΔMo). Résultats Les liquides articulaires prélevés au cours de l’inflammation microcristalline (goutte et PPC) avaient une concentration en IL-1β et IL-8 plus forte que les liquides d’arthrose. En revanche, leur osmolarité était plus basse et inversement corrélée à la concentration des cytokines. Dans le modèle murin, l’augmentation de l’osmolarité plasmatique par injection de mannitol diminuait l’inflammation induite par les cristaux, en termes d’infiltrat cellulaire et de production des cytokines inflammatoires. L’inhibition des aquaporines par du chlorure de mercure (HgCl2) diminuait également l’inflammation microcristalline. In vitro, les cellules THP-1 et BDMD traitées par HgCl2 diminuaient de façon substantielle la production d’IL-1β et l’activation de l’inflammasome NLRP3 induites par les cristaux. De plus, la production de l’IL-1β était diminué lorsque les cellules étaient cultivées en milieu hypertonique et augmentées en milieu hypotonique. Les cristaux induisaient une augmentation du volume cellulaire suivi d’un retour au volume initial, mimant le phénomène de RVD induit par une stimulation hypotonique. Celle-ci induisait la production d’un courant chlorure sortant suggérant l’implication du canal LRRC8. Ce phénomène de RVD et le courant chlorure n’étaient plus observés dans les cellules dont l’expression de LRRC8A est délétée ou dans les cellules traitées par DCPIB. In vivo, l’inflammation induite par les cristaux d’UMS et de PPC était diminuée chez les souris Lrrc8AΔMo par aux souris Lrrc8Acont. Les BMDM isolés souris Lrrc8AΔMo produisaient moins d’IL-1β par rapport aux BDMD isolés des souris Lrrc8Acont. Enfin, nous observons que les cristaux d’UMS et de PPC induisaient une libération extracellulaire d’ATP via le canal LRRC8. Cette libération d’ATP précédait le phénomène de RVD et était nécessaire à l’activation de l’inflammasome NLRP3 et de la production de l’IL-1β via les récepteurs purinergiques P2Y et le calcium intracellulaire. Conclusion Nos résultats mettent en évidence de nouveaux mécanismes de l’inflammation microcristalline avec un rôle central du canal osmo-sensible LRRC8 et des récepteurs purinergiques P2Y (Fig. 1) au cours des crises microcristallines, les cristaux et l’hypo-osmolarité du liquide articulaire induisent un gonflement des macrophages, ce qui active le canal anionique régulateur de volume (VRAC) LRRC8 aboutissant à la libération de l’ATP. L’ATP par action paracrine et autocrine active les récepteurs purinergiques P2Y2 et P2Y6 qui via la phospholipase C (PLC) et le calcium intracellulaire participe à l’activation de l’inflammasome NLRP3 et à la production de l’IL-1β.

Research on biomaterials typically starts with cytocompatibility evaluation, using the ISO 10993-5 standard as a reference that relies on extract tests to determine whether the material is safe (cell metabolic activity should exceed 70 %). However, the generalized approach within the standard may not accurately reflect the material's behavior in direct contact with cells, raising concerns about its effectiveness. Calcium phosphates (CaPs) are a group of materials that, despite being highly biocompatible and promoting bone formation, still exhibit inconsistencies in basic cytotoxicity evaluations. Hence, in order to test the cytocompatibility dependence on different experimental setups and material -cell interactions, we used amorphous calcium phosphate, alpha-tricalcium phosphate, hydroxyapatite, and octacalcium phosphate (0.1 mg/mL to 5 mg/mL) with core cell lines of bone microenvironment: mesenchymal stem cells, osteoblast-like and endothelial cells. All materials have been characterized for their physicochemical properties before and after cellular contact and once in vitro assays were finalized, groups identified as 'cytotoxic' were further analyzed using a modified Annexin V apoptosis assay to accurately determine cell death. The obtained results showed that indirect contact following ISO standards had no sensitivity of tested cells to the materials, but direct contact tests at physiological concentrations revealed decreased metabolic activity and viability. In summary, our findings offer valuable guidelines for handling biomaterials, especially in powder form, to better evaluate their biological properties and avoid false negatives commonly associated with the traditional standard approach.

Silicate-based bioactive glass nano/microspheres hold significant promise for bone substitution by facilitating osteointegration through the release of biologically active ions and the formation of a biomimetic apatite layer. Cu-doping enhances properties such as pro-angiogenic and antibacterial behavior. While sol-gel methods usually yield homogeneous spherical particles for pure silica or binary glasses, synthesizing poorly aggregated Cu-doped ternary glass nano/microparticles without a secondary CuO crystalline phase remains challenging. This article introduces an alternative method for fabricating Cu-doped ternary microparticles using sol-gel chemistry combined with spray-drying. The resulting microspheres exhibit well-defined, poorly aggregated particles with spherical shapes and diameters of a few microns. Copper primarily integrates into the microspheres as Cu0 nanoparticles and as Cu2 + within the amorphous network. This doping affects silica network connectivity, as calcium and phosphorus are preferentially distributed in the glass network (respectively as network modifiers and formers) or involved in amorphous calcium phosphate nano-domains depending on the doping rate. These differences affect the interaction with simulated body fluid. Network depolymerization, ion release (SiO4 4-, Ca2 + , PO4 3-, Cu2 + ), and apatite nanocrystal layer formation are impacted, as well as copper release. The latter is mainly provided by the copper involved in the silica network and not from metal nanoparticles, most of which remain in the microspheres after interaction. This understanding holds promising implications for potential therapeutic applications, offering possibilities for both short-term and long-term delivery of a tunable copper dose.

The present work reports the adsorption, release, antibacterial properties, and in vitro cytotoxicity of sodium fusidate (SF) associated with a carbonated calcium phosphate bone cement. The adsorption study of SF on cement powder compared to stoichiometric hydroxyapatite and nanocrystalline carbonated apatite was investigated to understand the interaction between this antibiotic and the calcium phosphate phases involved in the cement formulation and setting reaction. The adsorption data revealed a fast kinetic process. However, the evolution of the amount of adsorbed SF was well described by a Freundlich -type isotherm characterized by a low adsorption capacity of the materials toward the SF molecule. The in vitro release results indicated a prolonged and controlled SF release for up to 34 days. The SF amounts eluted daily were at a therapeutic level (0.5 -2 mg/L) and close to the antibiotic minimum inhibitory concentration (0.1 -0.9 mg/L). Furthermore, the release data fitting and modeling suggested that the drug release occurred mainly by a diffusion mechanism. The antibacterial activity showed the effectiveness of SF released from the formulated cements against Staphylococcus aureus . Furthermore, the biological in vitro study demonstrated that the tested cements didn 't show any cytotoxicity towards human peripheral blood mononuclear cells and did not significantly induce inflammation markers like IL -8.

The present work focuses on the development of composite cements based on dicalcium phosphate dihydrate (DCPD), calcium carbonate CaCO3, sodium alginate (AG), and sodium fusidate (FS). The effect of AG, setting accelerator (0.5 M of Na2HPO4), and antibacterial agent (FS) on the features (setting ability, injectability, cohesion, and compressive strength) of DCPD-CaCO3-based cement was investigated. The reference and composite cements are composed of a nanocrystalline carbonated apatite, similar to bone mineral, and an excess of unreacted vaterite (CaCO3). The incorporation of AG increased the composite cement's total porosity compared to the reference cement (CR). The evaluation of the injectability and cohesion properties showed that adding 10 wt % of AG resulted in a total extrusion of the paste with an improvement in the cohesion of the cement paste. The compressive strength of the cements raised from 3.2 for CR up to 7 MPa with the addition of 10 % of AG and Na2HPO4 . The setting time is significantly reduced by introducing Na2HPO4, resulting in appropriate values (<= 30 min) for clinical use. Moreover, incorporating 3 wt % of FS in the composite cements had no significant effect on their features. The release study of FS-loaded composites showed sustained and controlled release profiles, with daily released amounts at the therapeutic level. The antibacterial activity of the designed FS-loaded composites demonstrated the effectiveness of the specimens in inhibiting the growth of S. Aureus. Furthermore, the in vitro biological tests did not show any toxicity of the tested cements towards hPBMCs, thereby confirming their biocompatibility.

Deposition of monosodium urate and calcium pyrophosphate (MSU and CPP) micro-crystals is responsible for painful and recurrent inflammation flares in gout and chondrocalcinosis. In these pathologies, the inflammatory reactions are due to the activation of macrophages responsible for releasing various cytokines including IL-1 beta. The maturation of IL-1 beta is mediated by the multiprotein NLRP3 inflammasome. Here, we find that activation of the NLRP3 inflammasome by crystals and concomitant production of IL-1 beta depend on cell volume regulation via activation of the osmo-sensitive LRRC8 anion channels. Both pharmacological inhibition and genetic silencing of LRRC8 abolish NLRP3 inflammasome activation by crystals in vitro and in mouse models of crystal-induced inflammation. Activation of LRRC8 upon MSU/CPP crystal exposure induces ATP release, P2Y receptor activation and intracellular calcium increase necessary for NLRP3 inflammasome activation and IL-1 beta maturation. We identify a function of the LRRC8 osmo-sensitive anion channels with pathophysiological relevance in the context of joint crystal-induced inflammation. Formation of urate and calcium pyrophosphate micro-crystals is responsible for painful inflammatory flares in gout and chondrocalcinosis. Here the authors show that the osmo-sensitive LRRC8 anion channel is involved with macrophage inflammasome activation by crystals involving cell volume regulation and ATP release leading to P2Y receptor activation.

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 β) 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.

A chitosan gel additive modulates the initial vaterite dissolution–recrystallisation in injectable aragonite-based composite cement and promotes its in vitro bioactivity.

Medical implants have improved the quality of life of many patients. However, surgical intervention may eventually lead to implant microbial contamination. The aims of this research were to develop an easy, robust, quantitative assay to assess surface antimicrobial activities, especially the anti-nascent biofilm activity, and to identify control surfaces, allowing for international comparisons. Using new antimicrobial assays to assess the inhibition of nascent biofilm during persistent contact or after transient contact with bacteria, we show that the 5 cent Euro coin or other metal-based antibacterial coins can be used as positive controls, as more than 4 log reduction on bacterial survival was observed when using either S. aureus or P. aeruginosa as targets. The methods and controls described here could be useful to develop an easy, flexible and standardizable assay to assess relevant antimicrobial activities of new implant materials developed by industries and academics.