Biomimetic apatite-based composite materials obtained by spark plasma sintering (SPS): physicochemical and mechanical characterizations.

Nanocrystalline calcium phosphate apatites are biomimetic compounds analogous to bone mineral and are at the origin of the bioactivity of most biomaterials used as bone substitutes. Their unique surface reactivity originates from the presence of a hydrated layer containing labile ions (mostly divalent ones). So the setup of 3D biocompatible apatite-based bioceramics exhibiting a high reactivity requests the development of «low¼ temperature consolidation processes such as spark plasma sintering (SPS), in order to preserve the characteristics of the hydrated nanocrystals. However, mechanical performances may still need to be improved for such nanocrystalline apatite bioceramics, especially in view of load-bearing applications. The reinforcement by association with biopolymers represents an appealing approach, while preserving the advantageous biological properties of biomimetic apatites. Herein, we report the preparation of composites based on biomimetic apatite associated with various quantities of microcrystalline cellulose (MCC, 1-20 wt%), a natural fibrous polymer. The SPS-consolidated composites were analyzed from both physicochemical (X-ray diffraction, Fourier transform infrared, solid state NMR) and mechanical (Brazilian test) viewpoints. The preservation of the physicochemical characteristics of apatite and cellulose in the final material was observed. Mechanical properties of the composite materials were found to be directly related to the polymer/apatite ratios and a maximum crushing strength was reached for 10 wt% of MCC.

Sodium fusidate loaded apatitic calcium phosphates: Adsorption behavior, release kinetics, antibacterial efficacy, and cytotoxicity assessment.

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.

Challenging applicability of ISO 10993-5 for calcium phosphate biomaterials evaluation: Towards more accurate in vitro cytotoxicity assessment.

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, α-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.

Interaction between a bisphosphonate, tiludronate, and biomimetic nanocrystalline apatites.

Bisphosphonates (BPs) are well established as successful antiresorptive agents for the prevention and treatment of bone diseases such as osteoporosis and Paget's disease. The aim of this work was to clarify the reaction mechanisms between a BP molecule, tiludronate, and the nanocrystalline apatite surface. The adsorption of tiludronate on well-characterized synthetic biomimetic nanocrystalline apatites with homogeneous but different compositions and surface characteristics was investigated to determine the effect of the nanocrystalline apatite substrate on the adsorption behavior. The results show that the adsorption of tiludronate on nanocrystalline biomimetic apatite surfaces varies over a large range. The most immature apatitic samples exhibited the highest affinity and the greatest amount adsorbed at saturation. Maturation of the nanocrystals induces a decrease of these values. The amount of phosphate ion released per adsorbed BP molecule varied, depending on the nanocrystalline substrate considered. The adsorption mechanism, although associated with a release of phosphate ions, cannot be considered as a simple ion exchange process involving one or two phosphate ions on the surface. A two-step process is proposed consisting of a surface binding of BP groups to calcium ions associated with a proton release inducing the protonation of surface orthophosphate ions and their eventual solubilization.

Composition and properties of silver-containing calcium carbonate-calcium phosphate bone cement.

The introduction of silver, either in the liquid phase (as silver nitrate solution: Ag(L)) or in the solid phase (as silver phosphate salt: Ag(S)) of calcium carbonate-calcium phosphate (CaCO3-CaP) bone cement, its influence on the composition of the set cement (C-Ag(L) and C-Ag(S) cements with a Ca/Ag atomic ratio equal to 10.3) and its biological properties were investigated. The fine characterisation of the chemical setting of silver-doped and reference cements was performed using FTIR spectroscopy. We showed that the formation of apatite was enhanced from the first hours of maturation of C-Ag(L) cement in comparison with the reference cement, whereas a longer period of maturation (about 10 h) was required to observe this increase for C-Ag(S) cement, although in both cases, silver was present in the set cements mainly as silver phosphate. The role of silver nitrate on the setting chemical reaction is discussed and a chemical scheme is proposed. Antibacterial activity tests (S. aureus and S. epidermidis) and in vitro cytotoxicity tests (human bone marrow stromal cells (HBMSC)) showed that silver-loaded CaCO3-CaP cements had antibacterial properties (anti-adhesion and anti-biofilm formation) without a toxic effect on HBMSC cells, making C-Ag(S) cement a promising candidate for the prevention of bone implant-associated infections.

Exploring the Formation Kinetics of Octacalcium Phosphate from Alpha-Tricalcium Phosphate: Synthesis Scale-Up, Determination of Transient Phases, Their Morphology and Biocompatibility.

Even with decades of research studies behind octacalcium phosphate (OCP), determination of OCP phase formation has proved to be a cumbersome challenge. Even though obtaining a large quantity of OCP is important for potential clinical uses, it still remains a hindrance to obtain high yields of pure OCP. Taking that into consideration, the purpose of this study was to scale-up OCP synthesis for the first time and to use a multi-technique approach to follow the phase transformation pathway at multiple time points. In the present study, OCP has been synthesized from α-tricalcium phosphate (α-TCP), and subsequently scaled-up tenfold and hundredfold (100 mg → 10 g). The hydrolysis mechanism has been followed and described by using XRD and FTIR spectroscopy, as well as Raman and SEM. Gradual transformation into the OCP phase transpired through dicalcium phosphate dihydrate (brushite, DCPD, up to ~36%) as an intermediary phase. Furthermore, the obtained transitional phases and final OCP phases (across all scale-up levels) were tested with human bone marrow-derived mesenchymal stem cells (hBMSCs), in order to see how different phase mixtures affect the cell viability, and also to corroborate the safety of the scaled-up product. Twelve out of seventeen specimens showed satisfactory percentages of cell viability and confirmed the prospective use of scaled-up OCP in further in vitro studies. The present study, therefore, provides the first scale-up process of OCP synthesis, an in depth understanding of the formation pathway, and investigation of the parameters able to contribute in the OCP phase formation.

Modeling Pkd1 gene-targeted strategies for correction of polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) causes renal cysts and leads to end-stage renal disease in midlife due mainly to PKD1 gene mutations. Virtually no studies have explored gene therapeutic strategies for long-term effective treatment of PKD. Toward this aim, the severely cystic Pkd1-null mouse model was targeted with a series of transgene transfers using genomic Pkd1 under its regulatory elements (Pkd1wt), a kidney-targeted Pkd1 gene (SBPkd1), or Pkd1Minigene. The introduced Pkd1wt gene constructs with ∼8-fold overexpression display similar endogenous cellular profiles and full complementation of Pkd1-/- phenotype and establish the referral Pkd1 genomic length for proper regulation. SBPkd1 transgene transfer expressing 0.6- or 7-fold Pkd1 endogenous levels is sufficient to correct glomerular and proximal tubular cysts and to markedly postpone cysts in other tubular segments as well, showing that the small SB elements appreciably overlap with Pkd1 promoter/5' UTR regulation. Renal-targeted Pkd1Minigene at high copy numbers conveys an expression level similar to that of the endogenous Pkd1 gene, with widespread and homogeneous weak Pkd1 cellular signal, partially rescuing all cystic tubular segments. These transgene transfers determine that Pkd1 intragenic sequences regulate not only expression levels but also spatiotemporal patterns. Importantly, our study demonstrates that Pkd1 re-expression from hybrid therapeutic constructs can ameliorate, with considerably extended lifespan, or eliminate PKD.

Biomimetic apatite-based biomaterials: on the critical impact of synthesis and post-synthesis parameters.

Nanocrystalline apatites are major constituents of hard tissues, and attempts are made worldwide to prepare synthetic analogs. However the impact of synthesis/postsynthesis parameters is often disregarded. Based on an updated knowledge on such compounds, we inspected the effects of synthesis parameters (maturation time, temperature, pH, nature of counter-ions) and post-treatments (re-immersion in aqueous media, thermal treatment) on physicochemical characteristics. Great modifications were noticed during the 3 first days of maturation, where a progressive evolution of the apatite phase (localized in the core of the nanocrystals) toward stoichiometry was observed at the expense of the non-apatitic surface layer which progressively disappears. Similar trends were also evidenced for maturation run under increasing temperatures, studied here in the range 20-100 °C. pH impacted more specifically the chemical composition. The nature of the counter-ion in the starting phosphate salt influenced composition and nonstoichiometry, depending on its (in)ability to be incorporated in the lattice. Freeze-drying allowed to preserve a high surface reactivity, although further evolutions were noticed after re-immersion. Effects of a thermal treatment of dried samples were unveiled, suggesting a denaturation of the hydrated layer on the nanocrystals. This work underlines the necessity of a good control of synthesis/postsynthesis parameters for the production of biomimetic apatites.

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.

Antibacterial Electrodeposited Copper-Doped Calcium Phosphate Coatings for Dental Implants.

Dental implants provide a good solution for the replacement of tooth roots. However, the full restoration of tooth functions relies on the bone-healing period before positioning the abutment and the crown on the implant, with the associated risk of post-operative infection. This study aimed at developing a homogeneous and adherent thin calcium phosphate antibacterial coating on titanium dental implants by electrodeposition to favor both implant osseointegration and to limit peri-implantitis. By combining global (XRD, FTIR-ATR, elemental titration) and local (SEM, Raman spectroscopy on the coating surface and thickness) characterization techniques, we determined the effect of electrodeposition time on the characteristics and phases content of the coating and the associated mechanism of its formation. The 1-min-electrodeposited CaP coating (thickness: 2 ± 1 µm) was mainly composed of nano-needles of octacalcium phosphate. We demonstrated its mechanical stability after screwing and unscrewing the dental implant in an artificial jawbone. Then, we showed that we can reach a high copper incorporation rate (up to a 27% Cu/(Cu+Ca) molar ratio) in this CaP coating by using an ionic exchange post-treatment with copper nitrate solution at different concentrations. The biological properties (antibiofilm activity and cytotoxicity) were tested in vitro using a model of mixed bacteria biofilm mimicking peri-implantitis and the EN 10993-5 standard (direct contact), respectively. An efficient copper-doping dose was determined, providing an antibiofilm property to the coating without cytotoxic side effects. By combining the electrodeposition and copper ionic exchange processes, we can develop an antibiofilm calcium phosphate coating on dental implants with a tunable thickness and phases content.

Injectable bone cement containing carboxymethyl cellulose microparticles as a silver delivery system able to reduce implant-associated infection risk.

In the challenging quest for a solution to reduce the risk of implant-associated infections in bone substitution surgery, the use of silver ions is promising regarding its broad spectrum on planktonic, sessile as well as multiresistant bacteria. In view of controlling its delivery in situ at the desired dose, we investigated its encapsulation in carboxymethyl cellulose (CMC) microparticles by spray-drying and included the latter in the formulation of a self-setting calcium phosphate bone cement. We implemented an original step-by-step methodology starting from the in vitro study of the antibacterial properties and cytotoxicity of two silver salts of different solubility in aqueous medium and then in the cement to determine the range of silver loading able to confer anti-biofilm and non-cytotoxic properties to the biomaterial. A dose-dependent efficiency of silver was demonstrated on the main species involved in bone-implant infection (S. aureus and S. epidermidis). Loading silver in microspheres instead of loading it directly inside the cement permitted to avoid undesired silver-cement interactions during setting and led to a faster release of silver, i.e. to a higher dose released within the first days combining anti-biofilm activity and preserved cytocompatibility. In addition, a combined interest of the introduction of about 10% (w/w) silver-loaded CMC microspheres in the cement formulation was demonstrated leading to a fully injectable and highly porous (77%) cement, showing a compressive strength analogous to cancellous bone. This injectable silver-loaded biomimetic composite cement formulation constitutes a versatile bone substitute material with tunable drug delivery properties, able to fight against bone implant associated infection. STATEMENT OF SIGNIFICANCE: This study is based on two innovative scientific aspects regarding the literature: i) Choice of silver ions as antibacterial agent combined with their way of incorporation: Carboxymethylcellulose has never been tested into bone cement to control its drug loading and release properties. ii) Methodology to formulate an antibacterial and injectable bone cement: original and multidisciplinary step-by-step methodology to first define, through (micro)biological tests on two silver salts with different solubilities, the targeted range of silver dose to include in carboxymethylcellulose microspheres and, then optimization of silver-loaded microparticles processing to fulfill requirements (encapsulation efficiency and size). The obtained fully injectable composite controls the early delivery of active dose of silver (from 3 h and over 2 weeks) able to fight against bone implant-associated infections.

Mineral maturity and crystallinity index are distinct characteristics of bone mineral.

The purpose of this study was to test the hypothesis that mineral maturity and crystallinity index are two different characteristics of bone mineral. To this end, Fourier transform infrared microspectroscopy (FTIRM) was used. To test our hypothesis, synthetic apatites and human bone samples were used for the validation of the two parameters using FTIRM. Iliac crest samples from seven human controls and two with skeletal fluorosis were analyzed at the bone structural unit (BSU) level by FTIRM on sections 2-4 mum thick. Mineral maturity and crystallinity index were highly correlated in synthetic apatites but poorly correlated in normal human bone. In skeletal fluorosis, crystallinity index was increased and maturity decreased, supporting the fact of separate measurement of these two parameters. Moreover, results obtained in fluorosis suggested that mineral characteristics can be modified independently of bone remodeling. In conclusion, mineral maturity and crystallinity index are two different parameters measured separately by FTIRM and offering new perspectives to assess bone mineral traits in osteoporosis.

A soft-chemistry approach to the synthesis of amorphous calcium ortho/pyrophosphate biomaterials of tunable composition.

The development of amorphous phosphate-based materials is of major interest in the field of biomaterials science, and especially for bone substitution applications. In this context, we herein report the synthesis of gel-derived hydrated amorphous calcium/sodium ortho/pyrophosphate materials at ambient temperature and in water. For the first time, such materials have been obtained in a large range of tunable orthophosphate/pyrophosphate molar ratios. Multi-scale characterization was carried out thanks to various techniques, including advanced multinuclear solid state NMR. It allowed the quantification of each ionic/molecular species leading to a general formula for these materials: [(Ca2+y Na+z H+3+x-2y-z)(PO43-)1-x(P2O74-)x](H2O)u. Beyond this formula, the analyses suggest that these amorphous solids are formed by the aggregation of colloids and that surface water and sodium could play a role in the cohesion of the whole material. Although the full comprehension of mechanisms of formation and structure is still to be investigated in detail, the straightforward synthesis of these new amorphous materials opens up many perspectives in the field of materials for bone substitution and regeneration. STATEMENT OF SIGNIFICANCE: The metastability of amorphous phosphate-based materials with various chain length often improves their (bio)chemical reactivity. However, the control of the ratio of the different phosphate entities has not been yet described especially for small ions (pyrophosphate/orthophosphate) and using soft chemistry, whereas it opens the way for the tuning of enzyme- and/or pH-driven degradation and biological properties. Our study focuses on elaboration of amorphous gel-derived hydrated calcium/sodium ortho/pyrophosphate solids at 70 °C with a large range of orthophosphate/pyrophosphate ratios. Multi-scale characterization was carried out using various techniques such as advanced multinuclear SSNMR (31P, 23Na, 1H, 43Ca). Analyses suggest that these solids are formed by colloids aggregation and that the location of mobile water and sodium could play a role in the material cohesion.

Calcium phosphate coatings elaborated by the soaking process on titanium dental implants: Surface preparation, processing and physical-chemical characterization.

OBJECTIVE: Dental implant manufacturers are looking for new surfaces to improve osseointegration. It is accepted that calcium phosphate coatings favor bone healing. Among all the techniques, the soaking process seems attractive because of its ability in producing a bioactive coating at low temperature. The objective of this study is to improve the titanium implant surface roughness and chemistry by optimizing the surface preparation and the soaking process parameters to produce a bioactive and adherent calcium phosphate coating. METHODS: Titanium samples were sandblasted and acid etched. Coatings were realized by an alternate soaking process including a centrifugation step to create a phosphate solution thin film on the implant that reacts with the calcium of the second bath. We performed a characterization of the sample surface with complementary physical and physico-chemical techniques to assess the effect of surface preparation and coating process operating parameters on coating formation and characteristics. RESULTS: Surface preparation led to a roughness around 1.6µm, micro-porosities, high surface wettability and removed the embedded sandblasting particles. We showed that the centrifugation step is critical and determines the coating formation, coverage and thickness. A thin coating (∼2µm) composed of apatite analogous to bone mineral was deposited. The coating adhesion was demonstrated by screwing/unscrewing test in an artificial jawbone. SIGNIFICANCE: The titanium dental implant pre-treatment and coating developed in this study is expected to favor early implant osseointegration through coating dissolution in vivo and could be associated with biological active agents to confer additional functionality to the coating.

Adsorption of Proteins on m-CPPD and Urate Crystals Inhibits Crystal-induced Cell Responses: Study on Albumin-crystal Interaction.

The biological effects and cellular activations triggered by monosodium urate (MSU) and calcium pyrophosphate dihydrate (monoclinic: m-CPPD) crystals might be modulated by protein coating on the crystal surface. This study is aimed at: (i) Identifying proteins adsorbed on m-CPPD crystals, and the underlying mechanisms of protein adsorption, and (ii) to understand how protein coating did modulate the inflammatory properties of m-CPPD crystals. The effects of protein coating were assessed in vitro using primary macrophages and THP1 monocytes. Physico-chemical studies on the adsorption of bovine serum albumin (BSA) upon m-CPPD crystals were performed. Adsorption of serum proteins, and BSA on MSU, as well as upon m-CPPD crystals, inhibited their capacity to induce interleukin-1-ß secretions, along with a decreased ATP secretion, and a disturbance of mitochondrial membrane depolarization, suggesting an alteration of NLRP3 inflammasome activation. Proteomic analysis identified numerous m-CPPD-associated proteins including hemoglobin, complement, albumin, apolipoproteins and coagulation factors. BSA adsorption on m-CPPD crystals followed a Langmuir-Freundlich isotherm, suggesting that it could modulate m-CPPD crystal-induced cell responses through crystal/cell-membrane interaction. BSA is adsorbed on m-CPPD crystals with weak interactions, confirmed by the preliminary AFM study, but strong interactions of BSA molecules with each other occurred favoring crystal agglomeration, which might contribute to a decrease in the inflammatory properties of m-CPPD crystals. These findings give new insights into the pathogenesis of crystal-related rheumatic diseases and subsequently may open the way for new therapeutic approaches.

TEVAR in patients with late complications of aortic coarctation repair.

PURPOSE: To review the use of thoracic endovascular aortic repair (TEVAR) for late pseudoaneurysm formation after surgical repair of aortic coarctation. METHODS: From May 2001 to May 2005, 8 patients (5 men; mean age 47.6 years, range 18-73) with a history of aortic coarctation repairs 17 to 40 years prior were referred to our institution for an anastomotic thoracic pseudoaneurysm. TEVAR was performed successfully in 7 patients; 1 died of suspected aneurysm rupture before the scheduled procedure. A carotid-subclavian bypass was performed in 3 patients. RESULTS: All the procedures were immediately successful. No type I endoleaks were seen on the final control angiogram, but 2 of the patients with carotid-subclavian bypasses required additional left subclavian artery embolization due to type II endoleak. One of these patients died before embolotherapy on the 5th postoperative day from presumed aneurysm rupture (14% 30-day mortality rate). Over a follow-up period ranging from 15 to 72 months (mean 37), all the false aneurysms have remained thrombosed and the mean diameter has decreased from 44 to 23 mm. No endograft-related complications have occurred, and no further interventions have so far been necessary. CONCLUSION: TEVAR is a feasible alternative treatment for patients who have already undergone surgical repair of aortic coarctation. Technical issues regarding the endovascular strategy should be discussed with a multidisciplinary team to define the correct interventional plan.

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.

Time-domain THz spectroscopy of the characteristics of hydroxyapatite provides a signature of heating in bone tissue.

Because of the importance of bone in the biomedical, forensic and archaeological contexts, new investigation techniques are constantly required to better characterize bone ultrastructure. In the present paper, we provide an extended investigation of the vibrational features of bone tissue in the 0.1-3 THz frequency range by time-domain THz spectroscopy. Their assignment is supported by a combination of X-ray diffraction and DFT-normal modes calculations. We investigate the effect of heating on bone tissue and synthetic calcium-phosphates compounds with close structure and composition to bone mineral, including stoichiometric and non-stoichiometric hydroxyapatite (HA), tricalcium phosphate, calcium pyrophosphate and tetracalcium phosphate. We thus demonstrate that the narrow vibrational mode at 2.1 THz in bone samples exposed to thermal treatment above 750 °C arises from a lattice mode of stoichiometric HA. This feature is also observed in the other synthetic compounds, although weaker or broader, but is completely smeared out in the non-stoichiometric HA, close to natural bone mineral composition, or in synthetic poorly crystalline HA powder. The THz spectral range therefore provides a clear signature of the crystalline state of the investigated bone tissue and could, therefore be used to monitor or identify structural transitions occurring in bone upon heating.

Preparation, physical-chemical characterisation and cytocompatibility of calcium carbonate cements.

The feasibility of calcium carbonate cements involving the recrystallisation of metastable calcium carbonate varieties has been demonstrated. Calcium carbonate cement compositions presented in this paper can be prepared straightforwardly by simply mixing water (liquid phase) with two calcium carbonate phases (solid phase) which can be easily obtained by precipitation. An original cement composition was obtained by mixing amorphous calcium carbonate and vaterite with an aqueous medium. The cement set and hardened within 2h at 37 degrees C in an atmosphere saturated with water and the final composition of the cement consisted mostly of aragonite. The hardened cement was microporous and showed poor mechanical properties. Cytotoxicity tests revealed excellent cytocompatibility of calcium carbonate cement compositions. Calcium carbonates with a higher solubility than the apatite formed for most of the marketed calcium phosphate cements might be of interest to increase biomedical cement resorption rates and to favour its replacement by bone tissue.