Impact of the chemical composition of poly-substituted hydroxyapatite particles on the in vitro pro-inflammatory response of macrophages

International audienceTo improve the biological properties of calcium phosphate (CaP) bone substitute, new chemical compositions are under development. In vivo such materials are subject to degradation that could lead to particles release and inflammatory reactions detrimental to the bone healing process. This study aimed at investigating the interactions between a murine macrophage cell line (RAW 264.7) and substituted hydroxyapatite particles presenting promising biological properties. Micron size particles of stoichiometric and substituted hydroxyapatites (CO 3 substitution for PO 4 and OH; SiO 4 substitution for PO 4; CO 3 and SiO 4 co-substitution) were obtained by aqueous precipitation followed by spray drying. Cells, incubated with four doses of particles ranging from 15 to 120 µg/mL, revealed no significant LDH release or ROS production, indicating no apparent cytotoxicity and no oxidative stress. TNF-α production was independent of the chemistry of the particles; however the particles elicited a significant dose-dependent pro-inflammatory response. As micron size particles of these hydroxyapatites could be at the origin of inflammation, attention must be paid to the degradation 2 behavior of substituted hydroxyapatite bone substitute in order to limit, in vivo, the generation of particulate debris

Élaboration et fonctionnalisation de biocéramiques phosphocalciques

La préparation de céramiques phosphocalciques dopées au silicium (Si) a été étudiée sur la base de la formule hypothétique Ca3 x(PO4)2 2x(SiO4)x du phosphate tricalcique partiellement substitué par des groupements silicates. Des poudres d apatite déficitaire en calcium ont été synthétisées en présence de Si par précipitation en milieu aqueux à pH 7 et 30C. L augmentation de la teneur en Si accroît la vitesse de réaction de précipitation de l apatite. Le temps de maturation requis pour obtenir, après calcination des précipités, une poudre constituée d une phase cristalline unique de phosphate tricalcique beta (b-TCP) diminue avec cette teneur. Des matériaux denses ont été obtenus après frittage naturel à 1100C pendant 2 h. Le silicium n est que partiellement incorporé dans la structure du b-TCP, l excédent se retrouve dans des grains de silice amorphe qui ralentissent le processus de densification. In vitro, des tests d immersion en solutions salines ont révélé que la présence de Si permettait d augmenter la bioactivité des céramiques. La biocompatibilité des matériaux a été démontrée par la prolifération des cellules MC3T3-E1 et la différenciation des cellules souches ST-2 a lieu. Le processus de minéralisation osseuse, étudié à l aide des cellules SaOs-2, est observé sur tous les matériaux et est amélioré en présence de silicium. Dans l optique d une fonctionnalisation de surface par des biomolécules, un protocole préliminaire de greffage d une quantité contrôlée d organo-alkoxysilane sur la surface d hydroxyapatite silicatée a été mis en place. Différents facteurs influents dont le pH du milieu réactionnel et la charge de surface des poudres ont ainsi été révélés.The preparation of silicon doped calcium phosphate ceramics was studied on the basis of the hypothetical formula Ca3 x(PO4)2 2x(SiO4)x of tricalcium phosphate partially substituted by silicate groups. Calcium deficient apatite powders have been synthesised in the presence of silicon by a wet precipitation method at pH 7 and 30C. The increase of silicon content increased the reaction rate of apatite precipitation. The maturation time required to obtain, after calcination of the precipitate, a powder consisting of a beta tricalcium phosphate (b-TCP) as unique crystalline phase decreased with this content. Dense materials were obtained after natural sintering at 1100C for 2 h. Silicon was only partially incorporated in the crystal structure of b TCP, the remaining silicon was in the form of grains of amorphous silica that slow down the densification process. In vitro, immersion tests in saline solutions showed that the bioactivity of the ceramics was improved by silicon. The materials were biocompatible through the MC3T3-E1 culture and the differentiation of ST-2 stem cells occurred well. The bone mineralisation process, evaluated through SaOs-2 cells culture, was observed on all materials and increased in the presence of silicon. For further surface functionalisation with biomolecules, a preliminary study of grafting a controlled amount of organo-alkoxysilane on silicated hydroxyapatite surface was proposed. Different influencing factors including the pH of the system and the charge of the powders surface were highlighted.LIMOGES-BU Sciences (870852109) / SudocSudocFranceCote d'IvoireFRC

Élaboration de biocéramiques macroporeuses d’architectures complexes en hydroxyapatites polysubstituées

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Macroporous ceramics of carbonated hydroxyapatite for bone grafting applications

International audienceCalcium phosphate based materials, specifically hydroxyapatite (HA) and tricalcium phosphate (TCP), have been extensively used as bone graft substitutes for various applications (e.g. bone augmentation procedure). They temporarily substitute for bone while simultaneously supporting its regeneration. However, they present inappropriate resorption rate. Indeed, ideally a bone substitute should degrade only after the regenerated tissue has been remodelled at least once in the natural remodelling cycle [1]. Thus, the solubility of HA is too slow and that of TCP too fast to foster successful bone re-growth. To further improve the properties of calcium phosphate ceramics, ionic substitutions could be used. One way to modulate their resorption rate would be to substitute carbonate ions (CO3 2-) for phosphate ones (PO43-) into the HA structure [2]. Thus, the aim of the present work was to elaborate interconnected macroporous scaffold of pure carbonated hydroxyapatite (CHA). REFERENCES: 1 M. K. Heljak et al. (2012) Int. J. Numer. Meth. Biomed. Engng. 28:789-800. 2 Y. Doi et al. (1998) J. Biomed. Mater. Res. 39:603-610

Elaboration and characterization of macroporous carbonated hydroxyapatite for bone tissue engineering

International audienceThe mineral of bone is a biological calcium phosphate material which contains various ions in substitution (magnesium, carbonate, sodium...). Therefore, due to their chemical composition, calcium phosphate based materials such as hydroxyapatite (Ca10(PO4)6(OH)2, HA) or tricalcium phosphate (Ca3(PO4)2, TCP) are currently used to elaborate scaffold for bone grafting applications. These bioceramics allow the creation of a strong bond between the material and the surrounding tissues. However, they present inappropriate in vivo resorption rate especially for bone tissue engineering applications: too slow for HA and too fast for TCP. Ideally, a bone substitute should degrade only after the regenerated tissue has been remodelled at least once in the natural remodelling cycle [1]. One way to modulate the resorption rate of calcium phosphate ceramics would be to substitute carbonate ions (CO32-) for phosphate ones (PO43-) into the HA structure [2]. From this basis, the goal of this work was to elaborate pure carbonated hydroxyapatite (CHA) macroporous scaffold for bone tissue engineering applications. Thereby, granules of CHA were elaborated and their physico-chemical and biochemical properties were studied and compared to pure HA and TCP. To evaluate the biochemical properties of the granules, in vitro assays have been performed to study their dissolution rate. The powders were synthesised by a wet precipitation method. Materials with macroporous architecture were shaped by a replica method. The latter involved the optimisation of the slurry properties. The preparation of the granules was then completed by their sintering under controlled atmosphere and their granulation. To assess the phase composition of the material, X-ray diffraction and Fourier transformed infra-red analysis were performed. The chemical composition of the granules was confirmed by elemental analyses (e.g. inductively coupled plasma atomic emission spectroscopy). The solubility product of the ceramics was then determined through standard experiments and compared to common bioceramics. [1] Evolutionary design of bone scaffolds with reference to material selection, M. K. Heljak et al., Int. J. Numer. Meth. Biomed. Engng. 2012; 28:789-800 [2] Effect of Carbonate Content and Crystallinity on the Metastable Equilibrium Solubility Behavior of Carbonated Apatites, A.A. Baig et al., J. Colloid Interface Sci. 1996; 179:608-61

Investigation of microwave sintering of B-type carbonated hydroxyapatite bioceramics

International audienceB-type carbonated hydroxyapatite (CBHA) is potentially an excellent biodegradable bioceramic for bone repair. However, conventional sintering results in formation of undesired phases. Therefore, microwave sintering of CBHA was investigated to assess the possibility to reduce formation of unwanted phases. Pellets with 0.8 mol% of B-type carbonate were sintered in a multimode instrumented cavity under static air with short thermal cycles. They were prepared from a CBHA powder alone and from a mixture of CBHA and carbon powder to generate a local in-situ CO2 atmosphere. XRD, FT-IR, SEM and BET analyses indicated that CBHA densification with increase temperature lead to decomposition into apatite. The addition of carbon powder to the CBHA that generate a CO2-rich atmosphere around the samples did not prevent the decomposition. Efficient control of temperature and atmosphere composition is required to improve microwave sintering of CBHA bioceramics

Elaboration and characterization of macroporous carbonated hydroxyapatite for bone tissue engineering

International audienceThe mineral of bone is a biological calcium phosphate material which contains various ions in substitution (magnesium, carbonate, sodium...). Therefore, due to their chemical composition, calcium phosphate based materials such as hydroxyapatite (Ca10(PO4)6(OH)2, HA) or tricalcium phosphate (Ca3(PO4)2, TCP) are currently used to elaborate scaffold for bone grafting applications. These bioceramics allow the creation of a strong bond between the material and the surrounding tissues. However, they present inappropriate in vivo resorption rate especially for bone tissue engineering applications: too slow for HA and too fast for TCP. Ideally, a bone substitute should degrade only after the regenerated tissue has been remodelled at least once in the natural remodelling cycle [1]. One way to modulate the resorption rate of calcium phosphate ceramics would be to substitute carbonate ions (CO32-) for phosphate ones (PO43-) into the HA structure [2]. From this basis, the goal of this work was to elaborate pure carbonated hydroxyapatite (CHA) macroporous scaffold for bone tissue engineering applications. Thereby, granules of CHA were elaborated and their physico-chemical and biochemical properties were studied and compared to pure HA and TCP. To evaluate the biochemical properties of the granules, in vitro assays have been performed to study their dissolution rate. The powders were synthesised by a wet precipitation method. Materials with macroporous architecture were shaped by a replica method. The latter involved the optimisation of the slurry properties. The preparation of the granules was then completed by their sintering under controlled atmosphere and their granulation. To assess the phase composition of the material, X-ray diffraction and Fourier transformed infra-red analysis were performed. The chemical composition of the granules was confirmed by elemental analyses (e.g. inductively coupled plasma atomic emission spectroscopy). The solubility product of the ceramics was then determined through standard experiments and compared to common bioceramics. [1] Evolutionary design of bone scaffolds with reference to material selection, M. K. Heljak et al., Int. J. Numer. Meth. Biomed. Engng. 2012; 28:789-800 [2] Effect of Carbonate Content and Crystallinity on the Metastable Equilibrium Solubility Behavior of Carbonated Apatites, A.A. Baig et al., J. Colloid Interface Sci. 1996; 179:608-61

Thermal stability and sintering of C_xSi_yHA ceramics for bone tissue engineering application

International audienceOne of the most important components of the bone tissue engineering is the scaffold on which the new tissue can grow. It has to provide the mechanical stability of the construct and a template for the three-dimensional new extracellular matrix organization. As the stiffness of scaffold as well as regenerated tissue has to match the stiffness of the native bone, scaffolds for bone engineering often contain calcium phosphates bioceramic. Among them, hydroxyapatite (HA, Ca10(PO4)6(OH)2) is commonly used as bone substitute due to its chemical and crystallographic similarities with bone mineral part, in spite of its non-adequate resorption rate. The apatitic lattice of HA is very tolerant with substitutions. Unfortunately, HA shows limited ability to stimulate the development of new bone tissue (bone apposition) and has a very low rate of biodegradation (slow osseointegration). One way to modulate bioactivity of synthetic HA is to incorporate ionic groups in its structure. Thus, carbonate improves resorption of HA closed to natural bone; and the soluble silicon (e.g. silicate ions) stimulates bone cells activity. This work is focused on the elaboration of macroporous bioceramics of a co-substituted hydroxyapatite with a controlled amount of carbonate (x) and silicate groups (y) (CxSiyHA) for bone tissue engineering application. The work consisted first in the synthesis of CxSiyHA powders, followed by the scaffolds shaping by means of a replication process of PMMA skeleton. Finally, the green were debinded sintered and accurately characterized. CxSiyHA powders, of assumed composition Ca10-x+y(PO4)6-y-x(SiO4)y(CO3)x(OH)2-x+y with x>y and x+y2 atmosphere. The physico-chemical properties of scaffolds were evaluated by infrared spectroscopy (FTIR) and X-ray diffraction (XRD). Their morphology and topography were examined through Scanning Electron Microscopy (SEM) and X-ray microtomography. Finally, solubility constants were determined according to standard procedures

The key role of the A-site composition of oxy-hydroxyapatites in high-temperature solid-gas exchange reactions

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Biocéramique en hydroxyapatite carbo-silicatée: synthèse, élaboration et étude de biocompatibilité in vitro

National audienc