Edouard Jallot

Importance of hydroxyapatite particles characteristics on cytokines production by human monocytes in vitro

Calcium phosphate bioceramics have been applied as bone substitutes for several decades. Aseptic loosening after total joint arthroplasty is a major problem in orthopaedic surgery. Hydroxyapatite particles from materials wear have been reported as the main cause of implant failure. For this reason, an investigation into possible wear particles from materials used in the implant may lead to longevity after arthroplasty. Monocytes are among the first cells to colonize the inflammatory site. In the present study, we have evaluated the inflammatory response after exposition to particles with different characteristics (size, sintering temperature and shape). Our data demonstrate that the most important characteristic was the shape and the size of the particles. The needle shaped particles induced the larger production of TNF-alpha, IL-6 and IL-10 by cells. To a less manner, the smallest particles induced an increase of the expression and production of the cytokines studied (TNF-alpha, IL-6 and IL-10). The sintering temperature appeared to be a less important characteristic even though it was involved in the dissolution/precipitation process.

A new insight into the dissociating effect of strontium on bone resorption and formation.

Calcium phosphates are widely used as biomaterials and strontium (Sr) is known to have the ability to modify the bone balance towards osteosynthesis. In the present study we investigated the capacity of Sr-substituted sol-gel calcium phosphate to modify the expression of genes and proteins involved in extracellular matrix synthesis by primary bone cells. We first determined the most effective concentration of strontium using human primary bone cells. Sol-gel biphasic calcium phosphate (BCP) powders were then synthesised to obtain release of the optimal concentration of strontium. Finally, human osteoblasts obtained from explant cultures were cultured in the presence of sol-gel BCP, Sr-substituted BCP (5% Sr-substituted BCP, corresponding to a release of 5×10(-5)M [Sr(2+)] under the culture conditions (BCP(5%))) and medium containing strontium chloride (SrCl(2)). Viability, proliferation, cell morphology, protein production and protein activity were studied. We demonstrated that 5×10(-5)M SrCl(2) and BCP(5%) increased the expression of type I collagen and SERPINH1 mRNA and reduced the production of matrix metalloproteinases (MMP-1 and MMP-2) without modifying the levels of the tissue inhibitors of MMPs (TIMPs). Thus strontium has a positive effect on bone formation.

New Strontium-based Bioactive Glasses: Physicochemical Reactivity and Delivering Capability of Biologically Active Dissolution Products

The development of bone tissue regeneration calls for biomaterials able to release biologically active substances in a controlled manner after implantation. In this context, strontium-doped bioactive glasses are of major interest; their key property relies on the increased kinetics of surface reactions, along with the release of critical concentrations of ionic dissolution products capable of stimulating cellular responses. In this paper, we report a complete evaluation of the in vitro reactivity of new SiO2–CaO–SrO and SiO2–CaO–P2O5–SrO bioactive glasses. In contact with simulated acellular physiological fluids, these materials induce the formation of a calcium phosphate surface layer that closely resembles to the biological apatite present in bones. The two most commonly used materials shapes for clinical applications – glass particles and glass bulk – were studied and provide us with concordant results. Compared to strontium-free materials, the dissolution of SiO2–CaO–SrO and SiO2–CaO–P2O5–SrO glasses is reduced. However, the surface layer is more quickly transformed into a bone-like apatite phase, according to the kinetics of evolution of the Ca/P atomic ratio. Evidences of the presence of Sr at the glass/biological fluids interface were obtained, along with the demonstration that this element is released in physiological concentrations into the biological environment. Knowing the well-recognized beneficial effects of strontium on cell activity and bone remodeling, this crucial result gives high hopes for the development of innovative applications based on Sr-doped glasses in the treatment of osteoporosis and tissue engineering.

Resorption kinetics of osseous substitute: natural coral and synthetic hydroxyapatite

Coral and hydroxyapatite may be used as substitution biomaterials for bone grafts. In this work, we extracted the implants from the femora to study the kinetics of elementary mineral transformation of the osseous substitutes. The use of physical analysis methods such as PIXE (particle-induced X-ray emission) shows that coral and hydroxyapatite, after their implantation in vivo, reach a mineral composition comparable with that of bone. For the first time we have measured the concentration of mineral elements, at different time intervals after implantation, along a cross-section. The distribution according to mineral elements (Ca, P, Sr, Zn, Fe) in the implant, in the receiver site and also at the interface, showed that the kinetics of coral resorption was faster than that of hydroxyapatite; in the same way, the osseous attack was not global but, rather, centripetal.

Structural characterization of sol–gel derived Sr-substituted calcium phosphates with anti-osteoporotic and anti-inflammatory properties

Sol–gel chemistry has been successfully used to prepare un-doped and Sr-doped calcium phosphate ceramics exhibiting a porous structure. The samples composition is very close to the nominal one. All samples present phase mixtures of mainly hydroxyapatite (HAp) and tricalcium phosphate (β-TCP). Doping with Sr2+ ions has a clear effect on the proportions of the different phases, increasing the amount of β-TCP. An amorphous phase is also observed incorporating some 40% of the total amount of strontium. Strontium ions also substitute for calcium both in HAp and β-TCP in specific sites that have been determined from Rietveld refinement on synchrotron powder diffraction data. The soluble amorphous and TCP phases are responsible for a beneficial partial release of strontium ions in solution during interactions between the material and biological fluids. Preliminary in vitro study demonstrates anti-inflammatory effects of strontium for human monocytes cultured in contact with calcium phosphates.

Thorough analysis of silicon substitution in biphasic calcium phosphate bioceramics: a multi-technique study.

Four samples of composition Ca(10)(PO(4))(6-x)(SiO(4))(x)(OH)(2-x), with x=0.0, 0.1, 0.2 and 0.5, were prepared and characterized using powder X-ray and neutron powder diffraction, and (1)H, (31)P and (29)Si nuclear magnetic resonance (NMR) spectroscopy. The composition of the Si-substituted HAp phases was determined by joint Rietveld refinements from powder X-ray and powder neutron diffraction data. Taking into account electroneutrality, a chemical formula for the Si-substituted HAp phases with indication of the incorporated silicate amount is proposed. Solid-state (29)Si NMR confirms the presence of only Q(0) species, in good agreement with the presence of substituted HAp and beta-TCP phases only. Thanks to NMR spectroscopy, two types of protons in the Si-substituted HAp phase were identified, the new site corresponding to species engaged in hydrogen bonding with silicate anions. This allowed further refinement of the formulae for these phases with very good quantitative agreement for populations derived from the refinement and integration of NMR data.

87Sr solid-state NMR as a structurally sensitive tool for the investigation of materials: antiosteoporotic pharmaceuticals and bioactive glasses.

Strontium is an element of fundamental importance in biomedical science. Indeed, it has been demonstrated that Sr(2+) ions can promote bone growth and inhibit bone resorption. Thus, the oral administration of Sr-containing medications has been used clinically to prevent osteoporosis, and Sr-containing biomaterials have been developed for implant and tissue engineering applications. The bioavailability of strontium metal cations in the body and their kinetics of release from materials will depend on their local environment. It is thus crucial to be able to characterize, in detail, strontium environments in disordered phases such as bioactive glasses, to understand their structure and rationalize their properties. In this paper, we demonstrate that (87)Sr NMR spectroscopy can serve as a valuable tool of investigation. First, the implementation of high-sensitivity (87)Sr solid-state NMR experiments is presented using (87)Sr-labeled strontium malonate (with DFS (double field sweep), QCPMG (quadrupolar Carr-Purcell-Meiboom-Gill), and WURST (wideband, uniform rate, and smooth truncation) excitation). Then, it is shown that GIPAW DFT (gauge including projector augmented wave density functional theory) calculations can accurately compute (87)Sr NMR parameters. Last and most importantly, (87)Sr NMR is used for the study of a (Ca,Sr)-silicate bioactive glass of limited Sr content (only ~9 wt %). The spectrum is interpreted using structural models of the glass, which are generated through molecular dynamics (MD) simulations and relaxed by DFT, before performing GIPAW calculations of (87)Sr NMR parameters. Finally, changes in the (87)Sr NMR spectrum after immersion of the glass in simulated body fluid (SBF) are reported and discussed.

Role of magnesium during spontaneous formation of a calcium phosphate layer at the periphery of a bioactive glass coating doped with mgo

The prerequisite for bioactive glasses to bond to living bone is the formation of biologically active apatites on their surface in the body. Reactions and bioactivity mechanisms between bioactive glasses and bone depend on the glass composition. Our results demonstrate this property of bioactivity for the studied bioactive glass. The bioactive glass doped with MgO leads to the formation of a Ca–P–Mg rich layer on top of a pure Si layer. Ca, P and Mg elements diffuse through the Si rich layer which seems to act as a protective diffusion barrier leading to t1/2 leaching kinetics. This paper demonstrates for the first time, the presence of Mg in the Ca–P rich layer at the interface between the bioactive glass and bone. However, magnesium influences the formation and the evolution of the apatite layer. Magnesium has synergistic effects on the crystallinity and solubility of apatites. These phenomena can promote a greater dissolution of the apatite precipitates in the studied bioactive glass. Determination of supersaturations values permits to better understand growth and dissolution of precipitates forming the apatite-Mg layer. Measurements of supersaturations for Ca, P and Mg were consistent before 3 months and after 6 months. Before 3 months, the apatite-Mg layer grows and after 3 months this layer is in dissolution.

Structural characterization and biological fluid interaction of Sol-Gel-derived Mg-substituted biphasic calcium phosphate ceramics.

Sol-Gel chemistry has been used to prepare undoped and Mg-substituted biphasic calcium phosphate (BCP) ceramics composed of hydroxyapatite (HAp) and whitlockite (beta-TCP) phases. Different series of samples have been synthesized with different Mg-doping levels (from 0 to 5 atomic % of Ca atoms substituted) and different temperatures of calcination (from 500 to 1100 degrees C). All of the powdered samples were systematically treated by Rietveld refinement to extract the quantitative phase analysis and the structural and microstructural parameters, to locate the Mg crystallographic sites, and to refine the composition of the Mg-substituted phases. The temperature dependence of the weight amount ratio between HAp and beta-TCP is not monotonic because of the formation of minor phases such as Ca(2)P(2)O(7), CaO, MgO, and CaCO(3) and certainly an amorphous phase. On the other hand, the Mg stabilizing feature on the beta-TCP phase has been evidenced and explained. The mechanism of stabilization by small Mg(2+) is different from that by large Sr(2+). Nevertheless, in both cases, the beta-TCP stabilization is realized by an improvement of the environment of the Ca4 site unusually face-coordinated to a PO(4) tetrahedron. The substitution of a Mg atom in the Ca5 site allows considerable improvement of the bond valence sum of the unusual Ca4 polyhedron. The temperatures of calcination combined with the amount of Mg atoms introduced allow monitoring of the phase composition of the BCP ceramics as well as their microstructural properties. The bioactivity properties of the BCP samples are improved by the presence of Mg atoms in the structure of the beta-TCP phase. The mechanism of improvement is mainly attributed to an accelerated kinetic of precipitation of a calcium phosphate layer at the surface comprising HAp and/or beta-TCP phases.

The effect of zinc on hydroxyapatite-mediated activation of human polymorphonuclear neutrophils and bone implant-associated acute inflammation.

Hydroxyapatite (HA) is widely used as coating biomaterial for prosthesis metal parts and as bone substitute. The release of HA particles induces an inflammatory response and, if uncontrolled, could result in implant loss. At the inflamed site, the polymorphonuclear cells (PMNs) represent the earliest phagocytic cells that predominate the cellular infiltrate. We have recently proposed that HA wear debris activate polymorphonuclear cells (PMNs) initiating and/or amplifying thereby the acute inflammatory response. Previous studies have shown that activation of monocytes by HA could be modulated by supplementing this latter with the divalent cation, Zinc. The purpose of this work was to investigate the modulation of PMNs activation following exposure to zinc-substituted HA. Our study demonstrate that addition of zinc to HA particles resulted in decreased levels of the pro-inflammatory mediator interleukin-8 (IL-8) and the matrix metallo-proteinase-9. We also show that these changes involve IL-8 receptors (CXCR-1 and CXCR-2).