Frédéric Velard

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.

Inflammatory cell response to calcium phosphate biomaterial particles: an overview.

Bone is a metabolically active and highly organized tissue consisting of a mineral phase of hydroxyapatite (HA) and amorphous calcium phosphate (CaP) crystals deposited in an organic matrix. One objective of bone tissue engineering is to mimic the chemical and structural properties of this complex tissue. CaP ceramics, such as sintered HA and beta-tricalcium phosphate, are widely used as bone substitutes or prosthesis coatings because of their osteoconductive properties. These ceramic interactions with tissues induce a cell response that can be different according to the composition of the material. In this review, we discuss inflammatory cell responses to CaP materials to provide a comprehensive overview of mechanisms governing the integration or loosening of implants, which remains a major concern in tissue engineering. A focus on the effects of the functionalization of CaP biomaterials highlights potential ways to increase tissue integration and limit rejection processes.

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).

Cystic fibrosis transmembrane conductance regulator (CFTR) regulates the production of osteoprotegerin (OPG) and prostaglandin (PG) E2 in human bone

Bone loss is an important clinical issue in patients with cystic fibrosis (CF). Whether the cystic fibrosis transmembrane conductance regulator (CFTR) plays a direct role in bone cell function is yet unknown. In this study, we provide evidence that inhibition of CFTR-Cl(-) channel function results in a significant decrease of osteoprotegerin (OPG) secretion accompanied with a concomitant increase of prostaglandin (PG) E(2) secretion of primary human osteoblast cultures (n=5). Our data therefore suggest that in bone cells of CF patients, the loss of CFTR activity may result in an increased inflammation-driven bone resorption (through both the reduced OPG and increased PGE(2) production), and thus might contribute to the early bone loss reported in young children with CF.

Staphylococcus aureus vs. Osteoblast: Relationship and Consequences in Osteomyelitis

Bone cells, namely osteoblasts and osteoclasts work in concert and are responsible for bone extracellular matrix formation and resorption. This homeostasis is, in part, altered during infections by Staphylococcus aureus through the induction of various responses from the osteoblasts. This includes the over-production of chemokines, cytokines and growth factors, thus suggesting a role for these cells in both innate and adaptive immunity. S. aureus decreases the activity and viability of osteoblasts, by induction of apoptosis-dependent and independent mechanisms. The tight relationship between osteoclasts and osteoblasts is also modulated by S. aureus infection. The present review provides a survey of the relevant literature discussing the important aspects of S. aureus and osteoblast interaction as well as the ability for antimicrobial peptides to kill intra-osteoblastic S. aureus, hence emphasizing the necessity for new anti-infectious therapeutics.

Effect of strontium-substituted biphasic calcium phosphate on inflammatory mediators production by human monocytes

Calcium phosphate materials are widely used as bone substitutes because of their properties close to those of the mineral phase of bones. Nevertheless, after several months, calcium phosphate-based materials release particles that may be phagocytosed by monocytes, leading to an inflammatory reaction. Strontium is well known to counteract the osteoporosis process, but little is known about its effect on inflammatory processes. The purpose of this work was to study the effect of biphasic calcium phosphate (BCP) particles substituted with strontium on the inflammatory reaction. Human primary monocytes stimulated or not by lipopolysaccharide (LPS) were exposed to BCP particles containing strontium for 6 and 24 h. Inflammatory mediators (cytokines and matrix metalloproteinases (MMPs)) production was then quantified by ELISA and zymography. We observed that the presence of strontium had few effects on unstimulated cells, but it decreased the production of pro-inflammatory cytokines and the chemokine interleukin 8 in LPS-stimulated cell-conditioned medium. This work suggests for the first time that strontium may be involved in the control of inflammatory processes following BCP phagocytosis by human monocytes.

Polymorphonuclear neutrophil response to hydroxyapatite particles, implication in acute inflammatory reaction

Hydroxyapatite (HA) is widely used as a bone substitute or coating biomaterial in bone diseases or prosthesis metal parts. The release of HA particles induces an inflammatory response and, if uncontrolled, could result in implant loss. Among the hallmarks of such inflammatory response is early recruitment of the polymorphonuclear cells (PMNs). The purpose of this work is to investigate the response of PMNs following exposure to HA in terms of secreted mediators. Our study shows that HA particles increase the release of pro-inflammatory mediators such as interleukin-1alpha, as well as chemotactic factors such as interleukin-8, macrophage inflammatory protein-1alpha and macrophage inflammatory protein-1beta. HA also induces an increase in matrix metalloproteinase 9 expression. Taken together, our data demonstrate for the first time that HA is capable of activating PMNs, a phenomenon that could potentially contribute to the onset of implant-associated inflammation.

In vitro dissolution and corrosion study of calcium phosphate coatings elaborated by pulsed electrodeposition current on Ti6Al4V substrate

Calcium-deficient hydroxyapatite (Ca-def-HAP) coatings on titanium alloy (Ti6Al4V) substrates are elaborated by pulsed electrodeposition. In vitro dissolution/precipitation process is investigated by immersion of the coated substrate into Dulbecco’s Modified Eagle Medium (DMEM) from 1xa0h to 28xa0days. Calcium and phosphorus concentrations evolution in the biological liquid are determined by Induced Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) for each immersion time. Physical and chemical characterizations of the coating are performed by scanning electron microscopy (SEM) associated to Energy Dispersive X-ray Spectroscopy (EDXS) for X-ray microanalysis. Surface modifications are investigated by an original method based on the three-dimensional reconstruction of SEM images (3D-SEM). Moreover, corrosion measurements are carried out by potentiodynamic polarization experiments. The results show that the precipitation rate of the Ca-def HAP coating is more pronounced in comparison with that of stoichiometric hydroxyapatite (HAP) used as reference. The precipitated bone-like apatite coating is thick, homogenous and exhibits an improved link to the substrate. Consequently, the corrosion behaviour of the elaborated prosthetic material is improved.

Sulfadiazine resistance in Toxoplasma gondii: no involvement of overexpression or polymorphisms in genes of therapeutic targets and ABC transporters

Several treatment failures have been reported for the treatment of toxoplasmic encephalitis, chorioretinitis, and congenital toxoplasmosis. Recently we found three Toxoplasma gondii strains naturally resistant to sulfadiazine and we developed in vitro two sulfadiazine resistant strains, RH-RSDZ and ME-49-RSDZ, by gradual pressure. In Plasmodium, common mechanisms of drug resistance involve, among others, mutations and/or amplification within genes encoding the therapeutic targets dhps and dhfr and/or the ABC transporter genes family. To identify genotypic and/or phenotypic markers of resistance in T. gondii, we sequenced and analyzed the expression levels of therapeutic targets dhps and dhfr, three ABC genes, two Pgp, TgABC.B1 and TgABC.B2, and one MRP, TgABC.C1, on sensitive strains compared to sulfadiazine resistant strains. Neither polymorphism nor overexpression was identified. Contrary to Plasmodium, in which mutations and/or overexpression within gene targets and ABC transporters are involved in antimalarial resistance, T. gondii sulfadiazine resistance is not related to these toxoplasmic genes studied.

Induction of sulfadiazine resistance in vitro in Toxoplasma gondii

We induced sulfadiazine resistance in two sulfadiazine sensitive strains of Toxoplasma gondii, RH (Type I) and ME-49 (Type II) in vitro by using drug pressure. At first, sulfadiazine susceptibility of the two sensitive strains and two naturally resistant strains of T. gondii was evaluated on Vero cells using an enzyme-linked immunosorbent assay (ELISA). The IC(50) values of sulfadiazine were 77 μg/mL for RH, 51 μg/mL for ME-49 and higher than 1000 μg/mL for the two natural resistant strains. Secondly, induced resistance of the strains by gradually increase sulfadiazine concentration was verified by this test, which resulted IC(50) values at higher than 1000 μg/mL. In conclusion we developed in vitro two sulfadiazine resistant strains called RH-R(SDZ) and ME-49-R(SDZ). These strains resistant to sulfadiazine would be useful to characterize resistance mechanisms to sulfadiazine.