Effects of strontium-doped bioactive glass on the differentiation of cultured osteogenic cells.

There is accumulating evidence that strontium-containing biomaterials have positive effects on bone tissue repair. We investigated the in vitro effect of a new Sr-doped bioactive glass manufactured by the sol-gel method on osteoblast viability and differentiation. Osteoblasts isolated from foetal mouse calvaria were cultured in the presence of bioactive glass particles; particles were undoped (B75) or Sr-doped with 1 wt.% (B75-Sr1) and 5 wt.% (B75-Sr5). Morphological analysis was carried out by contrast-phase microscopy and scanning electron microscopy (SEM). Cell viability was evaluated by the MTS assay at 24 h, 48 h and 72 h. At 24 h, day 6 and day 12, osteoblast differentiation was evaluated by assaying alkaline phosphatase (ALP) activity, osteocalcin (OC) secretion and gene expression of various bone markers, using Real-Time-PCR. Alizarin Red staining and ALP histoenzymatic localisation were performed on day 12. Microscopic observations and MTS showed an absence of cytotoxicity in the three investigated bioactive glasses. B75-Sr5 particles in cell cultures, in comparison with those of B75 and B75-Sr1, resulted in a significant up-regulation of Runx2, Osterix, Dlx5, collagen I, ALP, bone sialoprotein (BSP) and OC mRNA levels on day 12, which was associated with an increase of ALP activity on day 6 and OC secretion on day 12. In conclusion, osteoblast differentiation of foetal mouse calvarial cells was enhanced in the presence of bioactive glass particles containing 5 wt.% strontium. Thus, B75-Sr5 may represent a promising bone-grafting material for bone regeneration procedures.

Influence of Mg doping on the early steps of physico-chemical reactivity of sol-gel derived bioactive glasses in biological medium.

The aim of this study is to highlight the influence of magnesium doping on the surface reactivity of binary (SiO(2)-CaO) and ternary (SiO(2)-CaO-P(2)O(5)) bioactive glasses, prepared by sol-gel chemistry, in biological conditions. These materials were produced in powder form, and their compositional and textural properties characterized. They were then soaked in biological fluids for different delays from 0 to 4 days. The surface changes were characterized using Particles Induced X-ray Emission (PIXE) associated with Rutherford Backscattering Spectroscopy (RBS), which are efficient methods for multi-elemental analysis. Elemental maps of major and trace elements were obtained at a micrometer scale and revealed the formation of a calcium phosphate-rich layer after a few days of interaction. We demonstrate that the presence of magnesium in the material has an impact on the development and the formation rate of the bone-like apatite layer.

Interfacial reactions of glasses for biomedical application by scanning transmission electron microscopy and microanalysis.

Short-term physico-chemical reactions at the interface between bioactive glass particles and biological fluids are studied for three glasses with different bioactive properties; these glasses are in the SiO(2)-Na(2)O-CaO-P(2)O(5)-K(2)O-Al(2)O(3)-MgO system. Our aim is to show the difference between the mechanisms of their surface reactions. The relation between the composition and the bioactive properties of these glasses is also discussed. The elemental analysis is performed at the submicrometer scale by scanning transmission electron microscopy associated with energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy. After different immersion times (ranging from 0 to 96 h) of bioactive glass particles in a simulated biological solution, results show the formation of different surface layers at the glass periphery in the case of two bioactive glasses (A9 and BVA). For the third glass (BVH) we do not observe any surface layer formation or any modification of the glass composition. For the two other glasses (A9 and BVA), we observe the presence of different layers: an already observed (Si, O, Al) rich layer at the periphery, a previously demonstrated thin (Si, O) layer formed on top of the (Si, O, Al) layer and a (Ca, P) layer. We determine the different steps of the mechanisms of the surface reactions, which appear to be similar in these glasses, and compare the physico-chemical reactions and kinetics using the different immersion times. The A9 glass permits the observation of all important steps of the surface reactions which lead to bioactivity. This study shows the important relationship between composition and bioactivity which can determine the medical applicability of the glass.

STEM and EDXS characterisation of physico-chemical reactions at the periphery of sol-gel derived Zn-substituted hydroxyapatites during interactions with biological fluids.

With its good properties of biocompatibility and bioactivity hydroxyapatite (HA) is highly used as bone substitutes and as coatings on metallic prostheses. In order to improve the bioactive properties of HA, we have elaborated Zn2+ doped hydroxyapatite. Zn2+ ions substitute for Ca2+ cations in the HA structure and four Zn concentrations (Zn/Zn+Ca) were prepared at 0.5, 1, 2 and 5 at.%. To study physico-chemical reactions at the materials periphery, we immersed the bioceramics into biological fluids for intervals from 1 day to 20 days. The surface changes were studied at the nanometer scale by scanning transmission electron microscopy associated with energy dispersive X-ray spectroscopy. After 20 days of immersion, we observed the formation of a calcium-phosphate layer at the periphery of the HA doped with 5% zinc. This layer contains magnesium and its thickness was around 200 nm. Formation of this Ca-P-Mg layer represents the bioactive properties of 5% Zn-substituted hydroxyapatite. This biologically active layer improves the properties of HA and will permit a chemical bond between the ceramic and bone.

Bioactive glass stimulates in vitro osteoblast differentiation and creates a favorable template for bone tissue formation.

In this study, we have investigated the behavior of fetal rat osteoblasts cultured on bioactive glasses with 55 wt% silica content (55S) and on a bioinert glass (60S) used either in the form of granules or in the form of disks. In the presence of Bioglass granules (55 wt% silica content), phase contrast microscopy permitted step-by-step visualization of the formation of bone nodules in contact with the particles. Ultrastructural observations of undecalcified sections revealed the presence of an electron-dense layer composed of needle-shaped crystals at the periphery of the material that seemed to act as a nucleating surface for biological crystals. Furthermore, energy dispersive X-ray (EDX) analysis and electron diffraction patterns showed that this interface contains calcium (Ca) and phosphorus (P) and was highly crystalline. When rat bone cells were cultured on 55S disks, scanning electron microscopic (SEM) observations revealed that cells attached, spread to all substrata, and formed multilayered nodular structures by day 10 in culture. Furthermore, cytoenzymatic localization of alkaline phosphatase (ALP) and immunolabeling with bone sialoprotein antibody revealed a positive staining for the bone nodules formed in cultures on 55S. In addition, the specific activity of ALP determined biochemically was significantly higher in 55S cultures than in the controls. SEM observations of the material surfaces after scraping off the cell layers showed that mineralized bone nodules remained attached on 55S surfaces but not on 60S. X-ray microanalysis indicated the presence of Ca and P in this bone tissue. The 55S/bone interfaces also were analyzed on transverse sections. The interfacial analysis showed a firm bone bonding to the 55S surface through an intervening apatite layer, confirmed by the X-ray mappings. All these results indicate the importance of the surface composition in supporting differentiation of osteogenic cells and the subsequent apposition of bone matrix allowing a strong bond of the bioactive materials to bone.

Integration of dense HA rods into cortical bone.

HA ceramics are daily used in human surgery for bone healing partly due to their ability to integrate into bone. They are generally used under a macroporous form. The behaviour of dense HA after implantation is not so well known. We implanted within cortical sheep femurs dense pure HA-ceramics cylinders for periods from 2 weeks to 18 months. The samples were then sectioned and examined using back-scattered and secondary SEM and the interface was analysed using EDS. Histomorphometry measurement was also performed using an image analysis device coupled to a light microscope. It appeared that the cylinders were in direct contact with immature bone after three weeks. The bone maturated within three months. The implant surface showed moderate signs of resorption and some grains were released from the surface. The resorption zone was only a few microm thick after 18 months. The bulk ceramic contained default zones of increased porosity. They can constitute fragile zone when located close to the surface in which the resorption rate is increased. We conclude that dense pure HA is poorly degraded when implanted in cortical bone. Degradation depends on the defaults found on the ceramic structure and the remodelling of bone surrounding the material.

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.

Influence of physicochemical reactions of bioactive glass on the behavior and activity of human osteoblasts in vitro.

Bioactive glasses are characterized by a bond to bone with a hydroxyl carbonate apatite layer. They enhance bone tissue formation and for this purpose are used in orthopedic surgery and in dental implantology. In the current work, we studied the biological response of human osteoblasts with a bioactive glass. This bioactive glass is based on 50% Si0(2), 20% Na(2)O, 16% CaO, 6% P(2)O(5), 5% K(2)0, 2% Al(2)O(3) and 1% MgO and designated A9. Cracks and irregularities were observed on the material surface when it was immersed in the culture medium. In addition, energy dispersive X-ray analyses highlighted a selective release of the elements at the surface of the bioactive glass, such as Na(+) and K(+) ions, released from the first day, contrary to the Si, Al, Ca, P, and Mg elements, which were released more slowly. Cell proliferation kinetics, total protein synthesis, and DNA content of the osteoblasts in contact with bioactive glass were similar to control cells. The morphological studies by light and scanning electron microscopy revealed an increasing cellular density in culture with bioactive glass without contact inhibition. The immunohistochemical studies highlighted the expression of types I, III, and V collagens by osteoblasts cultured in the presence of bioactive glass. The pH measurement of the culture medium in the presence of bioactive glass demonstrated a slight alkalinization. We thus conclude that human osteoblasts preserve their properties in the presence of bioactive glass (A9).

Correlation between hydroxyapatite osseointegration and Young's Modulus.

From the standpoint of hard tissue response to implant materials, calcium phosphate is probably the most compatible of materials known. During the last few years, much attention has been paid to hydroxyapatite and beta-tricalcium phosphate as potential biomaterials for a bone substitute. Good implantation of biomaterials in the skeleton is evidenced by an ability to reach full integration of the non-living implant with living bone. The aim of this study is to correlate hydroxyapatite osseointegration with Young's Modulus. Cylinders (5-6 mm in diameter) of these ceramics were packed into holes made in the femur diaphysis of a mature sheep. At 2, 4, 8, 12, 16, 20, 28, 36 and 48 weeks after the operation, samples of the bone/implant interface were embedded in polymethylmethacrylate. We used the PIXE method (Particles Induced X-rays Emission) to measure the distribution of mineral elements (Ca, P, Sr, Zn, Mn and Fe) at the bone/implant interface. At 4, 8, 12, 16, 20, 28, 36 and 48 weeks after implantation we studied Young's Modulus on a biopsy of the ceramic. Young's Modulus increased with time after implantation and is linked with biomaterials integration into cortical bone.

[Biomaterials in an osteo-articular environment. Report of 129 anatomoclinical cases]. / Biomatériaux en milieu ostéo-articulaire. A propos de 129 observations anatomocliniques.

Actually, there is a range of biomaterials which are synthetic or metallic (or the both). They are employed as prosthesis (biostability property) or as bone graft (bioresorbability property). To understand the interactions between cells and such materials, we studied with human bone cellular cultures the cytologycal, immunohistochemical, cytogenetical and ultrastructural aspects of biomaterials in cell cultures. This paper concerns bioceramics like Pyrost, coral, biosorb, oxbone and polymers like polyethylene and silicones. The aim of this work is to evaluate the efficiency of some biomaterials. We found that porosity is primordial to promote biodegradation of bone substitutes. In fact, the biomaterials is integrated and lead to an osteoconduction, an osteoformation and finally an osteoinduction. Our observations show the implant resorption and ossification occurring in the matrix which penetrate it.

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.

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.

Imaging physicochemical reactions occurring at the pore surface in binary bioactive glass foams by micro ion beam analysis.

In this work, the physicochemical reactions occurring at the surface of bioactive sol-gel derived 3D glass scaffolds via a complete PIXE characterization were studied. 3D glass foams in the SiO(2)-CaO system were prepared by sol-gel route. Samples of glass scaffolds were soaked in biological fluids for periods up to 2 days. The surface changes were characterized using particle induced X-ray emission (PIXE) associated to Rutherford backscattering spectroscopy (RBS), which are efficient methods to perform quantitative chemical maps. Elemental maps of major and trace elements at the glass/biological fluids interface were obtained at the micrometer scale for every interaction time. Results revealed interconnected macropores and physicochemical reactions occurring at the surface of pores. The micro-PIXE-RBS characterization of the pores/biological fluids interface shows the glass dissolution and the rapid formation of a Ca rich layer with the presence of phosphorus that came from biological fluids. After 2 days, a calcium phosphate-rich layer containing magnesium is formed at the surface of the glass scaffolds. We demonstrate that quantities of phosphorus provided only by the biological medium have a significant impact on the development and the formation of the phosphocalcic layer.

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.

Dissolution Kinetics, Selective Leaching, and Interfacial Reactions of a Bioglass Coating Enriched in Alumina.

Bioglass coatings are interesting for developing a direct bond between prostheses and bone. But the high solubility of these materials limits their application. The addition of alumina can be used to control their solubility, but may inhibit the bonding mechanisms. In this paper, we study a bioglass in the SiO(2)-Na(2)O-CaO-P(2)O(5)-K(2)O-Al(2)O(3)-MgO system. After delays of implantation from 2 to 12 months, the bioglass/bone interface is characterized by energy-dispersive X-ray spectroscopy coupled with scanning transmission electron microscopy. Bioglass dissolution can be decomposed into three steps with selective leaching. Results show that, at 2 months after implantation, the bioglass is composed of Al, Si, Ca, and P. Alumina addition increases the coating stability without inhibiting the bonding process. Complex physicochemical reactions take place at the bioglass periphery. The coating bonds to bone through a Ca-P layer on top of a pure Si-rich layer. These phenomena are associated with bioactivity properties, which occur for up to 6 months. After 12 months, the bioglass is composed of silicon. Copyright 2001 Academic Press.

In vitro effects of zirconia and alumina particles on human blood monocyte-derived macrophages: X-ray microanalysis and flow cytometric studies.

The cytocompatibility of two particulate bioceramics, zirconia and alumina, was studied using human blood monocytes driven to differentiate into mature macrophages with granulocyte macrophage-colony-stimulating factor. Changes in individual cell elemental composition, particularly sodium and potassium content, were assessed by X-ray microanalysis of ultrathin freeze-dried sections. Phagocytosis and respiratory burst of macrophages exposed to biomaterial for 7 days were analyzed under flow cytometry using uptake of fluorescent latex beads and 2'7'-dichlorofluorescien diacetate oxidation, respectively. Zirconia and alumina particles were found to decrease the intracellular potassium/sodium ratio (an index of cell vitality) significantly (p<.01) in 7-day-cultured macrophages compared to control cells cultured out of material. Phagocytosis of both ceramic particles by macrophages was followed by a concomitant decrease in cell phagocytic ability (27%) and a marked altered oxidative metabolism (>2 times reduced by zirconia and >5 times reduced by alumina). The present study clearly demonstrates that reduction of the phagocytic capacity of macrophages associated with altered oxidative metabolism caused by biomaterial particles is characterized by changes in intracellular elemental content. Thus, investigation of cellular homeostasis by electron probe microanalysis together with analysis of functional changes may improve estimation of biomaterial cytocompatibility.

Diffusion of mineral elements evaluated by PIXE at the bone-coral interface.

Substituting the tissue of human organs with biomaterials is problematic. However, its importance and relevance justify all the efforts made. An interdisciplinary approach is required. We report on our study of a product for bone substitution. Coral is a natural product, the interest of which we have already demonstrated in our previous work. Following sterilization, natural coral was implanted in sheep femurs. We regularly extracted the implants from the femurs to study the kinetics of elemental mineral transformation of the bone substitutes. For the first time ever, and thanks to the PIXE method (particles induced X-ray emission), we were able to measure the concentration of mineral elements at different time intervals after implantation over a whole cross-section. We found a discontinuity of the mineral elements (Ca, P, Sr, Zn, Fe) at the interface between the implant and the receiver. This shows that the osseous attack is not global but, on the contrary, centripetal. Moreover, the fit of the concentration time course indicates that the kinetics of ossification are different for each atomic element and characterize a distinct biological phenomenon. Our analyses confirm the biocompatibility and the ossification of the implanted coral.

Effect of hydroxyapatite sintering temperature on intracellular ionic concentrations of monocytes: a TEM-cryo-X-ray microanalysis study.

Hydroxyapatite used as bone replacement can lead to particle release in the implantation site. These particles interact with monocytes, which are the first immune cells to colonize the implant and an inflammatory site. Thanks to cryo-X-ray microanalysis, we can observe cells in a state close to the physiological one and we have access to diffusible ions. We paid particular attention to the potassium-to-sodium ratio, which is one of the best viability criteria. We used this method to study the interaction between three hydroxyapatite particles treated at three different temperatures (not treated, treated at 600 degrees C and 1180 degrees C), and monocytes. In the culture condition, the hydroxyapatite treated at 1180 degrees C underwent the least dissolution. We demonstrate that monocytes were altered by the three hydroxyapatite particles. The hydroxyapatite particules treated at 600 degrees C were found to be more toxic.

Micrometer level structural and chemical evaluation of electrodeposited calcium phosphate coatings on TA6V substrate by STEM-EDXS.

Hydroxyapatite (HA) coatings on titanium alloy substrates Ti6Al4V have been prepared in our laboratory by electrodeposition and hydrothermal synthesis. In this paper, the morphology, crystal size, porosity and Ca/P atomic ratio are investigated using scanning electron microscopy (SEM), scanning transmission electron microscopy (STEM), Raman microspectroscopy and X-ray energy dispersive spectroscopy (EDXS). The results obtained show that after being hydrothermally treated and calcined at high temperature, the electrodeposited brushite coating is converted into a stoichiometric hydroxyapatite having a crystal size which changes considerably from the surface to the substrate alloy. In addition, variation of the surface coating porosity as a function of the electrolyte temperature has also been carried out.