RESUMEN
Dopaminergic neuronal cell death, associated with intracellular α-synuclein (α-syn)-rich protein aggregates [termed "Lewy bodies" (LBs)], is a well-established characteristic of Parkinson's disease (PD). Much evidence, accumulated from multiple experimental models, has suggested that α-syn plays a role in PD pathogenesis, not only as a trigger of pathology but also as a mediator of disease progression through pathological spreading. Here, we have used a machine learning-based approach to identify unique signatures of neurodegeneration in monkeys induced by distinct α-syn pathogenic structures derived from patients with PD. Unexpectedly, our results show that, in nonhuman primates, a small amount of singular α-syn aggregates is as toxic as larger amyloid fibrils present in the LBs, thus reinforcing the need for preclinical research in this species. Furthermore, our results provide evidence supporting the true multifactorial nature of PD, as multiple causes can induce a similar outcome regarding dopaminergic neurodegeneration.
Asunto(s)
Enfermedad de Parkinson , alfa-Sinucleína , Amiloide/metabolismo , Animales , Humanos , Cuerpos de Lewy/química , Cuerpos de Lewy/metabolismo , Cuerpos de Lewy/patología , Enfermedad de Parkinson/metabolismo , PrimatesRESUMEN
The speciation of highly-diluted elements by X-ray absorption spectroscopy in a diverse range of materials is extremely challenging, especially in biological matrices such as articular cartilage. Here we show that using a high energy resolution fluorescence detected X-ray absorption spectroscopy (HERFD-XAS) technique coupled to an array of crystal analyzers, selenium speciation down to 400 ppb (µg kg-1) within articular cartilage can be demonstrated. This is a major advance in the speciation of highly-diluted elements through X-ray absorption spectroscopy and opens new possibilities to study the metabolic role of selenium and other elements in biological samples.
Asunto(s)
Cartílago Articular/química , Selenio/análisis , Animales , Bovinos , Fluorescencia , Humanos , Análisis de los Mínimos Cuadrados , Límite de Detección , Masculino , Análisis de Componente Principal , Espectroscopía de Absorción de Rayos X/métodosRESUMEN
Benefiting from the recent advances of synchrotron X-ray nanoprobes, we demonstrate three-dimensional (3D) correlative nanoimaging on malaria-infected human red blood cells. By combining X-ray fluorescence tomography and phase contrast nanotomography on the same cell with sub-100 nm pixel size, we establish a routine workflow from the data acquisition, data processing, to tomographic reconstruction. We quantitatively compare the elemental volumes obtained with different reconstruction methods, with the total variation minimization giving the most satisfactory results. We reveal elemental correlations in different cell compartments more reliably on reconstructions as opposed to 2D projections. Finally, we determine for the first time the 3D mass fraction maps of multiple elements at the subcellular level. The estimated total number of Fe atoms and the total mass of red blood cells show very good agreement with previously reported values.
Asunto(s)
Eritrocitos/parasitología , Plasmodium falciparum , Microtomografía por Rayos X/métodos , HumanosRESUMEN
The newly discovered unicellular micro-alga Coccomyxa actinabiotis proves to be highly radio-tolerant and strongly concentrates radionuclides, as well as large amounts of toxic metals. This study helps in the understanding of the mechanisms involved in the accumulation and detoxification of silver and cobalt. Elemental distribution inside Coccomyxa actinabiotis cells was determined using synchrotron nano X-ray fluorescence spectroscopy at the ID22 nano fluorescence imaging beamline of the European Synchrotron Radiation Facility. The high resolution and high sensitivity of this technique enabled the assessment of elemental associations and exclusions in subcellular micro-algae compartments. A quantitative treatment of the scans was implemented to yield absolute concentrations of each endogenous and exogenous element with a spatial resolution of 100 nm and compared to the macroscopic content in cobalt and silver determined using inductively coupled plasma-mass spectrometry. The nano X-ray fluorescence imaging was complemented by transmission electron microscopy coupled to X-ray microanalysis (TEM-EDS), yielding differential silver distribution in the cell wall, cytosol, nucleus, chloroplast and mitochondria with unique resolution. The analysis of endogenous elements in control cells revealed that iron had a unique distribution; zinc, potassium, manganese, molybdenum, and phosphate had their maxima co-localized in the same area; and sulfur, copper and chlorine were almost homogeneously distributed among the whole cell. The subcellular distribution and quantification of cobalt and silver in micro-alga, assessed after controlled exposure to various concentrations, revealed that exogenous metals were mainly sequestered inside the cell rather than on mucilage or the cell wall, with preferential compartmentalization. Cobalt was homogeneously distributed outside of the chloroplast. Silver was localized in the cytosol at low concentration and in the whole cell excluding the nucleus at high concentration. Exposure to low concentrations of cobalt or silver did not alter the localization nor the concentration of endogenous elements within the cells. To our knowledge, this is the first report on element co-localization and segregation at the sub-cellular level in micro-algae by means of synchrotron nano X-ray fluorescence spectroscopy.
Asunto(s)
Chlorophyta/metabolismo , Cobalto/metabolismo , Microalgas/metabolismo , Nanopartículas/química , Plata/metabolismo , Espectrometría por Rayos X/métodos , Sincrotrones , Chlorophyta/ultraestructura , Hierro/metabolismo , Microalgas/ultraestructura , Microscopía Electrónica , Nanopartículas/ultraestructura , Análisis de Componente Principal , Espectrofotometría Atómica , Fracciones Subcelulares/metabolismoRESUMEN
Manganese enhanced MRI (MEMRI) offers many possibilities such as tract tracing and functional imaging in vivo. Mn is however neurotoxic and may induce symptoms similar to those associated with Parkinson's disease (manganism). The mechanisms of Mn-induced neurotoxicity are not clear. In this study, we combine synchrotron X-ray fluorescence microprobe (SR-XRF) and MEMRI techniques to investigate spatial distribution of Mn within the rat hippocampus and how Mn interacts with Ca, Fe and Zn at a cellular level. Images were acquired in the rat hippocampus (n=23) and using two injection routes: intra-cerebral (MnCl(2): 50 mM, 10 µL) and intra-peritoneal (MnCl(2): 100 mM, 30 mg/kg). For both injection routes, Mn is found in dentate gyrus and in CA3: control: 2.5 ± 1.6, intra-peritoneal: 5.0 ± 2.4, and intra-cerebral: 25.1 ± 9.2 µg/g. Mn follows Zn distribution and has a negative impact on the total amount of Zn and Fe. The Mn-enhanced MRI contrast is well correlated with the total Mn amount measured with SR-XRF (R(2)=0.93; p<0.002). After intra-cerebral injection, the hippocampal fissure is found to accumulate a large amount of Mn and yields a hypointense MRI signal, which may be ascribed to a reduction in T2. This study shows that SR-XRF is well suited to investigate Mn distribution at a mesoscale and that MRI is sensitive to low Mn concentrations. As perturbations in metal homeostasis may alter brain function, the injected dose of Mn in MEMRI studies needs to be carefully adjusted to obtain reliable functional information.
Asunto(s)
Hipocampo/anatomía & histología , Hipocampo/metabolismo , Hierro/metabolismo , Cloruro de Magnesio/farmacología , Imagen por Resonancia Magnética/métodos , Zinc/metabolismo , Animales , Medios de Contraste/farmacocinética , Medios de Contraste/farmacología , Femenino , Hipocampo/efectos de los fármacos , Tasa de Depuración Metabólica , Ratas , Ratas Sprague-Dawley , Sincrotrones , Distribución Tisular , Microtomografía por Rayos X/métodosRESUMEN
Very little is known about the sub-cellular distribution of metal ions in cells. Some metals such as zinc, copper and iron are essential and play an important role in the cell metabolism. Dysfunctions in this delicate housekeeping may be at the origin of major diseases. There is also a prevalent use of metals in a wide range of diagnostic agents and drugs for the diagnosis or treatment of a variety of disorders. This is becoming more and more of a concern in the field of nanomedicine with the increasing development and use of nanoparticles, which are suspected of causing adverse effects on cells and organ tissues. Synchrotron-based X-ray and Fourier-transformed infrared microspectroscopies are developing into well-suited sub-micrometer analytical tools for addressing new problems when studying the role of metals in biology. As a complementary tool to optical and electron microscopes, developments and studies have demonstrated the unique capabilities of multi-keV microscopy: namely, an ultra-low detection limit, large penetration depth, chemical sensitivity and three-dimensional imaging capabilities. More recently, the capabilities have been extended towards sub-100nm lateral resolutions, thus enabling sub-cellular chemical imaging. Possibilities offered by these techniques in the biomedical field are described through examples of applications performed at the ESRF synchrotron-based microspectroscopy platform (ID21 and ID22 beamlines).
Asunto(s)
Tecnología Biomédica , Sincrotrones , Animales , Células 3T3 BALB , Neuronas Dopaminérgicas/metabolismo , Francia , Hepatocitos/metabolismo , Humanos , Masculino , Manganeso/metabolismo , Melaninas/metabolismo , Metales/metabolismo , Ratones , Microespectrofotometría/métodos , Células PC12 , Fósforo/metabolismo , Potasio/metabolismo , Ratas , Espectroscopía Infrarroja por Transformada de Fourier , Espermatozoides/metabolismo , Rayos XRESUMEN
INTRODUCTION: Many metals like iron (Fe), copper (Cu) or zinc (Zn) fulfil various essential biological functions and are thus vital for all living organisms. For instance, they play important roles in nervous tissue, participating in a wide range of processes such as neurotransmitter synthesis, myelination or synaptic transmission. STATE OF THE ART: As in other tissues, brain cells tightly control the concentration of metals but any excess or deficit can lead to deleterious responses and alter cognitive functions. Of note, certain metals such as Zn, Fe or Cu accumulate in specific brain structures over lifespan and several neurodegenerative diseases are associated with a dysregulation of the homeostatic mechanisms controlling the concentration of these cations. CONCLUSION AND PERSPECTIVES: This review will address some of the cellular and molecular processes controlling the entry and distribution of selected metals (mainly Zn and Fe) in the brain, as well as their roles in synaptic transmission, in the pathogenesis of some neurologic diseases such as Parkinson's disease and Alzheimer's disease, and their impact on cognitive functions.
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Química Encefálica/fisiología , Encéfalo/fisiología , Hierro/fisiología , Oligoelementos/metabolismo , Zinc/fisiología , Animales , Humanos , Hierro/metabolismo , Enfermedades del Sistema Nervioso/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Transmisión Sináptica/fisiología , Zinc/metabolismoRESUMEN
Four-coordinate (Pt(II)) platinum-based anticancer drugs are widely used in primary or palliative chemotherapy and produce considerable efficacy in certain clinical applications, for example testicular cancer. However, in many cancers the Pt(II) drugs are beset by poor efficacy mainly due to suboptimal pharmacokinetic properties. Consequently, the six-coordinate (Pt(IV)) class of Pt drugs were developed to improve platinum efficacy by (i) increasing stability, (ii) reducing reactivity, (iii) increasing lipophilicity, and (iv) nuclear targeting. However, comparatively little information is available on the pharmacokinetic properties of these compounds within solid tumour tissue. In the present study, the distribution and fluxes of [(14)C]-labelled [PtCl(2)(en)] (where en stands for ethane-1,2-diamine) and cis,trans-[PtCl(2)(OH)(2)(en)] drugs were determined in the multicell layer (MCL) tumour model comprising colon cancer cells. Flux data were analysed by mathematical modelling of drug diffusion and cellular uptake in the transport system. The flux of the Pt(IV) compound through the MCL was not significantly different to that of the Pt(II) drug nor were the diffusion coefficient or tissue uptake; the latter confirmed with elemental imaging analysis by synchrotron radiation induced X-ray emission. However, the flux of the Pt(IV) through the MCL was increased by hydrostatic pressure, thereby demonstrating the potential to target cancer cells further away from the vessels with six-coordinate platinum drugs.
Asunto(s)
Antineoplásicos/metabolismo , Neoplasias/metabolismo , Compuestos Organoplatinos/metabolismo , Transporte Biológico , Línea Celular Tumoral , Humanos , Cinética , Modelos BiológicosRESUMEN
In the present study, we characterized and compared the mineral phase deposited in the aortic wall of two different frequently used chronic renal failure rat models of vascular calcification. Vascular calcification was induced in rats by either a 4-week adenine treatment followed by a 10-week high-phosphate diet or 5/6 nephrectomy followed by 6 weeks of 0.25 microg/kg/day calcitriol treatment and a high-phosphate diet. Multi-element mapping for calcium and phosphate together with mineral identification was performed on several regions of aortic sections by means of synchrotron X-ray-mu-fluorescence and diffraction. Bulk calcium and magnesium content of the aorta was assessed using flame atomic absorption spectrometry. Based on the diffraction data the Von Kossa-positive precipitate in the aortic regions (N=38) could be classified into three groups: (1) amorphous precipitate (absence of any diffraction peak pattern, N=12); (2) apatite (N=16); (3) a combination of apatite and magnesium-containing whitlockite (N=10). The occurrence of these precipitates differed significantly between the two models. Furthermore, the combination of apatite and whitlockite was exclusively found in the calcitriol-treated animals. These data indicate that in adenine/phosphate-induced uremia-related vascular calcification, apatite is the main component of the mineral phase. The presence of magnesium-containing whitlockite found in addition to apatite in the vitamin D-treated rats, has to be seen in view of the well-known vitamin D-stimulated gastrointestinal absorption of magnesium.
Asunto(s)
Apatitas/metabolismo , Calcinosis/metabolismo , Insuficiencia Renal/complicaciones , Uremia/complicaciones , Enfermedades Vasculares/metabolismo , Animales , Aorta/metabolismo , Calcinosis/tratamiento farmacológico , Calcinosis/etiología , Calcitriol/uso terapéutico , Agonistas de los Canales de Calcio/uso terapéutico , Masculino , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Insuficiencia Renal/metabolismo , Espectrometría por Rayos X , Uremia/metabolismo , Enfermedades Vasculares/tratamiento farmacológico , Enfermedades Vasculares/etiología , Difracción de Rayos XRESUMEN
The abnormalities of metallochemical reactions may contribute to the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). In the present work, an investigation of the elemental composition of the gray matter, nerve cells and white matter from spinal cord tissues representing three ALS cases and five non-ALS controls was performed. This was done with the use of the synchrotron microbeam X-ray fluorescence technique (micro-SRXRF). The following elements were detected in the tissue sections: P, S, Cl, K, Ca, Fe, Cu, Zn and Br. A higher accumulation of Cl, K, Ca, Zn and Br was observed in the nerve cell bodies than in the surrounding tissue. Contrary to all other elements, Zn accumulation was lower in the white matter areas than in the gray matter ones. The results of quantitative analysis showed that there were no general abnormalities in the elemental accumulation between the ALS and the control group. However, for individual ALS cases such abnormalities were observed for the nerve cells. We also demonstrated differences in the elemental accumulation between the analyzed ALS cases.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Halógenos/análisis , Metales Alcalinotérreos/análisis , Metales Pesados/análisis , Fósforo/análisis , Potasio/análisis , Azufre/análisis , Humanos , Microscopía Fluorescente/métodos , Espectrometría por Rayos X/métodos , Médula Espinal/química , Sincrotrones , Rayos XRESUMEN
The ID22 beamline is dedicated to hard X-ray microanalysis allowing the combination of fluorescence, spectroscopy, diffraction and tomography techniques in a wide energy range from 6 to 70 keV. The recent installation of an in-vacuum undulator, a new sample stage and the adaptation of various focusing optics has contributed to a great improvement in the capabilities of the beamline, which is now accessed by a wide user community issued from medical, earth and environmental science, archaeology and material science. Many applications requiring low detection limits for localization/speciation of trace elements together with structural analysis have been developed at the beamline on the (sub)micrometer scale. The possibility of combining simultaneously different analytical probes offers the opportunity of a thorough study of a given sample or scientific problem. This paper presents a review of the recent developments of the beamline and a detailed description of its capabilities through examples from different fields of applications.
Asunto(s)
Ensayo de Materiales/instrumentación , Espectrometría por Rayos X/instrumentación , Sincrotrones , Difracción de Rayos X/instrumentación , Contaminantes Atmosféricos/análisis , Diseño de Equipo , Óptica y FotónicaRESUMEN
In this paper we describe the results of experiments using synchrotron radiation to trigger the Auger effect in living human cancer cells treated with a widely used chemotherapy drug: cis-diamminedichloroplatinum (II) (cisplatin). The experiments were carried out at the ID17 beamline of the European Synchrotron Radiation Facility, which produces a high-fluence monochromatic beam that is adjustable from 20 to 80 keV. Cisplatin was chosen as the carrier of platinum atoms in the cells because of its alkylating-like activity and the irradiation was done with monochromatic beams above and below the platinum K-shell edge (78.39 keV). Cell survival curves were comparable with those obtained for the same cells under conventional irradiation conditions. At a low dose of cisplatin (0.1 microM, 48 h), no difference was seen in survival when the cells were irradiated above and below the K-shell edge of platinum. Higher cisplatin concentrations were investigated to enhance the cellular platinum content. The results with 1 microM cisplatin for 12 h showed no difference when the cells were irradiated with beams above or below the platinum K-shell edge with the exception of the higher cell death resulting from drug toxicity. The intracellular content of platinum was significant, as measured macroscopically by inductively coupled plasma mass spectrometry. Its subcellular localization and particularly its presence in the cell nucleus were verified by microscopic synchrotron X-ray fluorescence. This was the first known attempt at K-shell edge photon activation of stable platinum in living cells with a platinum complex used for chemotherapy. Its evident toxicity in these cells leads us to put forth the hypothesis that cisplatin toxicity can mask the enhancement of cell death induced by the irradiation above the K-shell edge. However, K-shell edge photon activation of stable elements provides a powerful technique for the understanding of the biological effects of Auger processes. Further avenues of development are discussed.
Asunto(s)
Muerte Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Cisplatino/farmacología , Aceleradores de Partículas/instrumentación , Platino (Metal)/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Rayos X , Calibración , Ciclo Celular , Línea Celular , Ensayo de Unidades Formadoras de Colonias , Relación Dosis-Respuesta en la Radiación , Citometría de Flujo , Neoplasias de Cabeza y Cuello/radioterapia , Humanos , Fotones , Células Tumorales CultivadasRESUMEN
Hydroxypropylmethylcellulose (HPMC) is used as a ligand for a bioactive calcium phosphate ceramic (the filler) in a ready-to-use injectable sterilized biomaterial for bone and dental surgery. Light scattering experiments were usually used to study high water-soluble polymers and to determine the basic macromolecular parameters. In order to gain a deeper understanding of polymer/mineral interactions in this type of material, we have investigated the effect of divalent and trivalent ions (Ca(2+), PO(4)(3-)) and steam sterilization on dilute solutions of HPMC and hydroxyethylcellulose (HEC). The sterilization process may cause some degradation of HEC taking into account its high molecular weight and some rigidity of the polymer chain. Moreover, in the case of HPMC, the changes in the conformations rather than degradation process are supposed. These effects of degradation and flocculation are strengthened in alkaline medium. Experimental data suggested the formation of chelate complexes between Ca(2+) and HPMC which improve its affinity to the mineral blend and consolidate the injectable biomaterial even in the case of its hydration by biological fluid.
RESUMEN
Bisphosphonates have been widely used in the treatment of human bone pathologies including osteoporosis. In this case, bisphosphonates have been shown to reduce bone resorption, thereby increasing the mass and mechanical resistance of bone. Determining the effects of these molecules on the properties of the bone apatite crystals could provide a better insight into the mechanism of bisphosphonate/bone interaction. The aim of this study was to determine the ultrastructural effects of a third generation bisphosphonate (tiludronate) on the morphology, size, distribution, chemical composition, and structure of apatite crystals in bone (trabecular) in a rat osteoporotic model. Four groups of rats were studied: (1) sham operated, (2) untreated ovariectomized (OVX), (3) OVX rats which received 35 mg/kg of tiludronate, (4) OVX rats which received 160 mg/kg of tiludronate. The rats of groups 3 and 4 received tiludronate orally in 2 consecutive days every week for 1 year. Scanning electron microscopy (SEM), high and low resolution transmission electron microscopy (TEM), and electron microprobe analysis (EDX) were used for the ultrastructural characterization of the bone mineral. This study demonstrated that tiludronate slightly increased the width of bone apatite crystals without changing any other crystal characteristics.
Asunto(s)
Densidad Ósea/efectos de los fármacos , Calcificación Fisiológica/efectos de los fármacos , Difosfonatos/uso terapéutico , Vértebras Lumbares/ultraestructura , Osteoporosis/tratamiento farmacológico , Animales , Apatitas/análisis , Densidad Ósea/fisiología , Calcio/análisis , Difosfonatos/administración & dosificación , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Microanálisis por Sonda Electrónica , Femenino , Vértebras Lumbares/química , Vértebras Lumbares/efectos de los fármacos , Magnesio/análisis , Microscopía Electrónica de Rastreo , Osteoporosis/patología , Ovariectomía , Fósforo/análisis , Ratas , Ratas Sprague-DawleyRESUMEN
Bisphosphonates, potent inhibitors of bone resorption, have been used clinically to correct the continued loss of bone mass in osteoporosis and in other conditions. However, there has been some concern that long-term treatment with these compounds, as well as more recently developed drugs, may also decrease the rate of bone formation. Bisphosphonates, which are strongly bound to hydroxyapatite crystals, may alter the structure and reactivity of the crystals, interfere with new crystal nucleation and growth, as well as alter the short-range order of newly formed crystals. We have investigated the chemistry and structure of the solid calcium-phosphate mineral phase of lumbar vertebrae of ovariectomized, 6.5-month-old rats treated with bisphosphonates for 1 year after onset of osteopenia. Appropriate control groups were used for comparison. The techniques used to assess the mineral phase were chemical analyses, Fourier transform-infrared (FT-IR) and FT-Raman spectroscopy, FT-IR microspectroscopy, and phosphorus-31 magic-angle-sample spinning nuclear magnetic resonance spectroscopy ((31)P MAS NMR). The (31)P MAS NMR spectra of trabecular bone of lumbar vertebrae of control, ovariectomized, and treated animals were similar. However, there were several significant differences in the results obtained by FT-IR spectroscopy of the whole tissue samples, FT-IR microspectroscopy of sections of bone, and chemical analyses. For example, whereas chemical analyses demonstrated that the CO(3) content of the mineral phase of the ovariectomized animals was decreased compared with controls, FT-IR microspectroscopy of bone sections showed no changes in the relative CO(3) content, but some changes in the environment of the CO(3) groups. However, chemical analyses of the crystals, combined with data from all three spectroscopic methods and with data from serum analysis, did indicate small changes in the mineral phase after ovariectomy, corrected after treatment with bisphosphonates. In any event, the chemical and structural data in the present studies demonstrate that the bisphosphonate, tiludronate, does not significantly alter the mineral components of bone after 1 year of treatment during the course of which bone loss was reversed.
Asunto(s)
Enfermedades Óseas Metabólicas/fisiopatología , Difosfonatos/farmacología , Vértebras Lumbares/efectos de los fármacos , Ovariectomía , Animales , Apatitas/análisis , Enfermedades Óseas Metabólicas/inducido químicamente , Enfermedades Óseas Metabólicas/tratamiento farmacológico , Resorción Ósea/inducido químicamente , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/fisiopatología , Calcio/sangre , Colecalciferol/sangre , Modelos Animales de Enfermedad , Femenino , Vértebras Lumbares/fisiología , Espectroscopía de Resonancia Magnética , Hormona Paratiroidea/sangre , Fósforo/sangre , Radioisótopos de Fósforo , Ratas , Ratas Sprague-Dawley , Espectroscopía Infrarroja por Transformada de Fourier , Pérdida de PesoRESUMEN
Calcium phosphate ceramics (CaP) have recently been proposed as a potential matrix for a bioactive drug delivery system (DDS) in which the effect in situ of a released therapeutic agent is favored by the biocompatibility, osteoconductivity, and bioresorption of the ceramic material. Polymyxin B (PMB) is a polypeptidic antibiotic which undergoes thermodamage above 60 degrees C. The dynamic compaction method was developed to consolidate the drug load on CaP powder without external heating. Two projectile velocities (50 and 25 m/s) were used here to achieve powder consolidation. Among the different techniques used to associate therapeutic agents with CaP, wet adsorption was performed before the dynamic compaction process. The PMB release profile was measured by a capillary electrophoresis technique, CaP crystallography was studied by x-ray diffraction, and CaP physicochemical analysis was performed by infrared spectroscopy. The biological activities of PMB-loaded compacted CaP were determined by the effect of the antibiotic and monocyte/macrophage degradation on compact surfaces. PMB release began after 2-3 days of incubation for blocks compacted at 25 m/s velocity and on day 5 for those compacted at 50 m/s velocity. A discrepancy was noted between the amounts of PMB released (0.5-2.1 mg) and the amounts initially compacted (2-8 mg) with CaP powder. The biological activities (antibacterial activity and inhibited lipopolysaccharide effects on monocyte/macrophage CaP degradation) of PMB released from compacted calcium-deficient apatite were unaltered. Thus, dynamic compaction allows PMB to be used with CaP ceramics without any loss in its integrity and biological effects.
Asunto(s)
Antibacterianos/administración & dosificación , Antibacterianos/farmacocinética , Materiales Biocompatibles , Fosfatos de Calcio , Polimixina B/administración & dosificación , Polimixina B/farmacocinética , Apatitas/aislamiento & purificación , Materiales Biocompatibles/aislamiento & purificación , Fosfatos de Calcio/aislamiento & purificación , Células Cultivadas , Cerámica , Cristalografía por Rayos X , Sistemas de Liberación de Medicamentos , Humanos , Técnicas In Vitro , Macrófagos/metabolismo , Ensayo de Materiales , Monocitos/metabolismo , Espectrometría de Masa Bombardeada por Átomos Veloces , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de SuperficieRESUMEN
There is increasing evidence that noncollagenous matrix proteins initiate bone mineralization in vivo. Fibronectin, which is present during the early phases of mineralization, may contribute to this process in bone tissues. In this context, the mineralization potential of fibronectin was tested in an agarose gel precipitation system and a metastable calcium phosphate solution. The protein inhibited the precipitation of calcium phosphate crystals in solution but had no apparent effect in gel. Conversely, fibronectin stimulated crystal formation when apatite powder was used to seed crystal growth in gel. Although these results in vitro do not clearly indicate that fibronectin is involved in the mineralization process, they are consistent with in vivo events. Free fibronectin (e.g. in biological fluids) could inhibit crystal growth but might also activate the mineralization process when absorbed on apatite powder in a bone environment and areas of ectopic mineralization.
Asunto(s)
Durapatita/química , Fibronectinas/química , Animales , Huesos/química , Huesos/metabolismo , Fosfatos de Calcio/química , Bovinos , Precipitación Química , Cristalización , Cristalografía por Rayos X , Durapatita/metabolismo , Fibronectinas/metabolismo , Geles , Humanos , Técnicas In Vitro , Minerales/química , Minerales/metabolismo , Sefarosa , SolucionesRESUMEN
Heterotopic calcification induced after implantation of bone-marrow cells under the murine kidney capsule was used to study the mineral phases occurring during the mineralization process. Ossicles were found to contain numerous osteoblastic cells that produced an organic matrix closely associated with active hematopoietic tissue. During implantation of bone marrow, needle-shaped microcrystals were progressively deposited on collagen fibers. The mineral formed in the heterotopic calcification consisted mainly of calcium phosphate. The distribution and density of the microcrystals were heterogeneous after 6 weeks of implantation but became homogeneous and well-crystallized after 10 weeks. The Fourier transform infrared microspectroscopy provided important spatial data on the nature of the mineral formed and the changes in the mineral environment. Similarities were noted between young bone (bone callus) and 6-week heterotopic ossicles, and between adult bone and 10- or 12-week heterotopic ossicles. The study demonstrated that murine heterotopic calcification under the renal capsule can be a very useful model for studying bone apatite formation during the mineralization process.
Asunto(s)
Trasplante de Médula Ósea , Riñón/fisiopatología , Trasplante Heterotópico , Animales , Calcificación Fisiológica , Calcinosis , Femenino , Riñón/ultraestructura , Ratones , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Modelos Biológicos , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Fourier-transform infrared microspectroscopy (FT-IRM) was used to study bone mineralization processes in an in vivo model and in enamel in osteogenesis imperfecta. Finally, the ability of FT-IRM to map new bone formed in implanted macroporous calcium phosphate biomaterial from sections was reported for the first time. FT-IRM allowed the correlation of the microstructure of bone formation in the in vivo model with modifications in carbonate and phosphate environments of the mineral phases during maturation. FR-IRM analysis on enamel sections revealed changes in the mineral environment of carbonate and phosphate ions and probably in the size of enamel crystals. These modifications contributed to the fragility of enamel in osteogenesis imperfecta. The infrared functional group imaging of a part of implanted biomaterial and the bone ingrowth provided the visualization of chemical modifications occurring in biomaterial implants at 20 microns spatial resolution. The use of FT-IRM, in conjunction with appropriate sampling methods and data analysis should provide further insight into the molecular structure of mineral phases of calcified tissues and help to elucidate mineralization processes, skeletal disorders and properties of the biomaterials used as bone substitute.