RÉSUMÉ
Ozone (O3) is a short-lived molecule which can be produced in a controlled reaction when oxygen is exposed to electric discharge. In the last few decades, many publications dealing both with animals and humans reported beneficial effects of ozone administration linked to its immunomodulatory and protective role against cellular damage. This is the first work which brings insight into how ozone influences cells of neural lineage in vitro and hypothesizes the potential molecular and novel electromagnetic mechanisms behind its action. By using neural stem cells, we show that ozone, especially in concentrations of around 11⯵g/mL, significantly increases the speed of neural cell migration. With much lower effects, it also increases cell proliferation and cytokine production. Results of this study, at least partly, explain the observed beneficial effects of ozone in diseases of the nervous system tested on animal models and in human clinical trials. Therefore, here described effects of ozone on cellular level represent a firm basis for further investigation of possible applications of ozone in regeneration of the nervous system.
Sujet(s)
Prolifération cellulaire/effets des médicaments et des substances chimiques , Cellules souches neurales/effets des médicaments et des substances chimiques , Ozone/administration et posologie , Animaux , Cellules cultivées , SourisRÉSUMÉ
Bone and cartilage are tissues of a three-dimensional (3D) nature. Therefore, scaffolds for their regeneration should support cell infiltration and growth in all 3 dimensions. To fulfill such a requirement, the materials should possess large, open pores. Centrifugal spinning is a simple method for producing 3D fibrous scaffolds with large and interconnected pores. However, the process of bone regeneration is rather complex and requires additional stimulation by active molecules. In the current study, we introduced a simple composite scaffold based on platelet adhesion to poly-ε-caprolactone 3D fibers. Platelets were used as a natural source of growth factors and cytokines active in the tissue repair process. By immobilization in the fibrous scaffolds, their bioavailability was prolonged. The biological evaluation of the proposed system in the MG-63 model showed improved metabolic activity, proliferation and alkaline phosphatase activity in comparison to nonfunctionalized fibrous scaffold. In addition, the response of cells was dose dependent with improved biocompatibility with increasing platelet concentration. The results demonstrated the suitability of the system for bone tissue.
Sujet(s)
Plaquettes/métabolisme , Systèmes de délivrance de médicaments/méthodes , Protéines et peptides de signalisation intercellulaire/administration et posologie , Protéines et peptides de signalisation intercellulaire/pharmacologie , Polyesters/composition chimique , Ingénierie tissulaire/méthodes , Structures d'échafaudage tissulaires/composition chimique , Phosphatase alcaline/métabolisme , Animaux , Plaquettes/effets des médicaments et des substances chimiques , Plaquettes/ultrastructure , Lignée cellulaire tumorale , Prolifération cellulaire/effets des médicaments et des substances chimiques , Forme de la cellule/effets des médicaments et des substances chimiques , Humains , Cinétique , Ostéoblastes/cytologie , Ostéoblastes/effets des médicaments et des substances chimiques , Ostéoblastes/ultrastructure , Ostéogenèse/effets des médicaments et des substances chimiques , Adhésivité plaquettaire/effets des médicaments et des substances chimiques , Polyesters/pharmacologieRÉSUMÉ
BACKGROUND: The macroscopic structure of bone has been traditionally studied through x-ray radiography or x-ray tomography. However, the resolution limits of these techniques do not enable the reconstruction of the composite bone architecture at the nanometer level. Compared with histomorphometry, x-ray micro-CT has shown its efficiency in providing nondestructive and rapid 3D images and measurements on bone microstructure. Micro-CT higher resolution has been achieved with synchrotron radiation-based computed microtomography (SRµCT). PURPOSE: The aim of this study was a histological and SRµCT analysis of 2 porous titanium implants. MATERIALS AND METHODS: Two direct laser metal forming titanium implants were inserted in the posterior maxilla of a patient and retrieved after 2 months. One of these implants was treated to obtain thin ground sections, whereas the other underwent a SRµCT evaluation. RESULTS: The histological results, showing that the implant surface presented superficial debris and particle inclusions in the surrounding tissue close to the bone area, were confirmed by micro-CT investigations. CONCLUSIONS: SRµCT allowed high resolution with good sample penetration and depth of focus and an evaluation of the relative arrangement of structures that cannot be determined by 2D imaging.
Sujet(s)
Alliage dentaire/composition chimique , Implants dentaires , Conception de prothèse dentaire , Lasers , Maxillaire/anatomopathologie , Titane/composition chimique , Microtomographie aux rayons X/méthodes , Alliages , Régénération osseuse/physiologie , Remodelage osseux/physiologie , Humains , Traitement d'image par ordinateur/méthodes , Imagerie tridimensionnelle/méthodes , Mâle , Maxillaire/imagerie diagnostique , Maxillaire/chirurgie , Vidéomicroscopie/méthodes , Adulte d'âge moyen , Miniaturisation , Ostéo-intégration/physiologie , Ostéoblastes/anatomopathologie , Ostéocytes/anatomopathologie , Ostéogenèse/physiologie , Porosité , Propriétés de surface , SynchrotronsRÉSUMÉ
Mesenchymal stem cells deriving from dental pulp differentiate into osteoblasts capable of producing bone. In previous studies, we extensively demonstrated that, when seeded on collagen I scaffolds, these cells can be conveniently used for the repair of human mandible defects. Here, we assess the stability and quality of the regenerated bone and vessel network 3 years after the grafting intervention, with conventional procedures and in-line holotomography, an advanced phase-imaging method using synchrotron radiation that offers improved sensitivity toward low-absorbing structures. We found that the regenerated tissue from the graft sites was composed of a fully compact bone with a higher matrix density than control human alveolar spongy bone from the same patient. Thus, the regenerated bone, being entirely compact, is completely different from normal alveolar bone. Although the bone regenerated at the graft sites is not of the proper type found in the mandible, it does seem to have a positive clinical impact. In fact, it creates steadier mandibles, may well increase implant stability, and, additionally, may improve resistance to mechanical, physical, chemical, and pharmacological agents.
Sujet(s)
Mandibule/anatomopathologie , Régénération , Transplantation de cellules souches , Cellules souches/cytologie , Tomographie/méthodes , Composés azoïques , Biopsie , Densité osseuse , Éosine jaunâtre/métabolisme , Hématoxyline/métabolisme , Humains , Imagerie tridimensionnelle , Mandibule/imagerie diagnostique , Vert de méthyle , Radiographie , Coloration et marquage , SynchrotronsRÉSUMÉ
Recently published reports have described possible cellular therapy approaches to regenerate muscle tissues using arterial route delivery. However, the kinetic of distribution of these migratory stem cells within injected animal muscular dystrophy models is unknown. Using living X-ray computed microtomography, we established that intra-arterially injected stem cells traffic to multiple muscle tissues for several hours until their migration within dystrophic muscles. Injected stem cells express multiple traffic molecules, including VLA-4, LFA-1, CD44, and the chemokine receptor CXCR4, which are likely to direct these cells into dystrophic muscles. In fact, the majority of intra-arterially injected stem cells access the muscle tissues not immediately after the injection, but after several rounds of recirculation. We set up a new, living, 3D-imaging approach, which appears to be an important way to investigate the kinetic of distribution of systemically injected stem cells within dystrophic muscle tissues, thereby providing supportive data for future clinical applications.
Sujet(s)
Dystrophies musculaires/chirurgie , Transplantation de cellules souches/méthodes , Cellules souches/cytologie , Microtomographie aux rayons X/méthodes , Antigène AC133 , Animaux , Antigènes CD/sang , Antigènes CD/composition chimique , Cellules cultivées , Dextrane/composition chimique , Dextrane/pharmacocinétique , Modèles animaux de maladie humaine , Membres/imagerie diagnostique , Glycoprotéines/sang , Glycoprotéines/composition chimique , Humains , Agranulocytes/cytologie , Agranulocytes/métabolisme , Nanoparticules de magnétite/composition chimique , Souris , Souris SCID , Peptides/sang , Peptides/composition chimique , Réaction de polymérisation en chaine en temps réel , Recherche sur les cellules souches , Cellules souches/composition chimique , Distribution tissulaireRÉSUMÉ
Bone is a complex dynamic tissue undergoing a continuous remodeling process. Gravity is a physical force playing a role in the remodeling and contributing to the maintenance of bone integrity. This article reports an investigation on the alterations of the bone microarchitecture that occurred in wild type (Wt) and pleiotrophin-transgenic (PTN-Tg) mice exposed to a near-zero gravity on the International Space Station (ISS) during the Mice Drawer System (MDS) mission, to date, the longest mice permanence (91 days) in space. The transgenic mouse strain over-expressing pleiotrophin (PTN) in bone was selected because of the PTN positive effects on bone turnover. Wt and PTN-Tg control animals were maintained on Earth either in a MDS payload or in a standard vivarium cage. This study revealed a bone loss during spaceflight in the weight-bearing bones of both strains. For both Tg and Wt a decrease of the trabecular number as well as an increase of the mean trabecular separation was observed after flight, whereas trabecular thickness did not show any significant change. Non weight-bearing bones were not affected. The PTN-Tg mice exposed to normal gravity presented a poorer trabecular organization than Wt mice, but interestingly, the expression of the PTN transgene during the flight resulted in some protection against microgravity's negative effects. Moreover, osteocytes of the Wt mice, but not of Tg mice, acquired a round shape, thus showing for the first time osteocyte space-related morphological alterations in vivo. The analysis of specific bone formation and resorption marker expression suggested that the microgravity-induced bone loss was due to both an increased bone resorption and a decreased bone deposition. Apparently, the PTN transgene protection was the result of a higher osteoblast activity in the flight mice.
Sujet(s)
Remodelage osseux/génétique , Remodelage osseux/physiologie , Protéines de transport/génétique , Protéines de transport/physiologie , Cytokines/génétique , Cytokines/physiologie , Vol spatial , Impesanteur/effets indésirables , Animaux , Os et tissu osseux/cytologie , Os et tissu osseux/imagerie diagnostique , Os et tissu osseux/physiologie , Amorces ADN/génétique , Mâle , Souris , Souris de lignée C57BL , Souris transgéniques , Ostéocytes/cytologie , Ostéocytes/physiologie , Spiranes , Mise en charge/physiologie , Microtomographie aux rayons XRÉSUMÉ
The recent introduction of stem cells in cardiology provides new tools in understanding the regenerative processes of the normal and pathological heart and has opened a search for new therapeutic strategies. Recent published reports have contributed to identifying possible cellular therapy approaches to generate new myocardium, involving transcoronary and intramyocardial injection of progenitor cells. However, one of the limiting factors in the overall interpretation of clinical results obtained by cell therapy is represented by the lack of three-dimensional (3D) high-resolution methods for the visualization of the injected cells and their fate within the myocardium. This work shows that X-ray computed microtomography may offer the unique possibility of detecting, with high definition and resolution and in ex vivo conditions, the 3D spatial distribution of rat cardiac progenitor cells, labelled with iron oxide nanoparticles, inside the infarcted rat heart early after injection. The obtained 3D images represent a very innovative progress as compared to experimental two-dimensional (2D) histological analysis, which requires time-consuming energies for image reconstruction in order to provide the overall distribution of rat clonogenic cells within the heart. Through microtomography, we were able to observe in 3D the presence of these cells within damaged cardiac tissue, with important structural details that are difficult to visualize by conventional bidimensional imaging techniques. This new 3D-imaging approach appears to be an important way to investigate the cellular events involved in cardiac regeneration and represents a promising tool for future clinical applications.
Sujet(s)
Mouvement cellulaire , Imagerie tridimensionnelle/méthodes , Myoblastes cardiaques/cytologie , Infarctus du myocarde/anatomopathologie , Infarctus du myocarde/thérapie , Myocarde/anatomopathologie , Transplantation de cellules souches , Microtomographie aux rayons X/méthodes , Animaux , Cellules cultivées , Fluorescence , Protéines à fluorescence verte/métabolisme , Mâle , Infarctus du myocarde/imagerie diagnostique , Inclusion en paraffine , Rats , Rat WistarRÉSUMÉ
Advances in stem cell research have provided important understanding of the cell biology and offered great promise for developing new strategies for tissue regeneration. The beneficial effects of stem cell therapy depend also by the development of new approachs for the track of stem cells in living subjects over time after transplantation. Recent developments in the use of nanotechnologies have contributed to advance of the high-resolution in vivo imaging methods, including positron emission tomography (PET), single-photon emission tomography (SPECT), magnetic resonance (MR) imaging, and X-Ray computed microtomography (microCT). This review examines the use of nanotechnologies for stem cell tracking.
Sujet(s)
Suivi cellulaire/méthodes , Nanotechnologie/méthodes , Cellules souches/cytologie , Animaux , Humains , Imagerie par résonance magnétique , Radiographie , Cellules souches/imagerie diagnostique , Tomographie par émission monophotoniqueRÉSUMÉ
Spatiotemporal organized patterns of cell surface-associated and extracellular matrix (ECM)-embedded molecules play important roles in the development and functioning of tissues. ECM proteins interact with the surface of bioscaffold polymers and influence material-driven control of cell differentiation., Using X-ray phase-contrast micro computed tomography (microCT), we visualized the three-dimensional (3D) image of ECM organization after in vitro seeding of bone marrow-derived human and murine mesenchymal stem cells (MSCs) induced to myogenic differentiation, labelled with iron oxide nanoparticles, and seeded onto polyglycolic acid-polylactic acid scaffolds. X-ray microCT enabled us to detect with high spatial resolution the 3D structural organization of ECM within the bioscaffold and how the presence of cells modified the construct arrangement. Species-specific differences between the matrix produced by human and murine cells were observed. In conclusion, X-ray synchrotron radiation microCT analysis appeared to be a useful tool to identify the spatiotemporal pattern of organization of ECM fibers within a bioscaffold.
Sujet(s)
Matériaux biocompatibles/composition chimique , Protéines de la matrice extracellulaire/métabolisme , Protéines de la matrice extracellulaire/ultrastructure , Cellules souches mésenchymateuses/ultrastructure , Acide polyglycolique/composition chimique , Ingénierie tissulaire/méthodes , Animaux , Techniques de culture cellulaire/méthodes , Cellules cultivées , Cristallisation/méthodes , Matrice extracellulaire/composition chimique , Humains , Test de matériaux , Souris , Conformation moléculaire , Taille de particule , Propriétés de surface , Synchrotrons , TomodensitométrieRÉSUMÉ
Standard X-ray micro-computed tomography is a technique that allows a good visualization of the structure of mineralized tissues and biomaterials, but it fails to finely discern soft tissues. Here, we used X-ray synchrotron radiation pseudo-holotomography to visualize, at three-dimensional (3D) level, microvascular networks for the first time with no need for contrast agents, and to extract quantitative structural data in a bone-engineered construct implanted for 24 weeks in a mouse. When compared to standard histology, pseudo-holotomography allowed a previously unavailable 3D resolution of the vessels, which in turn appeared more clearly visible. Thus, pseudo-holotomography is an innovative technique that offers a promising powerful tool to investigate angio- and microvasculogenesis in advanced biomedical research areas such as regenerative medicine and antiangiogenic cancer therapies.
Sujet(s)
Holographie/méthodes , Amélioration d'image/méthodes , Cellules souches mésenchymateuses/cytologie , Cellules souches mésenchymateuses/physiologie , Microvaisseaux/cytologie , Néovascularisation physiologique/physiologie , Tomodensitométrie/méthodes , Animaux , Différenciation cellulaire , Cellules cultivées , Produits de contraste , Imagerie tridimensionnelle/méthodes , Microvaisseaux/physiologie , Projets pilotes , Ovis , Technique de soustraction , SynchrotronsRÉSUMÉ
Synchrotron radiation micro-computed tomography (SRmuCT) revealed the microstructure of a CEL2 glass-ceramic scaffold with macropores of several hundred microns characteristic length, in terms of the voxel-by-voxel 3D distribution of the attenuation coefficients throughout the scanned space. The probability density function of all attenuation coefficients related to the macroporous space inside the scaffold gives access to the tomograph-specific machine error included in the SRmuCT measurements (also referred to as instrumental resolution function). After Lorentz function-based clearing of the measured CT data from the systematic resolution error, the voxel-specific attenuation information of the voxels representing the solid skeleton is translated into the composition of the material inside one voxel, in terms of the nanoporosity embedded in a dense CEL2 glass-ceramic matrix. Based on voxel-invariant elastic properties of dense CEL2 glass-ceramic, continuum micromechanics allows for translation of the voxel-specific nanoporosity into voxel-specific elastic properties. They serve as input for Finite Element analyses of the scaffold structure. Young's modulus of a specific CT-scanned macroporous scaffold sample, predicted from a Finite Element simulation of a uniaxial compression test, agrees well with the experimental value obtained from an ultrasonic test on the same sample. This highlights the satisfactory predictive capabilities of the presented approach.
Sujet(s)
Os et tissu osseux/imagerie diagnostique , Tomodensitométrie/méthodes , Phénomènes biomécaniques , Élasticité , Modèles biologiques , Nanostructures , Porosité , Ingénierie tissulaire , Structures d'échafaudage tissulairesRÉSUMÉ
Environmental conditions likely affect physiology and behaviour of mice used for life sciences research on Earth or in Space. Here, we analysed the effects of cage confinement on the weightbearing musculoskeletal system, behaviour and stress of wild-type mice (C57BL/6JRj, 30 g b.wt., total n = 24) housed for 25 days in a prototypical ground-based and fully automated life support habitat device called "Mice in Space" (MIS). Compared with control housing (individually ventilated cages) the MIS mice revealed no significant changes in soleus muscle size and myofiber distribution (type I vs. II) and quality of bone (3-D microarchitecture and mineralisation of calvaria, spine and femur) determined by confocal and micro-computed tomography. Corticosterone metabolism measured non-invasively (faeces) monitored elevated adrenocortical activity at only start of the MIS cage confinement (day 1). Behavioural tests (i.e., grip strength, rotarod, L/D box, elevated plus-maze, open field, aggressiveness) performed subsequently revealed only minor changes in motor performance (MIS vs. controls). The MIS habitat will not, on its own, produce major effects that could confound interpretation of data induced by microgravity exposure during spaceflight. Our results may be even more helpful in developing multidisciplinary protocols with adequate scenarios addressing molecular to systems levels using mice of various genetic phenotypes in many laboratories.
Sujet(s)
Comportement animal/physiologie , Espaces restreints , Hébergement animal , Systèmes de maintien de vie , Appareil locomoteur/anatomie et histologie , Stress physiologique/physiologie , Analyse de variance , Animaux , Poids , Calcification physiologique/physiologie , Corticostérone/sang , Fèces/composition chimique , Immunohistochimie , Mâle , Souris , Souris de lignée C57BL , Muscles squelettiques/anatomie et histologie , Appareil locomoteur/imagerie diagnostique , Microtomographie aux rayons XRÉSUMÉ
Aggregation behavior of unsubstituted copper porphyrazine (CuPaz) on the water surface was studied by analysis of compression curves, Brewster angle microscopy (BAM), and optical spectroscopy. The structure and stability of the CuPaz aqua aggregates in the floating layers are determined by hydration degree that depends on initial surface concentration and surface pressure. Langmuir-Schaefer (LS) films of CuPaz were prepared by deposition of the variously structured floating layers and studied by X-ray scattering technique and optical spectroscopy. Stable and labile structures were detected and compared with the floating CuPaz aqua aggregates. Conditions of formation of the stable four-stacked nanoaggregates in LS films were determined. A model comprising both nucleation of CuPaz on the water surface and structural transformations in the solid films is proposed.
Sujet(s)
Cuivre/composition chimique , Métalloporphyrines/composition chimique , Nanostructures/composition chimique , SpectrophotométrieRÉSUMÉ
The surface of polyhydroxybutyrate (PHB) storage granules in bacteria is covered mainly by proteins referred to as phasins. The layer of phasins stabilizes the granules and prevents coalescence of separated granules in the cytoplasm and nonspecific binding of other proteins to the hydrophobic surfaces of the granules. Phasin PhaP1(Reu) is the major surface protein of PHB granules in Ralstonia eutropha H16 and occurs along with three homologues (PhaP2, PhaP3, and PhaP4) that have the capacity to bind to PHB granules but are present at minor levels. All four phasins lack a highly conserved domain but share homologous hydrophobic regions. To identify the region of PhaP1(Reu) which is responsible for the binding of the protein to the granules, N-terminal and C-terminal fusions of enhanced green fluorescent protein with PhaP1(Reu) or various regions of PhaP1(Reu) were generated by recombinant techniques. The fusions were localized in the cells of various recombinant strains by fluorescence microscopy, and their presence in different subcellular protein fractions was determined by immunodetection of blotted proteins. The fusions were also analyzed to determine their capacities to bind to isolated PHB granules in vitro. The results of these studies indicated that unlike the phasin of Rhodococcus ruber, there is no discrete binding motif; instead, several regions of PhaP1(Reu) contribute to the binding of this protein to the surface of the granules. The conclusions are supported by the results of a small-angle X-ray scattering analysis of purified PhaP1(Reu), which revealed that PhaP1(Reu) is a planar, triangular protein that occurs as trimer. This study provides new insights into the structure of the PHB granule surface, and the results should also have an impact on potential biotechnological applications of phasin fusion proteins and PHB granules in nanobiotechnology.
Sujet(s)
Protéines bactériennes/métabolisme , Cupriavidus necator/composition chimique , Hydroxy-butyrates/métabolisme , Lectines végétales/métabolisme , Polyesters/métabolisme , Séquence d'acides aminés , Fusion artificielle de gènes , Protéines bactériennes/composition chimique , Sites de fixation , Technique de Western , Fractionnement cellulaire , Cytoplasme/composition chimique , Gènes rapporteurs , Protéines à fluorescence verte/génétique , Protéines à fluorescence verte/métabolisme , Microscopie de fluorescence , Données de séquences moléculaires , Masse moléculaire , Lectines végétales/composition chimique , Liaison aux protéines , Structure secondaire des protéines , Protéines de fusion recombinantes/métabolisme , Diffusion aux petits anglesRÉSUMÉ
Recently our group demonstrated the myogenic capacity of human CD133(+) cells isolated from peripheral blood when delivered in vivo through the arterial circulation into the muscle of dystrophic scid/mdx mice. CD133(+) stem cells express the adhesion molecules CD44, LFA-1, PSGL-1, alpha4-integrins, L-selectin, and chemokine receptor CCR7. Moreover these cells adhere in vitro to VCAM-1 spontaneously and after stimulation with CCL19. Importantly, after muscle exercise, we found that the expression of VCAM-1 is strongly up-regulated in dystrophic muscle vessels, whereas the number of rolling and firmly adhered CD133(+) stem cells significantly increased. Moreover, human dystrophin expression was significantly increased when muscle exercise was performed 24 hours before the intra-arterial injection of human CD133(+) cells. Finally, treatment of exercised dystrophic mice with anti-VCAM-1 antibodies led to a dramatic blockade of CD133(+) stem cell migration into the dystrophic muscle. Our results show for the first time that the expression of VCAM-1 on dystrophic muscle vessels induced by exercise controls muscle homing of human CD133(+) stem cells, opening new perspectives for a potential therapy of muscular dystrophy based on the intra-arterial delivery of CD133(+) stem cells.
Sujet(s)
Muscles squelettiques/vascularisation , Muscles squelettiques/métabolisme , Dystrophie musculaire de l'animal/métabolisme , Dystrophie musculaire de l'animal/thérapie , Molécule-1 d'adhérence des cellules vasculaires/métabolisme , Antigène AC133 , Animaux , Antigènes CD/métabolisme , Adhérence cellulaire , Dystrophine/métabolisme , Glycoprotéines/métabolisme , Humains , Techniques in vitro , Injections artérielles , Souris , Souris de lignée mdx , Souris SCID , Peptides/métabolisme , Transplantation de cellules souches de sang périphérique , Récepteurs aux chimiokines/métabolisme , Transplantation hétérologueRÉSUMÉ
The chaperone-like activity and the oligomeric state of alphaB-crystallin were studied at different temperatures and in the presence of urea and thiocyanate. The activity, assessed measuring the ability of alphaB-crystallin to prevent the aggregation of denatured insulin, strongly depends on temperature. While a significant activity increase was detected at 42 degrees C, the presence of urea and thiocyanate does not affect the protein activity in an irreversible way. In-solution SAXS measurements performed in the same experimental conditions showed that alphaB-crystallin forms near-spherical, hollowed, polydisperse oligomers, whose dimensions change above 42 degrees C. Moreover, in the presence of urea and thiocyanate, a global fit analysis confirms the high stability of alphaB-crystallin assemblies in relationship with their variable quaternary structure. In particular, the changes in the inner radius as well as the thickness and dispersion of the protein shell, account for the preservation of the chaperone-like activity.