RESUMEN
Legionella risk assessment is nowadays based on the presence and concentration of either Legionella pneumophila or Legionella spp. Many species of Legionella can cause Legionnaires' disease, indeed about half of the known species have been associated with infection. The aim of this work was to develop a method to assess the composition of the Legionella species community in an environmental sample in order to have a better understanding of the contamination of the ecosystem by pathogenic strains. The method is based on the comparison of PCR-DGGE profile of DNA sample with a database consisting in DGGE profiles of Legionella species. Such a database includes all pathogenic Legionella strains. In order to homogenize and normalize the different DGGE fingerprint, a reference marker has been built and added during DGGE gel analysis. This study gives a valuable advance in the methods available for the understanding of Legionella contamination of water environments.
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Graphene-derived materials, such as graphene oxide (GO), have been widely explored for biomedical and biological applications, including cancer research. Despite some recent works proving that GO inhibits the migration and invasion of different cancer cells, so far most of these in vitro studies have been conducted using GO sheets dispersed in solution or as a planar film. On the contrary, little is known about cellular activities, such as cell viability, adhesion, and spreading, when cancer cells interface with GO functionalized hydrogel-based surfaces, biomechanically and structurally more similar to the tumor environment. Here, we evaluate the interactions of human breast cancer cells (MDA-MB-231) with alginate (Alg)/GO hydrogel-based substrates, and compare them with a cancer cell line from human osteosarcoma (HOS) and healthy murine fibroblasts (3T3). We observed that GO addition selectively inhibits malignant breast cancer cell adhesion efficiency and spreading area, while promotes HOS and 3T3 adhesive processes. Furthermore, we did not observe the same results over Alg substrates with GO nanosheets dispersed in the medium, without embedment into the Alg. This suggests that cancer (MDA-MB-231 and HOS) and healthy (3T3) cell adhesion efficacy does not depend on the cellular tumoral nature and it is driven by the topographical cues provided by the GO-based substrates, whose physical-mechanical characteristics better mimic those of the cell native tissue. We envision that this study can provide a rational for future design and use of graphene-based nanomaterials for cancer research by deepening the knowledge of graphene-cancer cell specific interactions.
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Neoplasias de la Mama/metabolismo , Grafito , Nanoestructuras/química , Animales , Neoplasias de la Mama/patología , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Grafito/química , Grafito/farmacocinética , Grafito/farmacología , Humanos , Ratones , Células 3T3 NIHRESUMEN
Articular chondrocytes derived from osteoarthritic tissues (OA HAC) show a severely reduced chondrogenic commitment. This impairment undermines their use for tissue-engineered cartilage repair, which relies on cell proliferation and growth to meet therapeutic needs, but also on efficient cell plasticity to recover the chondrogenic phenotype. Reversine (Rev), a 2,6-disubstituted purine inhibitor of spindle-assembly checkpoints, was described to convert differentiated mesenchymal cells to their undifferentiated precursors. We hypothesized that Rev exposure could divert OA HAC to more plastic cells, re-boosting their subsequent commitment. HAC were enzymatically released from OA cartilage specimens, expanded for 2 weeks and treated with 5 µm Rev in dimethylsulphoxide (DMSO) or with DMSO alone for 6 days. Cell growth was assessed using the AlamarBlueTM assay. Cytoskeletal structure, endoproliferation and caspase-3-immunopositivity were assayed by epifluorescence microscopy. The OA HAC chondrogenic performance was evaluated by quantitative reverse transcription-polymerase chain reaction (RT-PCR) for glyceraldehyde-3-phosphate dehydrogenase, Sox9, Aggrecan (Agg), type II collagen (Col2), Ki67, cyclinD1, transforming growth factor-ß1 (TGF-ß1), -2 and -3, interleukin-1ß (IL-1ß) and -6 , SMAD3 and -7, and vascular endothelial growth factor. Rev-treated OA HAC recovered polygonal morphology and reduced Ki67 expression and proliferation. Cell-cycle impairment accounted for altered cytoskeletal organization, endoproliferation and apoptosis, whereas a compensatory mechanism sustained the increased cyclinD1 transcript levels. Sox9, Agg and TGFs were overexpressed, but not Col2. IL transcripts were massively downregulated. These events were dose-related and transient. Overall, in spite of a higher Rev-induced transcriptional activity for extracellular matrix components and in spite of a Rev-treated cell phenotype closer to that of the three-dimensional native articular chondrocyte, Rev effects seem unleashed from a full regained chondrogenic potential.
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Forma de la Célula/efectos de los fármacos , Condrocitos/citología , Morfolinas/farmacología , Purinas/farmacología , Anciano , Anciano de 80 o más Años , Cartílago Articular , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Dimetilsulfóxido/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Masculino , Osteoartritis/patología , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Proteína smad3/genética , Proteína smad3/metabolismoRESUMEN
Chronic lymphocytic leukemia (CLL), the most common leukemia among adults in the western world, is characterized by a progressive accumulation of relatively mature CD5+ B cells in peripheral blood, lymph nodes and bone marrow. Despite much recent advancement in therapy, CLL is still incurable. Lymph nodes and bone marrow represent sanctuary sites preserving leukemic cells from spontaneous or drug-induced apoptosis, and infiltration of leukemic cells in these districts correlates with clinical stages and prognosis. The central role played by the microenvironment in the disease has become increasingly clear. Different chemokines (CXCL12, CXCL13, CCL19, CCL21) may in fact participate in attracting CLL cells into bone marrow and lymph nodes, where various factors, such as IL-15 and CXCL12, enhance leukemic cells survival. Recently, we have suggested that hepatocyte growth factor (HGF), produced by microenvironmental stromal cells, can contribute to CLL pathogenesis. We have demonstrated that HGF exerts a double effect on CLL B cells through the interaction with its receptor c- MET; HGF, infact, protects CLL B cells, which are c-MET+, from apoptosis, and also polarizes mono/macrophages towards the M2 phenotype, thus facilitating the evasion of the CLL clone from immune control. This double effect appears mediated by the activation of two major signaling pathways: STAT3TYR705 and AKT. The aim of this review is to summarize data on HGF and c-MET expression in normal B cells and in B cell malignancies, with a particular emphasis on our results obtained in CLL. Altogether, the observations described here suggest that the HGF/c-MET axis may have a prominent role in malignancy progression further indicating novel potential therapeutic options aimed to block HGF-induced signaling pathways in B lymphoproliferative disorders.
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Factor de Crecimiento de Hepatocito/metabolismo , Leucemia Linfocítica Crónica de Células B/etiología , Leucemia Linfocítica Crónica de Células B/metabolismo , Escape del Tumor , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Factor de Crecimiento de Hepatocito/genética , Humanos , Inmunomodulación , Leucemia Linfocítica Crónica de Células B/mortalidad , Leucemia Linfocítica Crónica de Células B/patología , Terapia Molecular Dirigida , Pronóstico , Unión Proteica , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal/efectos de los fármacos , Escape del Tumor/genética , Escape del Tumor/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
The body-machine interfaces (BMIs) map the subjects' movements into the low dimensional control space of external devices to reach assistive and/or rehabilitative goals. This work is a first proof of concept of this kind of BMI as tool for rehabilitation after stroke. We designed an exercise to improve the control of selective movements of the pelvis in stroke survivors, increasing the ability to decouple the motion in the sagittal and frontal planes and decreasing compensatory adjustments at the shoulder girdle. A Kinect sensor recorded the movements of the subjects. Subjects played different games by controlling the vertical and horizontal motion of a cursor on a screen with respectively the lateral tilt and the ante/retroversion of their pelvis. We monitored also the degrees of freedom not directly involved in cursor control, thus subjects could complete the task only with a correct posture. Our preliminary results highlight significant improvement not only in cursor control, but also in the Trunk Impairment Scale (TIS) and in the Five Times Sit to Stand Test (5xSST).
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Pelvis/fisiología , Rehabilitación de Accidente Cerebrovascular , Anciano , Ejercicio Físico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Movimiento/fisiología , Proyectos PilotoRESUMEN
Despite distal arm impairment after brain injury is an extremely disabling consequence of neurological damage, most studies on robotic therapy are mainly focused on recovery of proximal upper limb motor functions, routing the major efforts in rehabilitation to shoulder and elbow joints. In the present study we developed a novel therapeutic protocol aimed at restoring wrist functionality in chronic stroke patients. A haptic three DoFs (degrees of freedom) robot has been used to quantify motor impairment and assist wrist and forearm articular movements: flexion/extension (FE), abduction/adduction (AA), pronation/supination (PS). This preliminary study involved nine stroke patients, from a mild to severe level of impairment. Therapy consisted in ten 1-hour sessions over a period of five weeks. The novelty of the approach was the adaptive control scheme which trained wrist movements with slow oscillatory patterns of small amplitude and progressively increasing bias, in order to maximize the recovery of the active range of motion. The primary outcome was a change in the active RoM (range of motion) for each DoF and a change of motor function, as measured by the Fugl-Meyer assessment of arm physical performance after stroke (FMA). The secondary outcome was the score on the Wolf Motor Function Test (WOLF). The FMA score reported a significant improvement (average of 9.33±1.89 points), revealing a reduction of the upper extremity motor impairment over the sessions; moreover, a detailed component analysis of the score hinted at some degree of motor recovery transfer from the distal, trained parts of the arm to the proximal untrained parts. WOLF showed an improvement of 8.31±2.77 points, highlighting an increase in functional capability for the whole arm. The active RoM displayed a remarkable improvement. Moreover, a three-months follow up assessment reported long lasting benefits in both distal and proximal arm functionalities. The experimental results of th- s preliminary clinical study provide enough empirical evidence for introducing the novel progressive, adaptive, gentle robotic assistance of wrist movements in the clinical practice, consolidating the evaluation of its efficacy by means of a controlled clinical trial.
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Robótica/métodos , Rehabilitación de Accidente Cerebrovascular , Muñeca/fisiología , Adulto , Anciano , Algoritmos , Enfermedad Crónica , Diseño de Equipo , Femenino , Lateralidad Funcional/fisiología , Humanos , Masculino , Persona de Mediana Edad , Destreza Motora/fisiología , Movimiento , Proyectos Piloto , Rango del Movimiento Articular , Recuperación de la Función , Sobrevivientes , Resultado del TratamientoRESUMEN
Gait re-education is a primary rehabilitation goal after stroke. In this study, we used instrumented gait analysis for evaluating the outcomes of gait training assisted by an endpoint robot in a population of six chronic stroke survivors. The preliminary results, based on spatial-temporal and kinematic analysis, suggest that (a) self-placed walking speed increases, with an improvement of both length and duration of the stride, (b) balance increases during standing and walking, (c) the non-affected side becomes less involved in attempting to correct for the deficiencies of the affected side, thus reducing the importance of compensatory strategies.
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Terapia por Ejercicio/métodos , Marcha/fisiología , Robótica/métodos , Rehabilitación de Accidente Cerebrovascular , Caminata/fisiología , Adulto , Anciano , Trastornos Neurológicos de la Marcha/rehabilitación , Humanos , Masculino , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
OBJECTIVE: Autologous chondrocyte implantation is a cell-based treatment to repair articular cartilage defects, relying on the availability of expanded (de-differentiated) chondrocytes. Unfortunately, the expansion process causes several phenotypical changes, requiring re-establishment of the native chondrogenic phenotype to sustain proper repair. Among other proteins, transforming growth factor-ß (TGFß) is known to influence the chondrogenic re-differentiation of human articular chondrocytes (HACs) and their matrix deposition. Thus we investigated the effects of TGFß-depletion during the expansion phase. DESIGN: HACs were isolated from articular cartilage and expanded in the canonical serum-supplemented medium [fetal calf serum (FCS)] or in a chemically-defined (CD) medium, with or without anti-TGFß antibody administration. The re-differentiation potential of the cells was assessed by pellet cultures, gene expression analysis and histology. RESULTS: Cell proliferation proceeded more rapidly in CD-medium than in FCS-medium; it was not affected by the use of anti-TGFß antibody but was further increased by addition of exogenous TGFß1, via increased p-Smad1/5/8. Conversely, in FCS-medium, addition of anti-TGFß antibody decreased both proliferation and p-Smad1/5/8 level. Challenging either FCS- or CD-medium with anti-TGFß antibody during expansion enhanced chondrogenesis in the subsequent pellet cultures. Moreover, TGFß-depletion during expansion in CD-medium inhibited mRNA expression of hypertrophic markers, collagen type-X (COL10) and matrix metalloproteinase-13 (MMP-13). Interestingly, the TGFß1 level detected by enzyme-linked immunosorbent sandwich assay (ELISA) during cell expansion was correlated with COL10 mRNA expression after re-differentiation. CONCLUSION: TGFß-depletion during expansion improves the re-differentiation capacity of chondrocytes and inhibits hypertrophy. These results indicate the importance of the expansion medium composition to improve chondrogenic re-differentiation and to inhibit hypertrophy.
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Anticuerpos Bloqueadores/farmacología , Cartílago Articular/citología , Condrocitos/citología , Medios de Cultivo/química , Factor de Crecimiento Transformador beta/metabolismo , Benzamidas/farmacología , Biomarcadores/metabolismo , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Aumento de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Medio de Cultivo Libre de Suero , Humanos , Hipertrofia/inducido químicamente , Traumatismos de la Rodilla/metabolismo , Traumatismos de la Rodilla/patología , Traumatismos de la Rodilla/cirugía , Osteoartritis de la Rodilla/metabolismo , Osteoartritis de la Rodilla/patología , Osteoartritis de la Rodilla/cirugía , Pirazoles/farmacología , Pirimidinas/farmacología , Proteínas Smad/antagonistas & inhibidores , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
Cell-based cartilage resurfacing requires ex vivo expansion of autologous articular chondrocytes. Defined culture conditions minimize expansion-dependent phenotypic alterations but maintenance of the cells' differentiation potential must be carefully assessed. Transforming growth factor ß-1 (TGF ß-1) positively regulates the expression of several cartilage proteins, but its therapeutic application in damaged cartilage is controversial. Thus we evaluated the phenotypic outcomes of cultured human articular chondrocytes exposed to TGF ß-1 during monolayer expansion in a serum-free medium. After five doublings cells were transferred to micromass cultures to assess their chondrogenic differentiation, or replated in osteogenic medium. Immunocytostainings of micromasses of TGF-expanded cells showed loss of aggrecan and type II collagen. Positivity was evidenced for RAGE, IHH, type X collagen and for apoptotic cells, paralleling a reduction of BCL-2 levels, suggesting hypertrophic differentiation. TGF ß-1-exposed cells also evidenced increased mRNA levels for bone sialoprotein, osteopontin, matrix metalloproteinase-13, TIMP-3, VEGF and SMAD7, enhanced alkaline phosphatase activity and pyrophosphate availability. Conversely, SMAD3 mRNA and protein contents were reduced. After osteogenic induction, only TGF-expanded cells strongly mineralized and impaired p38 kinase activity, a contributor of chondrocytes' differentiation. To evaluate possible endochondral ossification progression, we seeded the chondrocytes on hydroxyapatite scaffolds, subsequently implanted in an in vivo ectopic setting, but cells failed to reach overt ossification; nonetheless, constructs seeded with TGF-exposed cells displayed blood vessels of the host vascular supply with enlarged diameters, suggestive of vascular remodeling, as in bone growth. Thus TGF-exposure during articular chondrocytes expansion induces a phenotype switch to hypertrophy, an undesirable effect for cells possibly intended for tissue-engineered cartilage repair.
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Cartílago Articular/crecimiento & desarrollo , Diferenciación Celular/efectos de los fármacos , Condrocitos/metabolismo , Hipertrofia/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Agrecanos/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Cartílago Articular/citología , Condrocitos/citología , Condrogénesis/efectos de los fármacos , Colágeno Tipo II/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Osteogénesis/efectos de los fármacos , Proteínas Smad/metabolismo , Ingeniería de Tejidos/métodos , Factor de Crecimiento Transformador beta1/administración & dosificaciónRESUMEN
The in vitro corrosion behavior and biocompatibility of two Zr alloys, Zr-2.5Nb, employed for the manufacture of CANDU reactor pressure tubes, and Zr-1.5Nb-1Ta (at%), for use as implant materials have been assessed and compared with those of Grade 2 Ti, which is known to be a highly compatible metallic biomaterial. The in vitro corrosion resistance was investigated by open circuit potential and electrochemical impedance spectroscopy (EIS) measurements, as a function of exposure time to an artificial physiological environment (Ringer's solution). Open circuit potential values indicated that both the Zr alloys and Grade 2 Ti undergo spontaneous passivation due to spontaneously formed oxide film passivating the metallic surface, in the aggressive environment. It also indicated that the tendency for the formation of a spontaneous oxide is greater for the Zr-1.5Nb-1Ta alloy and that this oxide has better corrosion protection characteristics than the ones formed on Grade 2 Ti or on the Zr-2.5Nb alloy. EIS study showed high impedance values for all samples, increasing with exposure time, indicating an improvement in corrosion resistance of the spontaneous oxide film. The fit obtained suggests a single passive film presents on the metals surface, improving their resistance with exposure time, presenting the highest values to the Zr-1.5Nb-1Ta alloy. For the biocompatibility analysis human osteosarcoma cell line (Saos-2) and human primary bone marrow stromal cells (BMSC) were used. Biocompatibility tests showed that Saos-2 cells grow rapidly, independently of the surface, due to reduced dependency from matrix deposition and microenvironment recognition. BMSC instead display a reduced proliferation, possibly caused by a reduced crosstalk with the metal surface microenvironment. However, once the substrate has been colonized, BMSC seem to respond properly to osteoinduction stimuli, thus supporting a substantial equivalence in the biocompatibility among the Zr alloys and Grade 2 titanium. In summary, high in vitro corrosion resistance together with satisfactory biocompatibility make the Zr-2.5Nb and Zr-1.5Nb-1Ta crystalline alloys promising biomaterials for surgical implants.
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Niobio/química , Tantalio/química , Circonio/química , Aleaciones/química , Materiales Biocompatibles , Células de la Médula Ósea/citología , Línea Celular Tumoral , Proliferación Celular , Técnicas Electroquímicas , Humanos , Osteosarcoma , Células del EstromaRESUMEN
A stem cell is defined as a cell able to self-renew and at the same time to generate one or more specialized progenies. In the adult organism, stem cells need a specific microenvironment where to reside. This tissue-specific instructive microenvironment, hosting stem cells and governing their fate, is composed of extracellular matrix and soluble molecules. Cell-matrix and cell-cell interactions also contribute to the specifications of this milieu, regarded as a whole unitary system and referred to as "niche". For many stem cell systems a niche has been identified, but only partially defined. In regenerative medicine and tissue engineering, biomaterials are used to deliver stem cells in specific anatomical sites where a regenerative process is needed. In this context, biomaterials have to provide informative microenvironments mimicking a physiological niche. Stem cells may read and decode any biomaterial and modify their behavior and fate accordingly. Any material is therefore informative in the sense that its intrinsic nature and structure will anyway transmit a signal that will have to be decoded by colonizing cells. We still know very little of how to create local microenvironments, or artificial niches, that will govern stem cells behavior and their terminal fate. Here we will review some characteristics identifying specific niches and some of the requirements allowing stem cells differentiation processes. We will discuss on those biomaterials that are being projected/engineered/manufactured to gain the informative status necessary to drive proper molecular cross-talk and cell differentiation; specific examples will be proposed for bone and cartilage substitutes.
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Materiales Biocompatibles , Diferenciación Celular , Regeneración , Nicho de Células Madre , Células Madre/fisiología , Ingeniería de Tejidos , Andamios del Tejido , Cartílago/fisiología , Comunicación Celular , Humanos , Células Madre/citología , Andamios del Tejido/químicaRESUMEN
We report on a pilot study of robot therapy with stroke patients. Patients were requested to track a continuously moving target according to a figure-of-eight. Assistance was provided by an attractive force field, whose magnitude was regulated according to a principle of minimal assistance and a principle of consolidation of the learned memory trace. From the analysis of the assistive forces, we show that subjects improve their degree of voluntary control.
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Trastornos del Movimiento/rehabilitación , Robótica/métodos , Rehabilitación de Accidente Cerebrovascular , Análisis y Desempeño de Tareas , Terapia Asistida por Computador/métodos , Humanos , Trastornos del Movimiento/diagnóstico , Trastornos del Movimiento/etiología , Desempeño Psicomotor , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/diagnóstico , Resultado del Tratamiento , VoliciónRESUMEN
Bone repair was addressed in a critical-sized defect model in sheep, combining a ceramic biomaterial and mesenchymal progenitor cells. The defects in the tibial mid-diaphysis were treated with autologous bone or with a silicon-stabilized tricalcium phosphate biomaterial, implemented or not by the addition of expanded bone marrow stromal cells. An internal locking compression plate and an external fixator were applied for stabilization. Radiographies were taken during the 8 months follow-up: the pixel grey levels of the lesion areas were determined to evaluate the repair process radiologically. Microradiography, histology and vascular density tests were performed. The autologous bone-treated group performed best, as assessed radiologically, within 20-24 weeks after surgery. Very limited healing was detected in the other experimental group: a partial bone deposition occurred at the periphery of the bony stumps only in the cell-seeded scaffolds. Interestingly, this effect ended within 20-24 weeks, as for the autologous bone, suggesting similar kinetics of the repair processes involved. Moreover, bone deposition was located where a significant reduction of the ceramic scaffold was detected. Faxitron microradiography and histology data confirmed these results. Vascular density analysis evidenced that cell-seeded scaffolds supported an increased vascular ingrowth. Thus, the interactions with the proper microenvironment and the oxygen and nutrient supply in the inner part of the constructs seem fundamental to initiate scaffold substitution and to improve cell performance in tissue-engineered approaches to bone repair.
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Células de la Médula Ósea/citología , Regeneración Ósea , Células del Estroma/citología , Animales , Femenino , OvinosRESUMEN
Resorbable porous ceramic constructs, based on silicon-stabilized tricalcium phosphate, were implanted in critical-size defects of sheep tibias, either alone or after seeding with bone marrow stromal cells (BMSC). Only BMSC-loaded ceramics displayed a progressive scaffold resorption, coincident with new bone deposition. To investigate the coupled mechanisms of bone formation and scaffold resorption, X-ray computed microtomography (muCT) with synchrotron radiation was performed on BMSC-seeded ceramic cubes. These were analyzed before and after implantation in immunodeficient mice for 2 or 6 months. With increasing implantation time, scaffold thickness significantly decreased while bone thickness increased. The muCT data evidenced that all scaffolds showed a uniform density distribution before implantation. Areas of different segregated densities were instead observed, in the same scaffolds, once seeded with cells and implanted in vivo. A detailed muX-ray diffraction analysis revealed that only in the contact areas between deposited bone and scaffold, the TCP component of the biomaterial decreased much faster than the HA component. This event did not occur at areas away from the bone surface, highlighting coupling and cell-dependency of the resorption and matrix deposition mechanisms. Moreover, in scaffolds implanted without cells, both the ceramic density and the TCP:HA ratio remained unchanged with respect to the pre-implantation analysis.
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Materiales Biocompatibles , Células de la Médula Ósea/citología , Sustitutos de Huesos , Animales , Fosfatos de Calcio , Cerámica , Estabilidad de Medicamentos , Femenino , Ensayo de Materiales , Modelos Animales , Oseointegración , Osteogénesis , Prótesis e Implantes , Ovinos , Silicio , Células del Estroma/citología , Factores de Tiempo , Ingeniería de Tejidos , Tomografía Computarizada por Rayos X , Difracción de Rayos XRESUMEN
The mechanism of mineralized matrix deposition was studied in a tissue engineering approach in which bone tissue is formed when porous ceramic constructs are loaded with bone marrow stromal cells and implanted in vivo. We investigated the local interaction between the mineral crystals of the engineered bone and the biomaterial by means of microdiffraction, using a set-up based on an x-ray waveguide. We demonstrated that the newly formed bone is well organized inside the scaffold pore, following the growth model of natural bone. Combining wide angle (WAXS) and small angle (SAXS) x-ray scattering with high spatial resolution, we were able to determine the orientation of the crystallographic c-axis inside the bone crystals, and the orientation of the mineral crystals and collagen micro-fibrils with respect to the scaffold. In this work we analysed six samples and for each of them two pores were studied in detail. Similar results were obtained in all cases but we report here only the most significant sample.
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Células de la Médula Ósea/citología , Células del Estroma/citología , Ingeniería de Tejidos/métodos , Anisotropía , Materiales Biocompatibles , Densidad Ósea , Células de la Médula Ósea/metabolismo , Huesos/metabolismo , Cerámica , Colágeno/química , Humanos , Procesamiento de Imagen Asistido por Computador , Imagenología Tridimensional , Dispersión de Radiación , Sincrotrones , Cloruro de Tolonio/farmacología , Difracción de Rayos X , Rayos XRESUMEN
OBJECTIVE: Proliferation and chondrogenic commitment of cultured articular chondrocytes are impaired when cells derive from aged donors. In those subjects the feasibility of cell-based therapies for articular surface repair is reduced. Moreover, the use of serum as medium supplement elicits non-physiological responses in cultured chondrocytes. This study was therefore undertaken to identify the expansion culture conditions needed to sustain growth and chondrogenic commitment of chondrocytes harvested from aged human subjects. DESIGN: Articular cartilage was obtained from aged (69-75 years) and from young adult subjects (27-35 years). Chondrocytes were isolated and cultured in serum-free (SF) or in serum-supplemented [fetal calf serum (FCS)] conditions. Chondrocytes were expanded in monolayer for five duplications and processed for RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The differentiation potential was assessed by micromass pellet cultures before and after expansion in either culture medium, or after a prolonged exposure to serum followed by a period in SF condition. RESULTS: Only SF-cultured chondrocytes reached five duplications within 25-35 days, maintaining the expression of some chondrogenic markers and without altering the levels of active matrix metalloproteinase 3 (MMP-3). Only the pellets derived from SF-expanded cultures positively stained for cartilage matrix deposition. On the contrary, exposure to serum diminished the proliferation capacities, abolished the differentiation potential in the same cells and elicited transcription of the MMP-3 gene. Shifting culture conditions from FCS to SF resumed growth rates but proper extracellular matrix deposition was only partially restored. CONCLUSIONS: The SF conditions have proven valuable to prime cell proliferation and to sustain proper commitment in chondrocytes from aged patients. This culturing approach may represent a therapeutic chance extendable to a range of patients normally excluded from clinical protocols based on autologous chondrocyte implantation (ACI).
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Técnicas de Cultivo de Célula/métodos , Condrocitos/trasplante , Adulto , Anciano , Cartílago Articular/citología , División Celular/fisiología , Senescencia Celular , Condrocitos/citología , Medio de Cultivo Libre de Suero , Matriz Extracelular/metabolismo , Femenino , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Ingeniería de Tejidos/métodosRESUMEN
Due to the postulated role of mitochondrial DNA damage in aging as well as in the pathogenesis of several chronic degenerative diseases, including tumors, there is a need for easy, safe, rapid and inexpensive methods for mitochondrial DNA isolation. We propose a simple, 1-day protocol based on alkaline extraction, which avoids time-consuming gradient centrifugations and use of toxic phenol. This procedure was used for the recovery of mitochondrial DNA from different rat organs (liver, kidney, heart, lung, brain and testis). The yield was quite high. Purity was sufficient enough to perform restriction analyses with several endonucleases. No changes were observed in the structure or methylation patterns of -CCGG- sites in liver mitochondrial DNA isolated from rats exposed whole-body to mainstream cigarette smoke or treated with the carcinogens benzo[alpha]pyrene, 2-acetylaminofluorene or diethylnitrosamine.
Asunto(s)
Carcinógenos/farmacología , Citosina/análogos & derivados , ADN Mitocondrial/metabolismo , Mitocondrias/química , 5-Metilcitosina , Animales , Fraccionamiento Celular/métodos , Citosina/metabolismo , Daño del ADN , ADN Mitocondrial/aislamiento & purificación , Concentración de Iones de Hidrógeno , Masculino , Métodos , Metilación , Ratas , Ratas Sprague-Dawley , Mapeo RestrictivoRESUMEN
DNA topoisomerase I activity (topo I) is known to be inhibited by poly(ADP-ribosyl)ation. Both poly(ADP-ribose)polymerase (pADPRP) and DNA topoisomerase I participate to major biological events, such as DNA transcription, repair and synthesis. It has been shown that thyroid hormones, such as 3,5,3'-triiodothyronine (T3), stimulate DNA transcription and down-regulate pADPRP activity. Using an in vitro model, we have studied the poly(ADP-ribosyl)ation of topo I, in vitro, in the presence of T3. T3 treatment of pADPRP inhibits the enzyme up to 75-80% of control activity. DNA topoisomerase I relaxing activity was determined on supercoiled plasmid DNA, and topoisomers were separated by agarose gel electrophoresis. Poly(ADP-ribosyl)ation completely inhibits the relaxing activity of topo I, with respect to non-ribosylated controls, but the activity remains unaffected when pADPRP is inactivated by heat or treated with specific inhibitors, such as 3-aminobenzamide (3ABA). In this study we show that treatment of pADPRP with T3 reduces the inhibition on topo I. In this system 10(-8) M T3 was effective in maintaining almost all topo I activity, even though modifications in processivity and distributivity of the reaction were noted. These data support a close relationship between pADPRP and topo I in hormone-stimulated DNA transcription.
Asunto(s)
ADN-Topoisomerasas de Tipo I/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/efectos de los fármacos , Triyodotironina/farmacología , ADN-Topoisomerasas de Tipo I/química , Poli(ADP-Ribosa) Polimerasas/químicaRESUMEN
The in vivo effect of the thyroidal state on poly(ADP-ribose) polymerase activity was investigated in eu- and hypothyroid rats after treatment with L-triiodothyronine. Untreated hypothyroids showed an increased basal rate of the enzyme. The treatment of both eu- and hypothyroid rats with L-triiodothyronine induced a prompt drop of the endogenous activity not due to a reduction of the catalytic protein. This decrease well evident 1 h after treatment was transient, returning to controls values within 8 h. In isolated liver nuclei from euthyroids the in vitro exposure to increasing L-triiodothyronine concentrations from 10(-18) to 10(-6) M resulted in a progressive inhibition of the enzyme. This loss in activity was not derived from a reduction of the total level of the catalytic protein. The pretreatment with the antagonist amiodarone suppressed the hormone effect, suggesting that nuclear receptors could mediate poly(ADP-ribose) polymerase activity.
Asunto(s)
Hipotiroidismo/enzimología , Hígado/enzimología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Glándula Tiroides/fisiología , Triyodotironina/farmacología , Amiodarona/farmacología , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Homeostasis , Cinética , Hígado/efectos de los fármacos , Masculino , Ratas , Ratas Wistar , Receptores de Hormona Tiroidea/fisiología , Valores de Referencia , Tiroidectomía , Factores de TiempoRESUMEN
To examine differences in perinatal health among nine Asian ethnic subgroups, a descriptive epidemiological study was conducted using linked birth/infant death certificates for 1982 to 1987. When compared with Whites, Asians had a lower proportion of young mothers, unmarried mothers, and women who received first trimester prenatal care; a higher proportion of foreign-born mothers; and a different birthweight distribution. A great deal of heterogeneity was found in risk factors and infant mortality rates among the various Asian ethnic subgroups. Paradoxically, although Asian ethnic subgroups had a higher perinatal risk profile, they had more favorable birth outcomes than did Whites.