RESUMEN
Fully aliphatic segmented poly(ether ester amide) copolymers with uniform hard segments prepared by melt polycondensation of α,ω-hydroxyl end-functionalized polytetrahydrofuran and short glycine or ß-alanine bisester-bisoxalamide units hold promise for biomedical applications. For polymers with the hard block contents varying from 10% to 27%, differential scanning calorimetry and atomic force microscopy reveal a highly phase-separated morphology, with ribbon-like nanocrystals dispersed in the soft segment matrix. To relate the polymer properties to the structure of the hard segment, the monomers were prepared and studied by optical and X-ray diffraction measurements. It was shown that the glycine and ß-alanine carbonyl ester groups are tilted away from the oxalamide plane, which can affect the degradation rate via hydrolysis of the ester bond.
Asunto(s)
Materiales Biocompatibles/química , Carbono/química , Nanopartículas/química , Nanopartículas/ultraestructura , Compuestos Orgánicos/química , Polímeros/química , Esterificación , Dureza , Enlace de Hidrógeno , Hidrólisis , Ensayo de MaterialesRESUMEN
Poly(trimethylene carbonate) (PTMC) macromers with molecular weights (M(n)) between 1000 and 41,000 g mol(-1) were prepared by ring opening polymerization and subsequent functionalization with methacrylate end groups. Flexible networks were obtained by radical photo-crosslinking reactions of these macromers. With increasing molecular weight of the macromer the networks obtained showed increasing swelling ratios in chloroform and decreasing glass transition temperatures, reaching a constant value of approximately -18°C, which is close to that of linear high molecular weight PTMC. For all prepared networks the creep resistance was high. However, the molecular weight of the macromer strongly influenced the tensile properties of the networks. With increasing molecular weight of the macromer the E-modulus of the networks decreased from 314 MPa (lowest M(n)) to 5 MPa (highest M(n)), while their elongation at break continuously increased, reaching a very high value of 1200%. The maximum tensile strength values of the networks were found to first decrease with increasing M(n), but to increase again at values above approximately 10,000gmol(-1), at which the networks started to show rubber-like behavior. The toughness (area under the stress-strain curves, W) determined in tensile testing experiments, in tear propagation experiments, and in suture retention strength measurements showed that PTMC networks prepared from the higher molecular weight macromers (M(n)>10,000 g mol(-1)) were tenacious materials. The mechanical properties of these networks compare favorably with those of linear high molecular weight PTMC and well-known elastomeric materials like silicone rubber (poly(dimethylsiloxane)) and natural latex rubber. Additionally they also compare well with those of native blood vessels, which may be of importance in the use of these materials for the tissue engineering of small diameter blood vessels.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Dioxanos/química , Elasticidad , Luz , Ensayo de Materiales/métodos , Polímeros/química , Animales , Vasos Sanguíneos/fisiología , Dioxanos/síntesis química , Vidrio/química , Espectroscopía de Resonancia Magnética , Metacrilatos/química , Peso Molecular , Docilidad , Polímeros/síntesis química , Ovinos , Sus scrofa , Resistencia a la Tracción/efectos de la radiación , Temperatura de Transición/efectos de la radiaciónRESUMEN
Cell-based cartilage repair strategies such as matrix-induced autologous chondrocyte implantation (MACI) could be improved by enhancing cell performance. We hypothesised that micro-aggregates of chondrocytes generated in high-throughput prior to implantation in a defect could stimulate cartilaginous matrix deposition and remodelling. To address this issue, we designed a micro-mould to enable controlled high-throughput formation of micro-aggregates. Morphology, stability, gene expression profiles and chondrogenic potential of micro-aggregates of human and bovine chondrocytes were evaluated and compared to single-cells cultured in micro-wells and in 3D after encapsulation in Dextran-Tyramine (Dex-TA) hydrogels in vitro and in vivo. We successfully formed micro-aggregates of human and bovine chondrocytes with highly controlled size, stability and viability within 24 hours. Micro-aggregates of 100 cells presented a superior balance in Collagen type I and Collagen type II gene expression over single cells and micro-aggregates of 50 and 200 cells. Matrix metalloproteinases 1, 9 and 13 mRNA levels were decreased in micro-aggregates compared to single-cells. Histological and biochemical analysis demonstrated enhanced matrix deposition in constructs seeded with micro-aggregates cultured in vitro and in vivo, compared to single-cell seeded constructs. Whole genome microarray analysis and single gene expression profiles using human chondrocytes confirmed increased expression of cartilage-related genes when chondrocytes were cultured in micro-aggregates. In conclusion, we succeeded in controlled high-throughput formation of micro-aggregates of chondrocytes. Compared to single cell-seeded constructs, seeding of constructs with micro-aggregates greatly improved neo-cartilage formation. Therefore, micro-aggregation prior to chondrocyte implantation in current MACI procedures, may effectively accelerate hyaline cartilage formation.
Asunto(s)
Cartílago/crecimiento & desarrollo , Agregación Celular , Condrocitos/citología , Regulación de la Expresión Génica , Análisis de la Célula Individual , Agrecanos/metabolismo , Animales , Cartílago/metabolismo , Bovinos , Trasplante de Células/métodos , Condrocitos/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Desnudos , Análisis por MicromatricesRESUMEN
The mechanical properties of individual collagen fibrils of approximately 200 nm in diameter were determined using a slightly adapted AFM system. Single collagen fibrils immersed in PBS buffer were attached between an AFM cantilever and a glass surface to perform tensile tests at different strain rates and stress relaxation measurements. The stress-strain behavior of collagen fibrils immersed in PBS buffer comprises a toe region up to a stress of 5 MPa, followed by the heel and linear region at higher stresses. Hysteresis and strain-rate dependent stress-strain behavior of collagen fibrils were observed, which suggest that single collagen fibrils have viscoelastic properties. The stress relaxation process of individual collagen fibrils could be best fitted using a two-term Prony series. Furthermore, the influence of different cross-linking agents on the mechanical properties of single collagen fibrils was investigated. Based on these results, we propose that sliding of microfibrils with respect to each other plays a role in the viscoelastic behavior of collagen fibrils in addition to the sliding of collagen molecules with respect to each other. Our finding provides a better insight into the relationship between the structure and mechanical properties of collagen and the micro-mechanical behavior of tissues.
Asunto(s)
Colágeno Tipo I/química , Fenómenos Mecánicos , Microscopía de Fuerza Atómica , Animales , Tampones (Química) , Bovinos , Reactivos de Enlaces Cruzados/farmacología , Elasticidad , Vidrio/química , Modelos Moleculares , Estrés Mecánico , Propiedades de Superficie , Resistencia a la Tracción , ViscosidadRESUMEN
For the treatment of cardiovascular disease, functional arterial blood vessel prostheses with an inner diameter less than 6 mm are needed. This article gives an overview of the preparation of such vascular grafts by means of tissue engineering.
Asunto(s)
Prótesis Vascular , Ingeniería de Tejidos , Implantes Absorbibles , Anastomosis Quirúrgica , Animales , Aterosclerosis/cirugía , Reactores Biológicos , Implantación de Prótesis Vascular , Células Cultivadas , Colágeno , Elastina , Matriz Extracelular , Humanos , Implantes Experimentales , Ensayo de Materiales , Polímeros , Flujo Pulsátil , Células Madre/citología , Ingeniería de Tejidos/métodos , Andamios del TejidoRESUMEN
Vascular endothelial cells form the inner lining of all blood vessels and play a central role in vessel physiology and disease. Endothelial cells are highly responsive to the mechanical stimulus of fluid shear stress that is exerted by blood flowing over their surface. In this study, the immediate micromechanical response of endothelial cells to physiological shear stress was characterized by tracking of ballistically injected, sub-micron, fluorescent particles. It was found that the mean squared displacement (MSD) of the particles decreases by a factor 1.5 within 10 min after the onset of shear stress. This decrease in particle motion is transient, since the MSD returns to control values within 15-30 min after the onset of shear. The immediate micromechanical stiffening is dependent on activation of the vascular endothelial growth factor receptor (VEGFR)-2, because inhibition of the receptor abrogates the micromechanical response. This work shows that the cytoskeleton is actively involved in the acute, functional response of endothelial cells to shear stress.
Asunto(s)
Células Endoteliales/metabolismo , Microesferas , Movimiento (Física) , Estrés Mecánico , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Actinas/metabolismo , Forma de la Célula , Células Cultivadas , Citoesqueleto/metabolismo , Células Endoteliales/citología , Hemorreología , Humanos , Inyecciones , Microfluídica , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
The physiology of vascular endothelial cells is strongly affected by fluid shear stress on their surface. In this study, a microfluidic assay was employed to analyze the alignment of actin filaments in endothelial cells in response to shear stress. When cells were cultured in microfluidic channels and subjected to shear stress, the alignment of filaments in the channel direction was significantly higher than in static cultures. By adding inhibitory drugs, the roles of several signaling proteins in the process of alignment were determined. Thus, it is shown how microfluidic technology can be employed to provide a mechanistic insight into cell physiology.
RESUMEN
Porous tubular poly(trimethylene carbonate) (PTMC) scaffolds for vascular tissue engineering, with an inner diameter of 3 mm and a wall thickness of 1 mm, were prepared by means of dip-coating and subsequent leaching of NaCl particles. The scaffolds, with an average pore size of 110 µm and a porosity of 85%, showed a smooth muscle cell (SMC) seeding efficiency of only 10%. To increase the efficiency of cell seeding, these scaffolds were coated with a microporous PTMC outer layer with a thickness of 0.1-0.4 mm, an average pore size of 28 µm, and a porosity of 65%. Coating of the scaffolds with the microporous outer layer did not influence the inner pore structure or the mechanical properties of the scaffolds to a significant extent. The intrinsic permeability of the scaffolds decreased from 60 × 10(-10) m(2) to approximately 5 × 10(-10) m(2) after coating with the microporous outer layer. The latter value is still relatively high indicating that these scaffolds may facilitate sufficient diffusion of nutrients and waste products during cell culturing. The efficiency of SMC seeding determined after 24 h cell adhesion in the scaffolds increased from less than 10% to 43% after coating with the microporous outer layer. The cells were homogeneously distributed in the scaffolds and cell numbers increased 60% during culturing for 7 days under stationary conditions. It is concluded that coating of porous tubular PTMC scaffolds with a microporous PTMC outer layer facilitates effective cell seeding in these scaffolds.
Asunto(s)
Materiales Biocompatibles/química , Técnicas de Cultivo de Célula , Dioxanos/química , Miocitos del Músculo Liso/metabolismo , Ingeniería de Tejidos , Andamios del Tejido/química , Animales , Vasos Sanguíneos/anatomía & histología , Vasos Sanguíneos/metabolismo , Células Cultivadas , Humanos , Ensayo de Materiales , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Porosidad , Ovinos , Porcinos , Resistencia a la Tracción , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodosRESUMEN
Polysaccharide hybrids consisting of hyaluronic acid (HA) grafted with a dextran-tyramine conjugate (Dex-TA) were synthesized and investigated as injectable biomimetic hydrogels for cartilage tissue engineering. The design of these hybrids (denoted as HA-g-Dex-TA) is based on the molecular structure of proteoglycans present in the extracellular matrix of native cartilage. Hydrogels of HA-g-Dex-TA were rapidly formed within 2 min via enzymatic crosslinking of the tyramine residues in the presence of horseradish peroxidase and hydrogen peroxide. The gelation time, equilibrium swelling and storage modulus could be adjusted by varying the degree of substitution of tyramine residues and polymer concentration. Bovine chondrocytes incorporated in the HA-g-Dex-TA hydrogels remained viable, as shown by the Live-dead assay. Moreover, enhanced chondrocyte proliferation and matrix production were observed in the HA-g-Dex-TA hydrogels compared to Dex-TA hydrogels. These results suggest that HA-g-Dex-TA hydrogels have a high potential as injectable scaffolds for cartilage tissue engineering.
Asunto(s)
Biomimética , Cartílago/química , Dextranos/química , Ácido Hialurónico/química , Hidrogeles/química , Ingeniería de Tejidos , Animales , Materiales Biocompatibles/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cartílago/patología , Bovinos , Proliferación Celular , Células Cultivadas , Condrocitos/química , Condrocitos/citología , Reactivos de Enlaces Cruzados/química , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Inyecciones , Ensayo de Materiales , Modelos Moleculares , Datos de Secuencia Molecular , Simpatomiméticos/química , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Tiramina/químicaRESUMEN
Injectable hydrogels based on hyaluronic acid (HA) and poly(ethylene glycol) (PEG) were designed as biodegradable matrices for cartilage tissue engineering. Solutions of HA conjugates containing thiol functional groups (HA-SH) and PEG vinylsulfone (PEG-VS) macromers were cross-linked via Michael addition to form a three-dimensional network under physiological conditions. Gelation times varied from 14min to less than 1min, depending on the molecular weights of HA-SH and PEG-VS, degree of substitution (DS) of HA-SH and total polymer concentration. When the polymer concentration was increased from 2% to 6% (w/v) in the presence of 100Uml(-1) hyaluronidase the degradation time increased from 3 to 15days. Hydrogels with a homogeneous distribution of cells were obtained when chondrocytes were mixed with the precursor solutions. Culturing cell-hydrogel constructs prepared from HA185k-SH with a DS of 28 and cross-linked with PEG5k-4VS for 3weeks in vitro revealed that the cells were viable and that cell division took place. Gel-cell matrices degraded in approximately 3weeks, as shown by a significant decrease in dry gel mass. At day 21 glycosaminoglycans and collagen type II were found to have accumulated in hydrogels. These results indicate that these injectable hydrogels have a high potential for cartilage tissue engineering.
Asunto(s)
Materiales Biocompatibles/síntesis química , Condrocitos/citología , Condrocitos/fisiología , Ácido Hialurónico/síntesis química , Hidrogeles/síntesis química , Polietilenglicoles/síntesis química , Animales , Cartílago Articular/lesiones , Cartílago Articular/cirugía , Bovinos , Células Cultivadas , Hidrogeles/administración & dosificación , Inyecciones Intraarticulares , Ensayo de MaterialesRESUMEN
In this study we have investigated the influence of the mechanism of microsphere degradation or erosion on the in vivo degradation of microsphere/calcium phosphate cement composites (microsphere CPCs) used in tissue engineering. Microspheres composed of poly(lactic-co-glycolic acid) (PLGA), gelatin and poly(trimethylene carbonate) (PTMC) were used as the model and the resulting microsphere CPCs were implanted subcutaneously for 4, 8 or 12weeks in the back of New Zealand white rabbits. Besides degradation, the soft tissue response to these formulations was evaluated. After retrieval, specimens were analyzed by physicochemical characterization and histological analysis. The results showed that all microsphere CPCs exhibited microsphere degradation after 12weeks of subcutaneous implantation, which was accompanied by decreasing compression strength. The PLGA microspheres exhibited bulk erosion simultaneously throughout the whole composite, whereas the gelatin type B microspheres were degradated from the outside to the center of the composite. High molecular weight PTMC microspheres exhibited surface erosion resulting in decreasing microsphere size. Furthermore, all composites showed a similar tissue response, with decreasing capsule thickness over time and a persistent moderate inflammatory response at the implant interface. In conclusion, microsphere CPCs can be used to generate porous scaffolds in an in vivo environment after degradation of microspheres by various degradation/erosion mechanisms.
Asunto(s)
Implantes Absorbibles , Materiales Biocompatibles/química , Cementos para Huesos/química , Andamios del Tejido , Animales , Análisis de Falla de Equipo , Femenino , Ensayo de Materiales , Microesferas , ConejosRESUMEN
Acceptance of microfluidic technology in everyday laboratory practice by biologists is still low. One of the reasons for this is that the technology combines poorly with standard cell biological and biochemical analysis tools. Flow cytometry is an example of a conventional analytical tool that is considered to be incompatible with microfluidic technology and its inherent small sample sizes. In this study, it is shown that properly designed microfluidic devices contain cell populations that are large enough to be analyzed by flow cytometry. To illustrate this, the uptake of fluorescent human low-density lipoprotein (LDL) by human endothelial cells that were cultured in a microfluidic channel was analyzed. It was found that the uptake of LDL by the cells increased linearly over time. Moreover, the uptake decreased when cells were pretreated with fluid shear stress inside the microfluidic devices. This study shows that microfluidic technology can be combined with conventional flow cytometry, while retaining the advantages of working with microfluidics such as low reagent use and dynamic cell culture conditions. This approach of combining microfluidic technology with conventional laboratory tools may contribute to greater acceptance of microfluidic devices in biological research.
Asunto(s)
Células Endoteliales/metabolismo , Citometría de Flujo/métodos , Lipoproteínas LDL/metabolismo , Células Endoteliales/citología , Humanos , Técnicas Analíticas Microfluídicas , Estrés MecánicoRESUMEN
Biocompatible and elastic porous tubular structures based on poly(1,3-trimethylene carbonate), PTMC, were developed as scaffolds for tissue engineering of small-diameter blood vessels. High-molecular-weight PTMC (M(n) = 4.37 x 10(5)) was cross-linked by gamma-irradiation in an inert nitrogen atmosphere. The resulting networks (50-70% gel content) were elastic and creep resistant. The PTMC materials were highly biocompatible as determined by cell adhesion and proliferation studies using various relevant cell types (human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) and mesenchymal stem cells (MSCs)). Dimensionally stable tubular scaffolds with an interconnected pore network were prepared by particulate leaching. Different cross-linked porous PTMC specimens with average pore sizes ranging between 55 and 116 microm, and porosities ranging from 59% to 83% were prepared. These scaffolds were highly compliant and flexible, with high elongations at break. Furthermore, their resistance to creep was excellent and under cyclic loading conditions (20 deformation cycles to 30% elongation) no permanent deformation occurred. Seeding of SMCs into the wall of the tubular structures was done by carefully perfusing cell suspensions with syringes from the lumen through the wall. The cells were then cultured for 7 days. Upon proliferation of the SMCs, the formed blood vessel constructs had excellent mechanical properties. Their radial tensile strengths had increased from 0.23 to 0.78 MPa, which is close to those of natural blood vessels.
Asunto(s)
Dioxanos/química , Elasticidad , Docilidad , Polímeros/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Células Cultivadas , Reactivos de Enlaces Cruzados/farmacología , Elasticidad/efectos de los fármacos , Elasticidad/efectos de la radiación , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/efectos de la radiación , Rayos gamma , Geles , Humanos , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/efectos de la radiación , Docilidad/efectos de los fármacos , Docilidad/efectos de la radiación , Porosidad/efectos de los fármacos , Porosidad/efectos de la radiación , Ovinos , Sus scrofa , Resistencia a la Tracción/efectos de los fármacos , Resistencia a la Tracción/efectos de la radiaciónRESUMEN
Vascular cell biology is an area of research with great biomedical relevance. Vascular dysfunction is involved in major diseases such as atherosclerosis, diabetes, and cancer. However, when studying vascular cell biology in the laboratory, it is difficult to mimic the dynamic, three-dimensional microenvironment that is found in vivo. Microfluidic technology offers unique possibilities to overcome this difficulty. In this review, an overview of the recent applications of microfluidic technology in the field of vascular biological research will be given. Examples of how microfluidics can be used to generate shear stresses, growth factor gradients, cocultures, and migration assays will be provided. The use of microfluidic devices in studying three-dimensional models of vascular tissue will be discussed. It is concluded that microfluidic technology offers great possibilities to systematically study vascular cell biology with setups that more closely mimic the in vivo situation than those that are generated with conventional methods.
Asunto(s)
Vasos Sanguíneos/fisiología , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Animales , Investigación Biomédica/instrumentación , Investigación Biomédica/métodos , Vasos Sanguíneos/citología , Humanos , Enfermedades Vasculares/patología , Enfermedades Vasculares/fisiopatologíaRESUMEN
Water-soluble chitosan derivatives, chitosan-graft-glycolic acid (GA) and phloretic acid (PA) (CH-GA/PA), were designed to obtain biodegradable injectable chitosan hydrogels through enzymatic crosslinking with horseradish peroxidase (HRP) and H2O2. CH-GA/PA polymers were synthesized by first conjugating glycolic acid (GA) to native chitosan to render the polymer soluble at pH 7.4, and subsequent modification with phloretic acid (PA). The CH-GA43/PA10 with a degree of substitution (DS, defined as the number of substituted NH2 groups per 100 glucopyranose rings of chitosan) of GA of 43 and DS of PA of 10 showed a good solubility at pH values up to 10. Short gelation times (e.g. 10 s at a polymer concentration of 3 wt%), as recorded by the vial tilting method, were observed for the CH-GA43/PA10 hydrogels using HRP and H2O2. It was shown that these hydrogels can be readily degraded by lysozyme. In vitro culturing of chondrocytes in CH-GA43/PA10 hydrogels revealed that after 2 weeks the cells were viable and retained their round shape. These features indicate that CH-GA/PA hydrogels are promising as an artificial extracellular matrix for cartilage tissue engineering.
Asunto(s)
Cartílago/metabolismo , Quitosano/química , Quitosano/metabolismo , Hidrogeles/química , Hidrogeles/metabolismo , Animales , Bovinos , Supervivencia Celular , Células Cultivadas , Concentración de Iones de Hidrógeno , Inyecciones , Estructura Molecular , Muramidasa/metabolismo , Reología , Factores de Tiempo , Ingeniería de Tejidos , Agua/químicaRESUMEN
Vascular endothelial cells have an extensive response to physiological levels of shear stress. There is evidence that the protein caveolin-1 is involved in the early phase of this response. In this study, caveolin-1 was downregulated in human endothelial cells by RNAi. When these cells were subjected to a shear stress of 15 dyn/cm(2) for 10 minutes, activation of Akt and ERK1/2 was significantly lower than in control cells. Moreover, activation of Akt and ERK1/2 in response to vascular endothelial growth factor was significantly lower in cells with low levels of caveolin-1. However, activation of integrin-mediated signaling during cell adhesion onto fibronectin was not hampered by lowered caveolin-1 levels. In conclusion, caveolin-1 is an essential component in the response of endothelial cells to shear stress. Furthermore, the results suggest that the role of caveolin-1 in this process lies in facilitating efficient VEGFR2-mediated signaling.
RESUMEN
Heat-induced apoptosis proceeds via mitochondria by permeabilization of the outer mitochondrial membrane (MOMP), resulting in the release of cytochrome c. This essential step is mediated by Bcl-2 family proteins, such as Bax. Recently, caspase-2 was assigned a prominent role in regulating Bax. Therefore, we studied the initiation of heat-induced apoptosis by monitoring Bcl-2 family members and the release of cytochrome c with or without caspase-2 inhibition. Three hematopoietic cell lines (HSB2, HL60 and Kasumi-1) were exposed to heat treatment and/or X-radiation. Expression and localization of Bax and Bcl-2 proteins was investigated by flow cytometry (FCM) and confocal microscopy respectively. Cytochrome c release was measured with FCM as evidence for MOMP. In addition, the role of caspase-2 in heat- and radiation-induced apoptosis was assessed using the specific caspase-2 inhibitor zVDVAD-fmk. Here we present evidence that heat treatment, and not irradiation, increases intracellular Bax protein expression and subsequently stimulates MOMP, resulting in the release of cytochrome c. Furthermore, by selective blocking of caspase-2 using zVDVAD-fmk less Bax was expressed and subsequently a significant decrease in cytochrome c release was observed. In conclusion, heat treatment of hematopoietic cells does require caspase-2 activation for the initiation of Bax-mediated MOMP.
Asunto(s)
Caspasa 2/metabolismo , Membranas Intracelulares/fisiología , Mitocondrias/fisiología , Proteína X Asociada a bcl-2/fisiología , Apoptosis , Línea Celular Tumoral , Citometría de Flujo , Calor , Humanos , Microscopía Confocal , Rayos XRESUMEN
PURPOSE: The effect of heat treatment in combination with X-irradiation was examined with regard to expression of p53, a tumor suppressor gene product, and Hsp70, a heat-shock protein, in association with the occurrence of programmed cell death (apoptosis). MATERIALS AND METHODS: Three hematopoietic cell lines (HSB2, HL60 and Kasumi-1), which differ in p53 status, were exposed to 42.5 degrees C during one hour and/or X-radiation (total dose 8 Gy). After exposure, both mRNA and protein expression levels of Hsp70 and p53 were investigated by real-time PCR (polymerase chain reaction) and Western blotting. Apoptosis was simultaneously analyzed by observation of cell morphology as well as flowcytometric determination of Annexin V binding to phosphatidylserine and propidium iodide exclusion. RESULTS: Both HL60 and HSB2 cell lines with a low p53 status and a quick response to heat treatment with Hsp70 over-expression are less susceptible to heat-induced apoptosis compared to Kasumi-1 cells with wild-type p53 protein and no Hsp70 response. The combination of first applying X-irradiation followed by heat treatment resulted in the most effective induction of apoptosis due to impairment of the Hsp70 response in all three cell lines. CONCLUSION: These results indicate that the Hsp70 response and p53 status mediate the susceptibility of hematopoietic cells to undergo heat-induced apoptosis. Therefore, these parameters can be used as markers to predict the effectiveness of hyperthermia in cancer treatment.
Asunto(s)
Apoptosis/efectos de la radiación , Regulación de la Expresión Génica/efectos de la radiación , Genes p53/efectos de la radiación , Proteínas HSP70 de Choque Térmico/efectos de la radiación , Sistema Hematopoyético/efectos de la radiación , Apoptosis/fisiología , Regulación de la Expresión Génica/fisiología , Genes p53/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Sistema Hematopoyético/citología , Sistema Hematopoyético/metabolismo , Calor , Humanos , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Factores de Tiempo , Células Tumorales Cultivadas , Rayos XRESUMEN
The tensile properties of segmented block copolymers with mono-disperse hard segments were studied with respect to the hard segment content (16-44 wt.%) and the temperature (20-110 °C). The copolymers were comprised of poly(tetramethylene oxide) segments with the molecular weights of 650-2,900 Da and of mono-disperse bisester-tetra-amide segments (T6A6T) based on adipic acid (A), terephthalic acid (T) and hexamethylene diamine (6). An increasing content of T6A6T gave rise to an increased modulus, yield stress and fracture stress. The modulus could be modeled by a composite model. Moreover, a strain-softening was observed well below the yield stress, due to the shearing of the T6A6T crystallites. At strains >200%, a strain-hardening of the PTMO segments took place and this even for PTMO segments that were amorphous in the isotropic state. The strain hardening increased the tensile properties. An increase in temperature had little effect on the modulus of the copolymers, but was found to lower the yield and fracture stresses. At temperatures above the melting temperature of the oriented PTMO, no strain-hardening took place. The yield stress as a function of temperature could be described by the Eyring relationship, but a modulus-yield stress relationship could not be established.