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1.
Cardiovasc Eng Technol ; 10(1): 173-180, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30141125

RESUMEN

Current protocols for mechanical preconditioning of tissue engineered heart valves have focused on application of pressure, flexure and fluid flow to stimulate collagen production, ECM remodeling and improving mechanical performance. The aim of this study was to determine if mechanical preconditioning with cyclic stretch could promote an intact endothelium that resembled the viability and morphology of a native valve. Confocal laser scanning microscopy was used to image endothelial cells on aortic valve strips subjected to static incubation or physiological strain regimens. An automated image analysis program was designed and implemented to detect and analyze live and dead cells in images captured of a live aortic valve endothelium. The images were preprocessed, segmented, and quantitatively analyzed for live/dead cell ratio, minimum neighbor distance and circularity. Significant differences in live/dead cellular ratio and the minimum distance between cells were observed between static and strained endothelia, indicating that cyclic strain is an important stimulus for maintaining a healthy endothelium. In conclusion, in vitro application of physiological levels of cyclic strain to tissue engineered heart valves seeded with autologous endothelial cells would be advantageous.


Asunto(s)
Válvula Aórtica/citología , Forma de la Célula , Células Endoteliales/fisiología , Animales , Supervivencia Celular , Femenino , Microscopía Confocal , Estrés Mecánico , Sus scrofa
2.
Bioengineered ; 5(3): 193-7, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24637710

RESUMEN

Due to the limited self-renewal capacity of cardiomyocytes, the mammalian heart exhibits impaired regeneration and insufficient ability to restore heart function after injury. Cardiovascular tissue engineering is currently considered as a promising alternative therapy to restore the structure and function of the failing heart. Recent evidence suggests that the epicardium may play critical roles in regulation of myocardial development and regeneration. One of the mechanisms that has been proposed for the restorative effect of the epicardium is the specific physiomechanical cues that this layer provides to the cardiac cells. In this article we explore whether a new generation of epicardium-mimicking, acellular matrices can be utilized to enhance cardiac healing after injury. The matrix consists of a dense collagen scaffold with optimized biomechanical properties approaching those of embryonic epicardium. Grafting the epicardial patch onto the ischemic myocardium--promptly after the incidence of infarct--resulted in preserved contractility, attenuated ventricular remodeling, diminished fibrosis, and vascularization within the injured tissue in the adult murine heart.


Asunto(s)
Colágeno/farmacología , Implantes Experimentales , Infarto del Miocardio/terapia , Técnicas de Cultivo de Tejidos/métodos , Ingeniería de Tejidos/métodos , Animales , Materiales Biomiméticos , Proliferación Celular , Colágeno/química , Módulo de Elasticidad , Embrión de Mamíferos , Células Epiteliales/citología , Células Epiteliales/fisiología , Fibrosis/prevención & control , Geles , Masculino , Ratones , Contracción Miocárdica/efectos de los fármacos , Infarto del Miocardio/fisiopatología , Infarto del Miocardio/cirugía , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiología , Neovascularización Fisiológica/efectos de los fármacos , Pericardio/citología , Pericardio/fisiología , Remodelación Ventricular/efectos de los fármacos
3.
Am J Transl Res ; 6(6): 724-35, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25628783

RESUMEN

Therapeutic delivery of cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs) represents a novel clinical approach to regenerate the injured myocardium. However, poor survival and contractility of these cells are a significant bottleneck to their clinical use. To better understand the role of cell-cell communication in enhancing the phenotype and contractile properties of hPSC-CMs, we developed a three-dimensional (3D) hydrogel composed of hPSC-CMs, human pluripotent stem cell-derived endothelial cells (hPSC-ECs), and/or human amniotic mesenchymal stem cells (hAMSCs). The objective of this study was to examine the role of multi-cellular interactions among hPSC-ECs and hAMSCs on the survival and long-term contractile phenotype of hPSC-CMs in a 3D hydrogel. Quantification of spontaneous contractility of hPSC-CMs in tri-culture demonstrated a 6-fold increase in the area of contractile motion after 6 weeks with characteristic rhythmic contraction frequency, when compared to hPSC-CMs alone (P < 0.05). This finding was supported by a statistically significant increase in cardiac troponin T protein expression in the tri-culture hydrogel construct at 6 weeks, when compared to hPSC-CMs alone (P < 0.001). The sustained hPSC-CM survival and contractility in tri-culture was associated with a significant upregulation in the gene expression of L-type Ca(2+) ion channel, Cav1.2, and the inward-rectifier potassium channel, Kir2.1 (P < 0.05), suggesting a role of ion channels in mediating these processes. These findings demonstrate that multi-cellular interactions modulate hPSC-CM phenotype, function, and survival, and they will have important implications in engineering cardiac tissues for treatment of cardiovascular diseases.

4.
Biomaterials ; 34(36): 9048-55, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23992980

RESUMEN

Regeneration of the damaged myocardium is one of the most challenging fronts in the field of tissue engineering due to the limited capacity of adult heart tissue to heal and to the mechanical and structural constraints of the cardiac tissue. In this study we demonstrate that an engineered acellular scaffold comprising type I collagen, endowed with specific physiomechanical properties, improves cardiac function when used as a cardiac patch following myocardial infarction. Patches were grafted onto the infarcted myocardium in adult murine hearts immediately after ligation of left anterior descending artery and the physiological outcomes were monitored by echocardiography, and by hemodynamic and histological analyses four weeks post infarction. In comparison to infarcted hearts with no treatment, hearts bearing patches preserved contractility and significantly protected the cardiac tissue from injury at the anatomical and functional levels. This improvement was accompanied by attenuated left ventricular remodeling, diminished fibrosis, and formation of a network of interconnected blood vessels within the infarct. Histological and immunostaining confirmed integration of the patch with native cardiac cells including fibroblasts, smooth muscle cells, epicardial cells, and immature cardiomyocytes. In summary, an acellular biomaterial with specific biomechanical properties promotes the endogenous capacity of the infarcted myocardium to attenuate remodeling and improve heart function following myocardial infarction.


Asunto(s)
Bioingeniería , Colágeno/farmacología , Corazón/efectos de los fármacos , Corazón/fisiopatología , Infarto del Miocardio/fisiopatología , Función Ventricular/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacos , Animales , Fenómenos Biomecánicos , Geles/farmacología , Pruebas de Función Cardíaca , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Miocardio/patología , Plásticos/farmacología , Ratas
5.
Biomech Model Mechanobiol ; 11(3-4): 355-61, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21604147

RESUMEN

Soft tissues, such as tendons, skin, arteries, or lung, are constantly subject to mechanical stresses in vivo. None more so than the aortic heart valve that experiences an array of forces including shear stress, cyclic pressure, strain, and flexion. Anisotropic biaxial cyclic stretch maintains valve homeostasis; however, abnormal forces are implicated in disease progression. The response of the valve endothelium to deviations from physiological levels has not been fully characterized. Here, we show the design and validation of a novel stretch apparatus capable of applying biaxial stretch to viable heart valve tissue, while simultaneously allowing for live en face endothelial cell imaging via confocal laser scanning microscopy (CLSM). Real-time imaging of tissue is possible while undergoing highly characterized mechanical conditions and maintaining the native extracellular matrix. Thus, it provides significant advantages over traditional cell culture or in vivo animal models. Planar biaxial tissue stretching with simultaneous live cell imaging could prove useful in studying the mechanobiology of any soft tissue.


Asunto(s)
Válvula Aórtica/patología , Prótesis Valvulares Cardíacas , Microscopía Confocal/métodos , Ingeniería de Tejidos/métodos , Anisotropía , Fenómenos Biomecánicos , Reactores Biológicos , Química Física/métodos , Endotelio/patología , Diseño de Equipo , Glucosa/química , Humanos , Concentración de Iones de Hidrógeno , Imagenología Tridimensional/métodos , Diseño de Prótesis , Estrés Mecánico , Factores de Tiempo
6.
J Heart Valve Dis ; 19(1): 86-95; discussion 96, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20329494

RESUMEN

BACKGROUND AND AIM OF THE STUDY: Although the vasoactive agents, angiotensin II (Ang II) and 5-hydroxytryptamine (5-HT) are implicated in aortic heart valve disease, it is unclear how these compounds alter the biomechanical properties of valve leaflet tissue. The study aim was to characterize temporal changes in the elastic modulus of tissues incubated with these compounds. METHODS: Valve leaflets were excised from fresh porcine aortic heart valves. Leaflet tissue was incubated with 10(-6) M 5-HT, or 10(-6) M Ang II. The stress and elongation of the tissue in the circumferential and radial directions was measured using a stepper motor-driven micromechanical testing machine at 0.5, 6, and 24 h, followed by calculations of strain and elastic modulus of each sample. RESULTS: Tissue samples incubated with Ang II showed a significant increase in stiffness with time in the radial direction, but not in the circumferential direction. Regression analysis showed a correlation between time and elastic modulus for the tissue (R2 = 0.84). Conversely, leaflets incubated in 5-HT did not show any significant change in elastic modulus over time in the radial direction; however, significant increases in stiffness were observed after 24 h in the circumferential direction. A strong correlation between the elastic modulus in the circumferential direction and time was also noted (R2 = 0.99). CONCLUSION: The study results showed that vasoactive agents are capable of increasing the elastic modulus of aortic valve tissue in a time-dependent manner. Furthermore, the biomechanical changes induced by vasoactive agents are direction-specific, indicating different modes of action.


Asunto(s)
Angiotensina II/farmacología , Válvula Aórtica/efectos de los fármacos , Válvula Aórtica/fisiología , Módulo de Elasticidad/fisiología , Serotonina/farmacología , Vasoconstrictores/farmacología , Animales , Fenómenos Biomecánicos , Técnicas In Vitro , Microscopía Confocal , Porcinos
7.
Biomech Model Mechanobiol ; 9(1): 117-25, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19636599

RESUMEN

Mechanical in vitro preconditioning of tissue engineered heart valves is viewed as an essential process for tissue development prior to in vivo implantation. However, a number of pro-inflammatory genes are mechanosensitive and their elaboration could elicit an adverse response in the host. We hypothesized that the application of normal physiological levels of strain to isolated valve interstitial cells would inhibit the expression of pro-inflammatory genes. Cells were subjected to 0, 5, 10, 15 and 20% strain. Expression of VCAM-1, MCP-1, GM-CSF and OPN was then measured using qRT-PCR. With the exception of OPN, all genes were significantly up regulated when no strain was applied. MCP-1 expression was significantly lower in the presence of strain, although strain magnitude did not affect the expression level. VCAM-1 and GM-CSF had the lowest expression levels at 15% strain, which represent normal physiological conditions. These findings were confirmed using confocal microscopy. Additionally, pSMAD 2/3 and IkappaBalpha expression were imaged to elucidate potential mechanisms of gene expression. Data showed that 15% strain increased pSMAD 2/3 expression and prevented phosphorylation of IkappaBalpha. In conclusion, cyclic strain reduces expression of pro-inflammatory genes, which may be beneficial for the in vitro pre-conditioning of tissue engineered heart valves.


Asunto(s)
Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Regulación de la Expresión Génica , Mediadores de Inflamación/metabolismo , Estrés Mecánico , Animales , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Microscopía Confocal , Osteopontina/genética , Osteopontina/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sus scrofa , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo
8.
J Heart Valve Dis ; 17(5): 571-7; discussion 578, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18980092

RESUMEN

BACKGROUND AND AIM OF THE STUDY: The endothelium of diseased heart valves is known to express the adhesion molecules VCAM-1, ICAM-1 and E-selectin, while healthy valves lack these pro-inflammatory proteins. The study aim was to determine if mechanical forces were responsible for the pro-inflammatory reaction in aortic valve endothelial cells. METHODS: Isolated porcine aortic valve endothelial cells (PAVEC) were cultured and seeded onto BioFlexTM culture plates. The cells were exposed to equibiaxial cyclic strains of 5, 10 and 20% for 24 h in a Flexcell FX-4000T Tension Plus system at 1 Hz. Pro-inflammatory protein expression was detected through the use of monoclonal antibodies via fluorescence-assisted cell sorting (FACS) and confocal laser scanning microscopy (CLSM). RESULTS: Pro-inflammatory protein expression was evident at cyclic strains of 5 and 20%, while a 10% strain did not elicit an inflammatory response. Confocal images indicated a disrupted endothelial monolayer, evidence of significant cell death, and the presence of all adhesion molecules at 5% strain. PAVEC exposed to 10% cyclic strain failed to express any of the pro-inflammatory proteins, while the cellular monolayer appeared near-confluent and characteristically similar to cellular images captured prior to cyclic stretching. CLSM images of PAVEC cyclically stretched by 20% demonstrated a similar proinflammatory reaction to those with 5% strain, while the cellular environment also showed decreased monolayer integrity. FACS data showed a significant up-regulation of the membrane-bound VCAM-1-, ICAM-1- and E-selectin-positive cells at 5% and 20% strain, compared to 10% strain and controls. CONCLUSION: The finding that equibiaxial cyclic strain regulates the pro-inflammatory response in PAVEC suggests that alterations in the mechanical environment of heart valves may contribute to valve pathogenesis.


Asunto(s)
Válvula Aórtica/fisiopatología , Selectina E/metabolismo , Células Endoteliales/fisiología , Molécula 1 de Adhesión Intercelular/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Animales , Fenómenos Biomecánicos , Muerte Celular/fisiología , Femenino , Citometría de Flujo , Técnicas In Vitro , Microscopía Confocal , Estrés Fisiológico/fisiología , Porcinos , Regulación hacia Arriba/fisiología , Vacio
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