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1.
Methods Mol Biol ; 2206: 57-66, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754811

RESUMO

The construction of vascular networks is essential for developing functional organ/tissue constructs in terms of oxygen and nutrient supply. Although recent advances in microfluidic techniques have allowed for the construction of microvascular networks using microfluidic devices, their structures cannot be maintained for extended periods of time due to a lack of perivascular cells. To construct long-lasting microvascular networks, it is important that perivascular cells are present to provide structural support to vessels, because in vivo microvessels are covered by perivascular cells and stabilized. Here, we describe a microfluidic cell culture platform for the construction of microvascular networks with supportive perivascular cells. Our results showed that microvascular networks covered by pericyte-like perivascular cells formed in a microfluidic device and their structures were maintained for at least 3 weeks in vitro.

2.
Biofabrication ; 12(4): 045008, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32644945

RESUMO

Vascular networks consist of hierarchical structures of various diameters and are necessary for efficient blood distribution. Recent advances in vascular tissue engineering and bioprinting have allowed us to construct large vessels, such as arteries, small vessels, such as capillaries and microvessels, and intermediate-scale vessels, such as arterioles, individually. However, little is known about the control of vessel diameters between small vessels and intermediate-scale vessels. Here, we focus on vascular remodeling, which creates lasting structural changes in the vessel wall in response to hemodynamic stimuli, to regulate vessel diameters in vitro. The purpose of this study is to control the vessel diameter at an intermediate scale by inducing outward remodeling of microvessels in vitro. Human umbilical vein endothelial cells and mesenchymal stem cells were cocultured in a microfluidic device to construct microvessels, which were then perfused with a culture medium to induce outward vascular remodeling. We successfully constructed vessels with diameters of 40-150 µm in perfusion culture, whereas vessels with diameters of <20 µm were maintained in static culture. We also revealed that the in vitro vascular remodeling was mediated by NO pathways and MMP-9. These findings provide insight into the regulation of diameters of tissue-engineered blood vessels. This is an important step toward the construction of hierarchical vascular networks within biofabricated three-dimensional systems.

3.
Anal Sci ; 36(8): 1015-1019, 2020 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-32201406

RESUMO

We report on a novel microdevice to tune the curvature of a cell-adhering surface by controlling the air-pressure and micro-slit. Human aortic smooth muscle cells were cultured on demi-cylindrical concaves formed on a microdevice. Their shape-adapting behavior could be tracked when the groove direction was changed to the orthogonal direction. This microdevice demonstrated live observation of cells responding to dynamic changes of the anisotropic curvature of the adhering surface and could serve as a new platform to pursue mechanobiology on curved surfaces.

4.
APL Bioeng ; 3(3): 036102, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31431938

RESUMO

Hemodynamic and biochemical factors play important roles in critical steps of angiogenesis. In particular, interstitial flow has attracted attention as an important hemodynamic factor controlling the angiogenic process. Here, we applied a wide range of interstitial flow magnitudes to an in vitro three-dimensional (3D) angiogenesis model in a microfluidic device. This study aimed to investigate the effect of interstitial flow magnitude in combination with the vascular endothelial growth factor (VEGF) concentration on 3D microvascular network formation. Human umbilical vein endothelial cells (HUVECs) were cultured in a series of interstitial flow generated by 2, 8, and 25 mmH2O. Our findings indicated that interstitial flow significantly enhanced vascular sprout formation, network extension, and the development of branching networks in a magnitude-dependent manner. Furthermore, we demonstrated that the proangiogenic effect of interstitial flow application could not be substituted by the increased VEGF concentration. In addition, we found that HUVECs near vascular sprouts significantly elongated in >8 mmH2O conditions, while activation of Src was detected even in 2 mmH2O conditions. Our results suggest that the balance between the interstitial flow magnitude and the VEGF concentration plays an important role in the regulation of 3D microvascular network formation in vitro.

5.
Biofabrication ; 11(3): 035007, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31025629

RESUMO

Recent advances in microfabrication technologies have enabled us to construct collagen gel microbeads, which can be cultured with hepatocytes. However, little is known about the hepatocyte-collagen gel microbead interactions. Here, we aimed to clarify the effects of the balance between cell-cell and cell-collagen gel microbead interactions on hepatocyte morphogenesis and functions. The magnitude of cell-microbead interactions was controlled by changing the size of the microbeads, which were smaller than, comparable to, and larger than hepatocytes. These small, medium, and large microbeads were cultured separately with primary hepatocytes. Phase-contrast and time-lapse imaging revealed that the medium microbeads significantly induced the construction of 3D structures composed of the microbeads and hepatocytes in a self-organizing manner, whereas hepatocytes formed 2D monolayers with the small or large microbeads. These results suggest that only the medium microbeads induced the 3D tissue formation of hepatocytes. Furthermore, liver-specific functions, such as albumin secretion and ammonia clearance, were significantly upregulated in the 3D structures. These findings are critical to understand how to control the construction of 3D hepatocyte tissues with hydrogel microbeads in the context of biofabrication.


Assuntos
Colágeno/farmacologia , Hepatócitos/citologia , Microesferas , Morfogênese , Animais , Bovinos , Células Cultivadas , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/fisiologia , Masculino , Morfogênese/efeitos dos fármacos , Ratos Sprague-Dawley , Suínos , Engenharia Tecidual
6.
Tissue Eng Part A ; 25(5-6): 499-510, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30234439

RESUMO

IMPACT STATEMENT: Construction of capillary networks is a fundamental challenge for the development of three-dimensional (3D) tissue engineering. However, it is not well understood how to construct stable capillary networks that maintain a luminal size similar to that of capillary structures in vivo (i.e., <10 µm diameter). In this study, we demonstrated the construction of stable capillary networks covered by pericyte-like perivascular cells using an in vitro 3D angiogenesis model by optimizing interactions between endothelial cells and perivascular cells. Our 3D angiogenesis model can be combined with 3D culture of epithelial cells in the context of vascularization of 3D tissue-engineered constructs.


Assuntos
Capilares/citologia , Pericitos/citologia , Engenharia Tecidual/métodos , Membrana Basal/metabolismo , Proliferação de Células , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Imageamento Tridimensional , Células-Tronco Mesenquimais/citologia , Microfluídica , Neovascularização Fisiológica
7.
Acta Biomater ; 95: 307-318, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30593886

RESUMO

There is a great deal of demand for the construction of transplantable liver grafts. Over the last decade, decellularization techniques have been developed to construct whole liver tissue grafts as potential biomaterials. However, the lack of intact vascular networks, especially sinusoids, in recellularized liver scaffolds leads to hemorrhage and thrombosis after transplantation, which is a major obstacle to the development of transplantable liver grafts. In the present study, we hypothesized that both mechanical (e.g., fluid shear stress) and chemical factors (e.g., fibronectin coating) can enhance the formation of hierarchical vascular networks including sinusoid-scale microvessels. We demonstrated that perfusion culture promoted formation of sinusoid-scale microvessels in recellularized liver scaffolds, which was not observed in static culture. In particular, perfusion culture at 4.7 ml/min promoted the formation of sinusoid-scale microvessels compared to perfusion culture at 2.4 and 9.4 ml/min. In addition, well-aligned endothelium was observed in perfusion culture, suggesting that endothelial cells sensed the flow-induced shear stress. Moreover, fibronectin coating of decellularized liver scaffolds enhanced the formation of sinusoid-scale microvessels in perfusion culture at 4.7 ml/min. This study represents a critical step in the development of functional recellularized liver scaffolds, which can be used not only for transplantation but also for drug screening and disease-modeling studies. STATEMENT OF SIGNIFICANCE: Decellularized liver scaffolds are promising biomaterials that allow production of large-scale tissue-engineered liver grafts. However, it is difficult to maintain recellularized liver grafts after transplantation due to hemorrhage and thrombosis. To overcome this obstacle, construction of an intact vascular network including sinusoid-scale microvessels is essential. In the present study, we succeeded in constructing sinusoid-scale microvessels in decellularized liver scaffolds via a combination of perfusion culture and surface coating. We further confirmed that endothelial cells in decellularized liver scaffolds responded to flow-derived mechanical stress by aligning actin filaments. Our strategy to construct sinusoid-scale microvessels is critical for the development of intact vascular networks, and addresses the limitations of recellularized liver scaffolds after transplantation.

8.
Microvasc Res ; 122: 60-70, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30472038

RESUMO

Every organ demonstrates specific vascular characteristics and functions maintained by interactions of endothelial cells (ECs) and parenchymal cells. Particularly, brain ECs play a central role in the formation of a functional blood brain barrier (BBB). Organ-specific ECs have their own morphological features, and organ specificity must be considered when investigating interactions between ECs and other cell types constituting a target organ. Here we constructed angiogenesis-based microvascular networks with perivascular cells in a microfluidic device setting by coculturing ECs and mesenchymal stem cells (MSCs). Furthermore, we analyzed endothelial barrier functions as well as fundamental morphology, an essential step to build an in vitro BBB model. In particular, we used both brain microvascular ECs (BMECs) and human umbilical vein ECs (HUVECs) to test if organ specificity of ECs affects the formation processes and endothelial barrier functions of an engineered microvascular network. We found that microvascular formation processes differed by the source of ECs. HUVECs formed more extensive microvascular networks compared to BMECs while no differences were observed between BMECs and HUVECs in terms of both the microvascular diameter and the number of pericytes peripherally associated with the microvasculatures. To compare the endothelial barrier functions of each type of EC, we performed fluorescence dextran perfusion on constructed microvasculatures. The permeability coefficient of BMEC microvasculatures was significantly lower than that of HUVEC microvasculatures. In addition, there were significant differences in terms of tight junction protein expression. These results suggest that the organ source of ECs influences the properties of engineered microvasculature and thus is a factor to be considered in the design of organ-specific cell culture models.


Assuntos
Barreira Hematoencefálica/metabolismo , Encéfalo/irrigação sanguínea , Permeabilidade Capilar , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células-Tronco Mesenquimais/metabolismo , Microvasos/metabolismo , Neovascularização Fisiológica , Pericitos/metabolismo , Barreira Hematoencefálica/citologia , Comunicação Celular , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Humanos , Dispositivos Lab-On-A-Chip , Microvasos/citologia , Fenótipo , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismo
9.
Methods Mol Biol ; 1905: 167-174, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30536099

RESUMO

Construction of three-dimensional (3D) hepatic tissue structures is important for in vitro tissue engineering of the liver, because 3D culture of hepatocytes is critical for the maintenance of liver-specific functions. Although conventional 3D culture methods are useful for constructing 3D hepatic tissue structures, the precise control of culture microenvironments is required to construct more physiological tissues in vitro. Recent advances in microfluidics technologies have allowed us to utilize microfluidic devices for hepatic cell culture, which opened the door for creating more physiological 3D culture models of the liver. Here, we describe the method for the construction of hepatic tissue structures using a microfluidic device which has a 3D gel region with adjacent microchannels. Primary rat hepatocytes are seeded into a microchannel in a microfluidic device. The cells are then cultured in interstitial flow conditions, which leads to the construction of 3D tissue structures.


Assuntos
Técnicas de Cultura de Células/instrumentação , Hepatócitos/citologia , Animais , Diferenciação Celular , Células Cultivadas , Desenho de Equipamento , Dispositivos Lab-On-A-Chip , Modelos Biológicos , Ratos , Engenharia Tecidual
10.
Sci Rep ; 7(1): 17349, 2017 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-29229920

RESUMO

Neurovascular unit (NVU) is a basic unit in the brain, including neurons, glial cells, blood vessels and extracellular matrix. This concept implies the importance of a three-dimensional (3D) culture model including these cell types for investigating brain functions. However, little is known about the construction of an in vitro 3D NVU model. In the present study, we aimed at constructing 3D neurovascular tissues by combining in vitro neurogenesis and angiogenesis models using a microfluidic platform, which is a critical step toward the NVU construction in vitro. Three gel conditions, which were fibrin gel, fibrin-Matrigel mixed gel and fibrin-hyaluronan mixed gel, were investigated to optimize the gel components in terms of neurogenesis and angiogenesis. First, fibrin-Matrigel mixed gel was found to promote neural stem cell (NSC) differentiation into neurons and neurite extension. In particular, 3D neural networks were constructed in 2-8 mg/ml fibrin-Matrigel mixed gel. Second, we found that capillary-like structures were also formed in the fibrin-Matrigel mixed gel by coculturing brain microvascular endothelial cells (BMECs) and human mesenchymal stem cells (MSCs). Finally, we combined both neural and vascular culture models and succeeded in constructing 3D neurovascular tissues with an optimized seeding condition of NSCs, BMECs and MSCs.


Assuntos
Encéfalo/citologia , Endotélio Vascular/citologia , Modelos Biológicos , Neovascularização Fisiológica , Células-Tronco Neurais/citologia , Neurogênese , Encéfalo/fisiologia , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Endotélio Vascular/fisiologia , Humanos , Técnicas Analíticas Microfluídicas , Morfogênese , Células-Tronco Neurais/fisiologia
11.
Biol Pharm Bull ; 40(10): 1646-1653, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28966236

RESUMO

The cross-linking of elastin by lysyl oxidase (LOX) family members is essential for the integrity and elasticity of elastic fibers, which play an important role in the characteristic resilience of various tissues. However, the temporal sequence of oxidation by LOX during elastic fiber formation is still incompletely understood. Here, we demonstrate that the cross-linking of tropoelastin molecules by LOX occurs concurrent with elastin deposition. Our data show that LOX deficiency or the inhibition of LOX enzyme activity leads to the loss of elastin deposition in skin fibroblast. Moreover, overexpression of LOX promotes the deposition and alignment of tropoelastin, whereas the addition of recombinant active-form of LOX in culture medium caused abnormal elastic fiber assembly. Immunoblotting and immunofluorescence show that LOX and tropoelastin are present together with fibronectin on the cell surface of preconfluent cultures. Further, fluorescence activated cell sorting (FACS) analysis for the localization of LOX on the cell surface reveals that the transfer of LOX to the extracellular space occurs in association with elastic fiber formation. In conclusion, our results support the view that LOX and tropoelastin are present on the cell surface and suggests the possibility that lysine oxidation by LOX precedes tropoelastin deposition onto microfibrils.


Assuntos
Proteína-Lisina 6-Oxidase/metabolismo , Tropoelastina/metabolismo , Aminoácido Oxirredutases/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Células HEK293 , Humanos , Lisina/metabolismo , Oxirredução , Proteína-Lisina 6-Oxidase/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tropoelastina/genética
12.
Integr Biol (Camb) ; 9(9): 762-773, 2017 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-28752870

RESUMO

Glioblastoma (GBM) is a highly invasive primary brain tumor that displays cellular heterogeneity, which is composed of glioma initiating cells (GICs) and their differentiated progeny. GICs play an important role in driving aggressive invasion. In particular, the interaction between GICs and blood vessels is critical because blood vessels are known to serve as routes for the invasion of GICs. However, the effect of endothelial cells on the three-dimensional (3D) invasion process of GICs as well as the spatial relationship between GICs and their differentiated progeny remains unclear. Here, we utilized a microfluidic device to recapitulate the 3D brain tumor microenvironments constituted by human umbilical vein endothelial cells (HUVECs) and type I collagen. Using the device, we found that HUVECs promoted the 3D invasion of heterogeneous glioma cell populations into type I collagen gel. The invasion induced by HUVECs was predominantly preceded by cells positive for nestin, a neural stem cell marker. In contrast, cells positive for tubulin ß3 (TUBB3), a differentiated cell marker, rarely preceded invasion. In addition, HUVECs induced the upregulation of TUBB3 in GICs. Finally, we found that the genes associated with invasion, such as integrins α2 and ß3, were significantly upregulated in the presence of HUVECs. These results as well as the experimental approach provide valuable knowledge for the development of effective therapeutic strategies targeting the aggressive invasion of GBM.


Assuntos
Neoplasias Encefálicas/patologia , Glioma/patologia , Animais , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/genética , Diferenciação Celular , Linhagem Celular Tumoral , Técnicas de Cocultura , Colágeno Tipo I/metabolismo , Glioblastoma/irrigação sanguínea , Glioblastoma/genética , Glioblastoma/patologia , Glioma/irrigação sanguínea , Glioma/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Integrinas/genética , Dispositivos Lab-On-A-Chip , Camundongos , Modelos Biológicos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Células-Tronco Neoplásicas/patologia , Células-Tronco Neurais/patologia , Regulação para Cima
13.
Stem Cells Dev ; 26(14): 1078-1085, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28446066

RESUMO

Oligodendrocytes are generated from oligodendrocyte precursor cells (OPCs). Mechanisms of OPC differentiation have been extensively examined with two-dimensional cell culture systems. However, these cellular events may be more accurately represented using a three-dimensional (3D) model. In this study, we report the development of a novel 3D OPC culture system using gels composed of a mixture of collagen and hyaluronan, wherein cultured rat primary OPCs can proliferate and differentiate into oligodendrocytes. Our data show that the gel concentration and cell-seeding density are critical factors for the numbers of OPCs and oligodendrocytes in our 3D culture system. In addition, Notch signaling, which supports cell-to-cell communication, may also be important for OPC function in our system because a Notch inhibitor DAPT suppressed OPC proliferation and differentiation. Taken together, cultured rat OPCs can grow in collagen-/hyaluronan-based gels, and our novel 3D OPC culture system may offer a useful platform for examining the mechanisms of OPC function in vitro.


Assuntos
Técnicas de Cultura de Células/métodos , Células Precursoras de Oligodendrócitos/citologia , Animais , Astrócitos/citologia , Contagem de Células , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Géis , Células Precursoras de Oligodendrócitos/metabolismo , Porosidade , Ratos Sprague-Dawley , Receptores Notch/metabolismo , Transdução de Sinais
14.
Biofabrication ; 8(3): 035014, 2016 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-27563029

RESUMO

We presented a new quantitative analysis for cell and extracellular matrix (ECM) interactions, using cell-coated ECM hydrogel microbeads (hydrobeads) made of type I collagen. The hydrobeads can carry cells as three-dimensional spheroidal forms with an ECM inside, facilitating a direct interaction between the cells and ECM. The cells on hydrobeads do not have a hypoxic core, which opens the possibility for using as a cell microcarrier for bottom-up tissue reconstitution. This technique can utilize various types of cells, even MDA-MB-231 cells, which have weak cell-cell interactions and do not form spheroids in conventional spheroid culture methods. Morphological indices of the cell-coated hydrobead visually present cell-ECM interactions in a quantitative manner.


Assuntos
Técnicas de Cultura de Células/métodos , Colágeno Tipo I/química , Matriz Extracelular/metabolismo , Caderinas/metabolismo , Técnicas de Cultura de Células/instrumentação , Linhagem Celular , Sobrevivência Celular , Células Hep G2 , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Concentração de Íons de Hidrogênio , Técnicas Analíticas Microfluídicas/métodos , Microscopia de Fluorescência , Viscosidade
15.
Genes Cells ; 20(12): 1077-87, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26610870

RESUMO

Vascular calcification increases the risk of cardiovascular mortality. We previously reported that expression of elastin decreases with progression of inorganic phosphorus (Pi)-induced vascular smooth muscle cell (VSMC) calcification. However, the regulatory mechanisms of elastin mRNA expression during vascular calcification remain unclear. MicroRNA-29 family members (miR-29a, b and c) are reported to mediate elastin mRNA expression. Therefore, we aimed to determine the effect of miR-29 on elastin expression and Pi-induced vascular calcification. Calcification of human VSMCs was induced by Pi and evaluated measuring calcium deposition. Pi stimulation promoted Ca deposition and suppressed elastin expression in VSMCs. Knockdown of elastin expression by shRNA also promoted Pi-induced VSMC calcification. Elastin pre-mRNA measurements indicated that Pi stimulation suppressed elastin expression without changing transcriptional activity. Conversely, Pi stimulation increased miR-29a and miR-29b expression. Inhibition of miR-29 recovered elastin expression and suppressed calcification in Pi-treated VSMCs. Furthermore, over-expression of miR-29b promoted Pi-induced VSMC calcification. RT-qPCR analysis showed knockdown of elastin expression in VSMCs induced expression of osteoblast-related genes, similar to Pi stimulation, and recovery of elastin expression by miR-29 inhibition reduced their expression. Our study shows that miR-29-mediated suppression of elastin expression in VSMCs plays a pivotal role in osteoblastic differentiation leading to vascular calcification.


Assuntos
Elastina/genética , MicroRNAs/genética , Músculo Liso Vascular/metabolismo , Osteoblastos/efeitos dos fármacos , Fósforo/farmacologia , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Regulação para Baixo , Regulação da Expressão Gênica , Humanos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Osteoblastos/metabolismo , Calcificação Vascular/etiologia
16.
Genes Cells ; 20(12): 982-91, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26419830

RESUMO

Vascular calcification is known to reduce the elasticity of aorta. Several studies have suggested that autophagy-lysosomal pathway (ALP) in vascular smooth muscle cells (VSMCs) is associated with vascular calcification. A major component of oxidized low-density lipoproteins, 7-ketocholesterol (7-KC), has been reported to promote inorganic phosphorus (Pi)-induced vascular calcification and induce ALP. The aim of this study was to unravel the relationship between ALP and the progression of calcification by 7-KC. Calcification of human VSMCs was induced by Pi stimulation in the presence or absence of 7-KC. FACS analysis showed that 7-KC-induced apoptosis at a high concentration (30 µM), but not at a low concentration (15 µM). Interestingly, 7-KC promoted calcification in VSMCs regardless of apoptosis. Immunoblotting and immunostaining showed that 7-KC inhibits not only the fusion of autophagosomes and lysosomes but also causes a swell of lysosomes with the reduction of cathepsin B and D. Moreover, lysosomal protease inhibitors exacerbated the apoptosis-independent calcification by 7-KC although inhibition of autophagosome formation by Atg5 siRNA did not. Finally, the 7-KC-induced progression of calcification was alleviated by the treatment with antioxidant. Taken together, our data showed that 7-KC promotes VSMC calcification through lysosomal-dysfunction-dependent oxidative stress.


Assuntos
Cetocolesteróis/farmacologia , Lisossomos/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Calcificação Vascular/induzido quimicamente , Apoptose/efeitos dos fármacos , Catepsina B/metabolismo , Catepsina D/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lisossomos/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Fósforo/farmacologia , Calcificação Vascular/metabolismo
17.
J Atheroscler Thromb ; 22(10): 1091-9, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25972025

RESUMO

AIM: von Willebrand factor (VWF) plays an important role in the regulation of hemostasis and thrombosis formation, particularly under a high shear rate. However, the adhesive force due to the molecular interaction between VWF and glycoprotein Ibα (GPIbα) has not been fully explored. Thus, we employed atomic force microscopy to directly measure the adhesive force between VWF and GPIbα. METHODS: We measured the adhesive force between VWF and GPIbα at the molecular level using an atomic force microscope (AFM). An AFM cantilever was coated with recombinant N-terminus VWF binding site of GPIbα, whereas a cover glass was coated with native VWF. RESULTS: The adhesive force at the molecular level was measured using an AFM. In the presence of 1 µg/mL VWF, the adhesion force was nearly 200 pN. As per the Gaussian fit analysis, the adhesive force of a single bond could have been 54 or 107 pN. CONCLUSION: Our consideration with the Gaussian fit analysis proposed that the adhesive force of a single bond could be 54 pN, which is very close to that obtained by optical tweezers (50 pN).


Assuntos
Plaquetas/metabolismo , Hemostasia/fisiologia , Microscopia de Força Atômica/métodos , Complexo Glicoproteico GPIb-IX de Plaquetas/química , Trombose/sangue , Fator de von Willebrand/química , Plaquetas/ultraestrutura , Humanos , Estrutura Molecular , Complexo Glicoproteico GPIb-IX de Plaquetas/ultraestrutura , Trombose/diagnóstico , Fator de von Willebrand/ultraestrutura
18.
Artigo em Inglês | MEDLINE | ID: mdl-26736271

RESUMO

Construction of stable capillary networks is required to provide sufficient oxygen and nutrients to the deep region of thick tissues, which is important in the context of 3D tissue engineering. Although conventional in vitro culture models have been used to investigate the mechanism of capillary formation, recent advances in microfluidics technologies allowed us to control biophysical and biochemical culture environments more precisely, which led to the construction of functional and stable capillary networks. In this study, endothelial cells and mesenchymal stem cells were co-cultured in microfluidic devices to construct stable capillary networks, which resulted in the construction of luminal structures covered by pericytes. Interactions between endothelial cells and mesenchymal stem cells are also discussed in the context of capillary formation.


Assuntos
Capilares , Técnicas de Cocultura/instrumentação , Dispositivos Lab-On-A-Chip , Diferenciação Celular , Técnicas de Cocultura/métodos , Desenho de Equipamento , Células Endoteliais da Veia Umbilical Humana , Humanos , Células-Tronco Mesenquimais/citologia , Pericitos/citologia , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos
19.
J Tissue Eng Regen Med ; 9(3): 247-56, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23086892

RESUMO

In liver sinusoids, hepatic stellate cells (HSCs) locate the outer surface of microvessels to form a functional unit with endothelia and hepatocytes. To reconstruct functional liver tissue in vitro, formation of the HSC-incorporated sinusoidal structure is essential. We previously demonstrated capillary formation of endothelial cells (ECs) in tri-culture, where a polyethylene terephthalate (PET) microporous membrane was intercalated between the ECs and hepatic organoids composed of small hepatocytes (SHs), i.e. hepatic progenitor cells, and HSCs. However, the high thickness and low porosity of the membranes limited heterotypic cell-cell interactions, which are essential to form HSC-EC hybrid structures. Here, we focused on the effective use of the thin and highly porous poly( d, l-lactide-co-glycolide) (PLGA) microporous membranes in SH-HSC-EC tri-culture to reconstruct the HSC-incorporated liver capillary structures in vitro. First, the formation of EC capillary-like structures was induced on Matrigel-coated PLGA microporous membranes. Next, the membranes were stacked on hepatic organoids composed of small SHs and HSCs. When the pore size and porosity of the membranes were optimized, HSCs selectively migrated to the EC capillary-like structures. This process was mediated in part by platelet-derived growth factor (PDGF) signalling. In addition, the HSCs were located along the outer surface of the EC capillary-like structures with their long cytoplasmic processes. In the HSC-incorporated capillary tissues, SHs acquired high levels of differentiated functions, compared to those without ECs. This model will provide a basis for the construction of functional, thick, vascularized liver tissues in vitro.


Assuntos
Materiais Revestidos Biocompatíveis/química , Células Endoteliais/metabolismo , Células Estreladas do Fígado/metabolismo , Hepatócitos/metabolismo , Ácido Láctico/química , Membranas Artificiais , Ácido Poliglicólico/química , Animais , Células Cultivadas , Técnicas de Cocultura/métodos , Células Endoteliais/citologia , Células Estreladas do Fígado/citologia , Hepatócitos/citologia , Masculino , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Porosidade , Ratos , Ratos Sprague-Dawley
20.
J Biomed Mater Res B Appl Biomater ; 103(6): 1180-7, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25303239

RESUMO

This article presents the concept of an implantable cage system that can house and protect implanted biomedical sensing and therapeutic devices in the body. Cylinder-shaped cages made of porous polyvinyl alcohol (PVA) sheets with an 80-µm pore size and/or stainless steel meshes with 0.54-mm openings were implanted subcutaneously in the dorsal region of rats for 5 weeks. Analysis of the explanted cages showed the formation of fibrosis tissue around the cages. PVA cages had fibrotic tissue growing mostly along the outer surface of cages, while stainless steel cages had fibrotic tissue growing into the inside surface of the cage structure, due to the larger porosity of the stainless steel meshes. As the detection of target molecules with short time lags for biosensors and mass transport with low diffusion resistance into and out of certain therapeutic devices are critical for the success of such devices, we examined whether the fibrous tissue formed around the cages were permeable to molecules of our interest. For that purpose, bath diffusion and microfluidic chamber diffusion experiments using solutions containing the target molecules were performed. Diffusion of sodium, potassium and urea through the fibrosis tissue was confirmed, thus suggesting the potential of these cylindrical cages surrounded by fibrosis tissue to successfully encase implantable sensors and therapeutic apparatus.


Assuntos
Implantes Experimentais , Álcool de Polivinil , Aço Inoxidável , Telas Cirúrgicas , Animais , Fibrose/etiologia , Fibrose/fisiopatologia , Masculino , Porosidade , Ratos , Ratos Sprague-Dawley
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