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1.
PLoS One ; 12(9): e0185125, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28934329

RESUMO

Extracellular matrix plays a role in differentiation and phenotype development of its resident cells. Although cardiac extracellular matrix from the contractile tissues has been studied and utilized in tissue engineering, extracellular matrix properties of the pacemaking sinoatrial node are largely unknown. In this study, the biomechanical properties and biochemical composition and distribution of extracellular matrix in the sinoatrial node were investigated relative to the left ventricle. Extracellular matrix of the sinoatrial node was found to be overall stiffer than that of the left ventricle and highly heterogeneous with interstitial regions composed of predominantly fibrillar collagens and rich in elastin. The extracellular matrix protein distribution suggests that resident pacemaking cardiomyocytes are enclosed in fibrillar collagens that can withstand greater tensile strength while the surrounding elastin-rich regions may undergo deformation to reduce the mechanical strain in these cells. Moreover, basement membrane-associated adhesion proteins that are ligands for integrins were of low abundance in the sinoatrial node, which may decrease force transduction in the pacemaking cardiomyocytes. In contrast to extracellular matrix of the left ventricle, extracellular matrix of the sinoatrial node may reduce mechanical strain and force transduction in pacemaking cardiomyocytes. These findings provide the criteria for a suitable matrix scaffold for engineering biopacemakers.


Assuntos
Matriz Extracelular/metabolismo , Ventrículos do Coração/metabolismo , Nó Sinoatrial/metabolismo , Animais , Membrana Basal/química , Membrana Basal/metabolismo , Membrana Basal/ultraestrutura , Relógios Biológicos/fisiologia , Fenômenos Biomecânicos , Colágeno/metabolismo , Colágeno/ultraestrutura , Elasticidade , Elastina/metabolismo , Elastina/ultraestrutura , Matriz Extracelular/química , Matriz Extracelular/ultraestrutura , Fibronectinas/metabolismo , Fibronectinas/ultraestrutura , Imunofluorescência , Ventrículos do Coração/química , Ventrículos do Coração/ultraestrutura , Espectrometria de Massas , Microscopia de Força Atômica , Microscopia Eletroquímica de Varredura , Miócitos Cardíacos/química , Miócitos Cardíacos/metabolismo , Proteoma , Proteômica , Nó Sinoatrial/química , Nó Sinoatrial/ultraestrutura , Suínos , Resistência à Tração
2.
Stem Cells ; 34(11): 2670-2680, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27434649

RESUMO

Insights into the expression of pacemaker-specific markers in human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte subtypes can facilitate the enrichment and track differentiation and maturation of hiPSC-derived pacemaker-like cardiomyocytes. To date, no study has directly assessed gene expression in each pacemaker-, atria-, and ventricular-like cardiomyocyte subtype derived from hiPSCs since currently the subtypes of these immature cardiomyocytes can only be identified by action potential profiles. Traditional acquisition of action potentials using patch-clamp recordings renders the cells unviable for subsequent analysis. We circumvented these issues by acquiring the action potential profile of a single cell optically followed by assessment of protein expression through immunostaining in that same cell. Our same-single-cell analysis for the first time revealed expression of proposed pacemaker-specific markers-hyperpolarization-activated cyclic nucleotide-modulated (HCN)4 channel and Islet (Isl)1-at the protein level in all three hiPSC-derived cardiomyocyte subtypes. HCN4 expression was found to be higher in pacemaker-like hiPSC-derived cardiomyocytes than atrial- and ventricular-like subtypes but its downregulation over time in all subtypes diminished the differences. Isl1 expression in pacemaker-like hiPSC-derived cardiomyocytes was initially not statistically different than the contractile subtypes but did become statistically higher than ventricular-like cells with time. Our observations suggest that although HCN4 and Isl1 are differentially expressed in hiPSC-derived pacemaker-like relative to ventricular-like cardiomyocytes, these markers alone are insufficient in identifying hiPSC-derived pacemaker-like cardiomyocytes. Stem Cells 2016;34:2670-2680.


Assuntos
Potenciais de Ação/fisiologia , Átrios do Coração/metabolismo , Sistema de Condução Cardíaco/metabolismo , Ventrículos do Coração/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular , Linhagem da Célula/genética , Eletrofisiologia , Expressão Gênica , Átrios do Coração/citologia , Sistema de Condução Cardíaco/citologia , Ventrículos do Coração/citologia , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miócitos Cardíacos/citologia , Especificidade de Órgãos , Canais de Potássio/genética , Canais de Potássio/metabolismo , Análise de Célula Única/métodos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
J Biomed Mater Res B Appl Biomater ; 103(1): 39-46, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24757041

RESUMO

Within the past two decades polylactic-co-glycolic acid (PLGA) has gained considerable attention as a biocompatible and biodegradable polymer that is suitable for tissue engineering and regenerative medicine. In this present study, we have investigated the potential of PLGA, collagen I (ColI), and polyurethane (PU) scaffolds for ligament tissue regeneration. Two different ratios of PLGA (50:50 and 85:15) were used to determine the effects on mechanical tensile properties and cell adhesion. The Young's modulus, tensile stress at yield, and ultimate tensile strain of PLGA(50:50)-ColI-PU scaffolds demonstrated similar tensile properties to that of ligaments found in the knee. Whereas, scaffolds composed of PLGA(85:15)-ColI-PU had lower tensile properties than that of ligaments. Furthermore, we investigated the effect of fiber orientation on mechanical properties and our results indicate that aligned fiber scaffolds demonstrate higher tensile properties than scaffolds with random fiber orientation. Also, human fibroblasts attached and proliferated with no need for additional surface modifications to the presented electrospun scaffolds in both categories. Collectively, our investigation demonstrates the effectiveness of electrospun PLGA scaffolds as a suitable candidate for regenerative medicine, capable of being manipulated and combined with other polymers to create three-dimensional microenvironments with adjustable tensile properties to mimic native tissues.


Assuntos
Colágeno Tipo I/química , Ácido Láctico/química , Ligamentos , Ácido Poliglicólico/química , Poliuretanos/química , Engenharia Tecidual , Tecidos Suporte/química , Módulo de Elasticidade , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Teste de Materiais , Copolímero de Ácido Poliláctico e Ácido Poliglicólico
4.
J Biomed Mater Res B Appl Biomater ; 102(8): 1730-9, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24687591

RESUMO

We examined the effects of the microenvironment on vascular differentiation of murine cardiovascular progenitor cells (CPCs). We isolated CPCs and seeded them in culture exposed to the various extracellular matrix (ECM) proteins in both two-dimensional (2D) and 3D culture systems. To better understand the contribution of the microenvironment to vascular differentiation, we analyzed endothelial and smooth muscle cell differentiation at both day 7 and day 14. We found that laminin and vitronectin enhanced vascular endothelial cell differentiation while fibronectin enhanced vascular smooth muscle cell differentiation. We also observed that the effects of the 3D electrospun scaffolds were delayed and not noticeable until the later time point (day 14), which may be due to the amount of time necessary for the cells to migrate to the interior of the scaffold. The study characterized the contributions of both ECM proteins and the addition of a 3D culture system to continued vascular differentiation. Additionally, we demonstrated the capability bioengineer a CPC-derived vascular graft.


Assuntos
Diferenciação Celular , Microambiente Celular , Células Endoteliais/metabolismo , Miocárdio/metabolismo , Miócitos de Músculo Liso/metabolismo , Células-Tronco/metabolismo , Animais , Prótese Vascular , Células Cultivadas , Células Endoteliais/citologia , Matriz Extracelular/química , Camundongos , Miocárdio/citologia , Miócitos de Músculo Liso/citologia , Células-Tronco/citologia , Tecidos Suporte/química
5.
Biomaterials ; 33(7): 2032-40, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22169822

RESUMO

While stem cell niches in vivo are complex three-dimensional (3D) microenvironments, the relationship between the dimensionality of the niche to its function is unknown. We have created a 3D microenvironment through electrospinning to study the impact of geometry and different extracellular proteins on the development of cardiac progenitor cells (Flk-1(+)) from resident stem cells and their differentiation into functional cardiovascular cells. We have investigated the effect of collagen IV, fibronectin, laminin and vitronectin on the adhesion and proliferation of murine ES cells as well as the effects of these proteins on the number of Flk-1(+) cells cultured in 2D conditions compared to 3D system in a feeder free condition. We found that the number of Flk-1(+) cells was significantly higher in 3D scaffolds coated with laminin or vitronectin compared to colIV-coated scaffolds. Our results show the importance of defined culture systems in vitro for studying the guided differentiation of pluripotent embryonic stem cells in the field of cardiovascular tissue engineering and regenerative medicine.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Vitronectina/metabolismo , Animais , Materiais Biocompatíveis/metabolismo , Proliferação de Células , Células Cultivadas , Colágeno Tipo IV/metabolismo , Células-Tronco Embrionárias/citologia , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Coração/embriologia , Humanos , Laminina/metabolismo , Camundongos , Miocárdio/citologia , Engenharia Tecidual/métodos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
6.
J Biomed Mater Res B Appl Biomater ; 99(1): 180-90, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21732530

RESUMO

Electrospinning using synthetic and natural polymers is a promising technique for the fabrication of scaffolds for tissue engineering. Numerous synthetic polymers are available to maximize durability and mechanical properties (polyurethane) versus degradability and cell adhesion (polycaprolactone). In this study, we explored the feasibility of creating scaffolds made of bicomponent nanofibers from both polymers using a coaxial electrospinning system. We used a core of poly(urethane) and a sheath of a mixture of poly(ε-caprolactone) and gelatin, all dissolved in 1,1,1,3,3,3-hexafluror-2-propanol. These nanofibrous scaffolds were then evaluated to confirm their core-sheath nature and characterize their morphology and mechanical properties under static and dynamic conditions. Furthermore, the antigenicity of the scaffolds was studied to confirm that there is no significant foreign body response to the scaffold itself that would preclude its use in vivo. The results show the advantages of combining both natural and synethic polymers to create a coaxial scaffold capable of withstanding dynamic culture conditions and encourage cellular migration to the interior of the scaffold for tissue-engineering applications. Also, the results show that there is no significant immunoreactivity in vivo to the components of the scaffolds.


Assuntos
Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Reação a Corpo Estranho/imunologia , Nanofibras/química , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Técnicas Eletroquímicas/métodos , Gelatina/química , Implantes Experimentais , Teste de Materiais , Camundongos , Células NIH 3T3 , Nanofibras/ultraestrutura , Poliésteres/química , Polímeros/síntese química , Polímeros/química , Polímeros/metabolismo , Poliuretanos/química , Porosidade , Estresse Mecânico
7.
Biomaterials ; 32(11): 2748-56, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21257198

RESUMO

Stem or progenitor cell populations are often established in unique niche microenvironments that regulate cell fate decisions. Although niches have been shown to be critical for the normal development of several tissues, their role in the cardiovascular system is poorly understood. In this study, we characterized the cardiovascular progenitor cell (CPC) niche in developing human and mouse hearts, identifying signaling pathways and extracellular matrix (ECM) proteins that are crucial for CPC maintenance and expansion. We demonstrate that collagen IV (ColIV) and ß-catenin-dependent signaling are essential for maintaining and expanding undifferentiated CPCs. Since niches are three-dimensional (3D) structures, we investigated the impact of a 3D microenvironment that mimics the in vivo niche ECM. Employing electrospinning technologies, 3D in vitro niche substrates were bioengineered to serve as culture inserts. The three-dimensionality of these structures increased mouse embryonic stem cell differentiation into CPCs when compared to 2D control cultures, which was further enhanced by incorporation of ColIV into the substrates. Inhibiting p300-dependent ß-catenin signals with the small molecule IQ1 facilitated further expansion of CPCs. Our study represents an innovative approach to bioengineer cardiac niches that can serve as unique 3D in vitro systems to facilitate CPC expansion and study CPC biology.


Assuntos
Sistema Cardiovascular/embriologia , Células-Tronco Embrionárias/citologia , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Matriz Extracelular/metabolismo , Feminino , Citometria de Fluxo , Imunofluorescência , Coração/embriologia , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , Microscopia Confocal , Gravidez , beta Catenina/metabolismo
8.
Biomaterials ; 30(27): 4665-75, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19524289

RESUMO

Synthetic polymers or naturally-derived extracellular matrix (ECM) proteins have been used to create tissue engineering scaffolds; however, the need for surface modification in order to achieve polymer biocompatibility and the lack of biomechanical strength of constructs built using proteins alone remain major limitations. To overcome these obstacles, we developed novel hybrid constructs composed of both strong biosynthetic materials and natural human ECM proteins. Taking advantage of the ability of cells to produce their own ECM, human foreskin fibroblasts were grown on silicon-based nanostructures exhibiting various surface topographies that significantly enhanced ECM protein production. After 4 weeks, cell-derived sheets were harvested and histology, immunochemistry, biochemistry and multiphoton imaging revealed the presence of collagens, tropoelastin, fibronectin and glycosaminoglycans. Following decellularization, purified sheet-derived ECM proteins were mixed with poly(epsilon-caprolactone) to create fibrous scaffolds using electrospinning. These hybrid scaffolds exhibited excellent biomechanical properties with fiber and pore sizes that allowed attachment and migration of adipose tissue-derived stem cells. Our study represents an innovative approach to generate strong, non-cytotoxic scaffolds that could have broad applications in tissue regeneration strategies.


Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Nanoestruturas/química , Medicina Regenerativa/métodos , Tecido Adiposo/citologia , Bioensaio , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/ultraestrutura , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fluorescência , Humanos , Fótons , Poliésteres/farmacologia , Porosidade/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/ultraestrutura , Tecidos Suporte
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