RESUMO
Extracellular nucleotide metabolism controls thrombosis and inflammation and may affect degeneration and calcification of aortic valve prostheses. We evaluated the effect of different decellularization strategies on enzyme activities involved in extracellular nucleotide metabolism. Porcine valves were tested intact or decellularized either by detergent treatment or hypotonic lysis and nuclease digestion. The rates of ATP hydrolysis, AMP hydrolysis, and adenosine deamination were estimated by incubation of aorta or valve leaflet sections with substrates followed by HPLC analysis. We demonstrated relatively high activities of ecto-enzymes on porcine valve as compared to the aortic wall. Hypotonic lysis/nuclease digestion preserved >80 % of ATP and AMP hydrolytic activity but reduced adenosine deamination to <10 %. Detergent decellularization completely removed (<5 %) all these activities. These results demonstrate high intensity of extracellular nucleotide metabolism on valve surface and indicate that various valve decellularization techniques differently affect ecto-enzyme activities that could be important in the development of improved valve prostheses.
Assuntos
Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Adenosina/metabolismo , Valva Aórtica/enzimologia , Bioprótese , Próteses Valvulares Cardíacas , Nucleotidases/metabolismo , Preservação de Tecido/métodos , Animais , Aorta/enzimologia , Valva Aórtica/citologia , Valva Aórtica/transplante , Cromatografia Líquida de Alta Pressão , Desaminação , Desoxirribonuclease I/metabolismo , Detergentes/química , Xenoenxertos , Hidrólise , Soluções Hipotônicas , Cinética , Ribonuclease Pancreático/metabolismo , Dodecilsulfato de Sódio/química , SuínosRESUMO
Herein we combine chemical and mechanical stimulation to investigate the effects of vascular endothelial growth factor (VEGF) and physiological shear stress in promoting the differentiation human adipose derived stem cells (ADSCs) into endothelial cells. ADSCs were isolated and characterized; endothelial differentiation was promoted by culturing confluent cells in 50 ng/ml VEGF under physiological shear stress for up to 14 days. Afterwards, endothelial cells were seeded onto collagen or acellular aortic valve matrices and exposed to four culture conditions: shear stress + VEGF; shear stress - VEGF; static + VEGF and static - VEGF. After 7 days, phenotype was investigated. ADSCs subjected to shear stress and VEGF express a comprehensive range of specific endothelial markers (vWF, eNOS and FLT-1 after 7 days and CD31, FLk-1 and VE-cadherin after 14 days) and maintain the phenotype when seeded onto scaffolds. Our protocol proved to be an efficient source of endothelial-like cells for tissue engineering based on autologous ADSC.
Assuntos
Adipócitos/citologia , Tecido Adiposo/patologia , Células Endoteliais/citologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Antígenos CD/metabolismo , Valva Aórtica/patologia , Caderinas/metabolismo , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas/citologia , Colágeno/metabolismo , Perfilação da Expressão Gênica , Humanos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fenótipo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Resistência ao Cisalhamento , Estresse Mecânico , Suínos , Engenharia Tecidual/métodos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator de von Willebrand/metabolismoRESUMO
Aortic valve (AV) performance is closely linked to its structural components. Glycosaminoglycans (GAGs) are thought to influence the time-dependent properties of living tissues. This study investigates the effect of GAGs on the viscoelastic behaviour of the AV. Fresh porcine AV cusps were either treated enzymatically to remove GAGs or left untreated (control). The specimens were tested for stress relaxation and tensile properties under equibiaxial load conditions. The stress relaxation curves were fitted using a double exponential decay equation and the early relaxation constant (τ(1)) and late relaxation constant (τ(2)) calculated for each specimen. Immunohistochemistry confirmed the successful depletion of both sulphated and non-sulphated GAGs from the AV cusps. A statistical increase in τ(1) was found for both the radial and circumferential directions between the control and -GAGs group (radial, control 17.37s vs. -GAGs 25.65 s; circumferential, control 21.47s vs. -GAGs 27.37 s). It was also found that τ(1) differed between the two directions for the control group but not after GAG depletion (control, radial 17.37s vs. circumferential 21.47 s; -GAGs, radial 25.65 s vs. circumferential 27.37s). No effect on stiffness was found. The results showed that the presence of GAGs influences the mechanical viscoelastic properties of the AV but has no effect on the stiffness. The natural anisotropy, which reflects the relaxation kinematics, is lost after GAG depletion.
Assuntos
Valva Aórtica , Fenômenos Biomecânicos , Glicosaminoglicanos/metabolismo , Animais , Imuno-Histoquímica , Suínos , TempoRESUMO
BACKGROUND: The extracellular matrix plays an important role in heart valve function. To improve the processing of porcine pulmonary valves for clinical use, we have studied the influence of cryopreservation, decellularization, and irradiation on extracellular matrix components. METHODS: Decellularization was carried out followed by DNAseI/RNAseA digestion and isotonic washout. Valves were cryopreserved in 10% DMSO/10% fetal bovine serum, and then subjected to 25-40 kGy γ-radiation. Extracellular matrix constituents were evaluated by histologic staining, immunohistochemistry, transmission electron microscopy, and liquid chromatography/mass spectrometry. RESULTS: Histologic, immunohistochemical, ultrastructural, and biochemical analyses demonstrated a marked reduction in the expression of extracellular matrix components particularly in the valves that had been γ-irradiated following decellularization and cryopreservation. In this group, histology and immunohistochemistry showed an obvious reduction in staining for chondroitin sulphates, versican, hyaluronan, and collagens. Transmission electron microscopy revealed the smallest fibril diameter of collagen, shortest D-period, and loss of compactness of collagen fiber packaging and fragmentation of elastic fibers. Biochemical analysis showed loss of collagen and elastin crosslinks. Decellularization followed by cryopreservation showed some reduction in staining for collagens and versican, smaller diameter, shorter D-period in collagen fibers, and ridges in elastic fibers. Cryopreservation alone showed minimal changes in ECM staining intensity, collagen, and elastin ultrastructure and biochemistry. CONCLUSION: γ-Irradiated valves that have been decellularized and cryopreserved produces significant changes in the expression of ECM components, thus providing useful information for improving valve preparation for clinical use and also some indication as to why irradiated human heart valves were not clinically successful.
Assuntos
Criopreservação/métodos , Matriz Extracelular/efeitos da radiação , Raios gama/efeitos adversos , Valva Pulmonar/efeitos da radiação , Valva Pulmonar/transplante , Animais , Colágeno/metabolismo , Seio Coronário/efeitos da radiação , Seio Coronário/ultraestrutura , Reagentes de Ligações Cruzadas/metabolismo , Elastina/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Glicosaminoglicanos/metabolismo , Humanos , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Miócitos de Músculo Liso/efeitos da radiação , Miócitos de Músculo Liso/ultraestrutura , Valva Pulmonar/ultraestrutura , Suínos , Transplante Heterólogo , Versicanas/metabolismoRESUMO
A key challenge in tissue engineering a heart valve is to reproduce the major tissue structures responsible for native valve function. Here we evaluated human adipose-derived stem cells (ADSCs) as a source of cells for heart valve tissue engineering investigating their ability to synthesize and process collagen and elastin. ADSCs were compared with human bone marrow mesenchymal stem cells (BmMSCs) and human aortic valve interstitial cells (hVICs). ADSCs and BmMSCs were stretched at 14% for 3 days and collagen synthesis determined by [(3)H]-proline incorporation. Collagen and elastin crosslinking was assessed by measuring pyridinoline and desmosine respectively, using liquid chromatography/mass spectrometry. Three-dimensional culture was obtained by seeding cells onto bovine collagen type I scaffolds for 2-20 days. Expression of matrix proteins and processing enzymes was assessed by Real Time-PCR, immunofluorescence and transmission electron microscopy. Stretch increased the incorporation of [(3)H]-proline in ADSCs and BmMSCs, however only ADSCs and hVICs upregulated COL3A1 gene. ADSCs produced collagen and elastin crosslinks. ADSCs uniformly populated collagen scaffolds after 2 days, and fibrillar-like collagen was detected after 20 days. ADSCs sense mechanical stimulation and produce and process collagen and elastin. These novel findings have important implications for the use of these cells in tissue engineering.
Assuntos
Tecido Adiposo/citologia , Matriz Extracelular/metabolismo , Próteses Valvulares Cardíacas , Células-Tronco/metabolismo , Engenharia Tecidual/métodos , Adulto , Aminoácidos/metabolismo , Forma Celular/efeitos dos fármacos , Colágeno/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Desmosina/metabolismo , Elastina/metabolismo , Matriz Extracelular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Pessoa de Meia-Idade , Fenótipo , Prolina/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/ultraestrutura , Estresse Mecânico , Alicerces Teciduais/químicaRESUMO
BACKGROUND AND AIM OF THE STUDY: Adipose tissue is a readily available source of multipotent adult stem cells for use in tissue engineering and regenerative medicine. Adipose-derived stem cells (ADSCs) are currently being investigated as a source of interstitial cells to populate tissue-engineered heart valve constructs. However, the ability of these cells to differentiate into endothelial cells that would be required to cover the surface of the valve cusps has not been fully investigated. METHODS: ADSCs were isolated and characterized using immunofluorescence and flow cytometry. Endothelial differentiation was promoted by culturing confluent cells in the presence of 2% fetal calf serum and 50 ng/ml vascular endothelial growth factor. Differentiation was evaluated by immunofluorescence staining for endothelial markers, and an analysis of acetylated low-density lipoprotein (Ac-LDL) uptake. An assessment of tubular formation was performed using an in vitro angiogenesis assay. RESULTS: Isolated ADSCs were positive for the mesenchymal markers CD105, CD73, CD29, CD90 and CD44, and negative for hematopoietic and endothelial markers. After a seven-day treatment period, approximately 15% of ADSCs expressed the endothelial marker von Willebrand factor, and 70% had lost the expression of smooth muscle a-actin. Treated cells also were able to incorporate Ac-LDL, and also to form tubular structures on Matrigel, unlike control cells. CONCLUSION: Based on these results, ADSCs are capable of differentiating into cells with phenotypic and functional features of endothelial cells. These predifferentiated cells provide new options for the tissue engineering of heart valves, based on autologous mesenchymal stem cells.
Assuntos
Células-Tronco Adultas/fisiologia , Diferenciação Celular , Células Endoteliais/fisiologia , Células-Tronco Mesenquimais/fisiologia , Gordura Subcutânea/fisiologia , Adulto , Células-Tronco Adultas/metabolismo , Animais , Transporte Biológico , Biomarcadores/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Citometria de Fluxo , Imunofluorescência , Regulação da Expressão Gênica , Humanos , Lipoproteínas LDL/metabolismo , Células-Tronco Mesenquimais/metabolismo , Microscopia de Fluorescência , Pessoa de Meia-Idade , Neovascularização Fisiológica , Fenótipo , Gordura Subcutânea/citologia , Gordura Subcutânea/metabolismo , Suínos , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto JovemRESUMO
OBJECTIVES: The aim of this study was to evaluate the role of valve endothelium in regulating the mechanical properties of aortic valve cusps. BACKGROUND: Mechanical properties of valve cusps are key to their function and durability; however, little is known about the regulation of valve biomechanics. METHODS: Mechanical properties of porcine aortic valve leaflets were evaluated in response to serotonin (5-hydroxytryptamine [5-HT]), with and without N-nitro-L-arginine-methyl-ester (L-NAME) or endothelial denudation, and endothelin (ET)-1, with and without cytochalasin-B. RESULTS: Under physiological loading conditions, 5-HT induced a decrease in the areal stiffness of the cusp (-25.0 +/- 4.0%; p < 0.01 vs. control), which was reversed by L-NAME or endothelial denudation (+17.5 +/- 5.3%, p = 0.07, and +14.7 +/- 1.8%, p < 0.05 vs. control, respectively). ET-1 caused an increase in stiffness (+34.4 +/- 13.8%; p < 0.05 vs. control), but not in the presence of cytochalasin-B (p = 0.29 vs. control). Changes in cusp stiffness were accompanied by aortic cusp relaxations to 5-HT (-0.29% +/- 0.1% change in load per 10-fold increase in 5-HT concentration; p = 0.03), which were reversed by endothelial denudation (+0.29 +/- 0.06% change in load per 10-fold increase in 5-HT concentration; p = 0.02) and by L-NAME (p < 0.05). Valve cusps contracted in response to ET-1 (+0.29 +/- 0.08% change in load per 10-fold increase in ET-1 concentration; p = 0.02), which was inhibited by cytochalasin-B. CONCLUSIONS: These data highlight the role of the endothelium in regulating the mechanical properties of aortic valve cusps and underline the importance of valve cellular integrity for optimal valve function.
Assuntos
Valva Aórtica/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Óxido Nítrico/fisiologia , Serotoninérgicos/farmacologia , Serotonina/farmacologia , Animais , Valva Aórtica/fisiologia , Endotélio Vascular/fisiologia , SuínosRESUMO
Elastin is a vital structural and regulatory matrix protein that plays an important role in conferring elasticity to blood vessel wall. Previous tissue engineering approaches to regenerate elastin in situ or within tissue engineering constructs are curtailed by innate poor elastin synthesis potential by adult vascular smooth muscle cells (SMCs). Currently, we seek to develop cellular cues to enhance tropoelastin synthesis and improve elastin matrix yield, stability, and ultrastructure. Our earlier studies attest to the elastogenic utility of hyaluronan (HA)-based cellular cues, though their effects are fragment size dependent and dose dependent, with HA oligomers deemed most elastogenic. We presently show transforming growth factor beta 1 (TGF-beta1) and HA oligomers, when provided concurrently, to synergistically and dramatically improve elastin matrix regeneration by adult vascular SMCs. Together, these cues suppress SMC proliferation, enhance synthesis of tropoelastin (8-fold) and matrix elastin protein (5.5-fold), and also improve matrix elastin yield (45% of total elastin vs. 10% for nonadditive controls), possibly by more efficient recruitment of tropoelastin for crosslinking. The density of desmosine crosslinks within the elastin matrix was itself attenuated, although the cues together modestly increased production and activity of the elastin crosslinking enzyme, lysyl oxidase. TGF-beta1 and HA oligomers together induced much greater assembly of mature elastin fibers than they did separately, and did not induce matrix calcification. The present outcomes might be great utility to therapeutic regeneration of elastin matrix networks in situ within elastin-compromised vessels, and within tissue-engineered vascular graft replacements.
Assuntos
Elastina/metabolismo , Matriz Extracelular/fisiologia , Ácido Hialurônico/farmacologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Regeneração/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Aminoácidos/análise , Animais , Proliferação de Células/efeitos dos fármacos , Elastina/genética , Elastina/ultraestrutura , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/ultraestrutura , Imunofluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Ácido Hialurônico/química , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Biossíntese de Proteínas , Estrutura Quaternária de Proteína , Proteína-Lisina 6-Oxidase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RatosRESUMO
BACKGROUND: Aortic valve calcification is a progressive process resembling ossification. Recent evidence indicates that the sympathetic nervous system plays an important role in regulating bone deposition and resorption through the beta2-adrenergic receptors (beta2-ARs). The aim of this study is to determine the level and pattern of expression of beta2-ARs in human valve interstitial cells (ICs) and assess their influence on differentiation of the cells into an osteoblast-like phenotype. METHODS AND RESULTS: Immunohistochemical analysis demonstrated a high expression of beta2-ARs, beta1-ARs, beta3-AR,s and receptor activator of nuclear factor-kappaB (RANK) in calcified aortic valves. The expression of beta2-ARs and beta1-ARs mRNA was assessed by real-time TaqMan PCR in cultures of human aortic valve ICs. Human valve ICs treated with the selective beta2-AR agonist, salmeterol, in the presence of osteogenic medium showed a significant 5-fold decrease in the alkaline phosphatase (ALP) activity in comparison to cells treated with osteogenic medium only (P<0.05). Immunocytochemical staining of the valve ICs showed a concomitant reduction in osteocalcin expression. In addition, other beta2-AR agonists caused a reduction in the protein expression of bone markers including ALP, Cbfa-1, and periostin. Human valve ICs treated with norepinephrine, in the presence of osteogenic medium, did not show a significant reduction in the ALP activity. CONCLUSIONS: These findings suggest an important role of the beta2-ARs in regulating valve calcification and may identify potential therapeutic targets.
Assuntos
Valva Aórtica/patologia , Valva Aórtica/fisiologia , Calcinose/patologia , Receptores Adrenérgicos beta/fisiologia , Adolescente , Agonistas Adrenérgicos beta/farmacologia , Agonistas Adrenérgicos beta/uso terapêutico , Idoso , Valva Aórtica/efeitos dos fármacos , Calcinose/tratamento farmacológico , Calcinose/genética , Criança , Humanos , Pessoa de Meia-IdadeRESUMO
BACKGROUND AND AIM OF THE STUDY: Cardiac valves perform highly sophisticated functions that depend upon the specific characteristics of the component interstitial cells (ICs). The ability of valve ICs to contribute to these functions may be related to the generation of different types of tension within the valve structure. The study aim was to characterize cellular morphology and the forces generated by valve ICs and to compare this with morphology and forces generated by other cell types. METHODS: Cultured human valve ICs, pericardial fibroblasts and vascular smooth muscle cells were seeded in 3-D collagen gels and placed in a device that accurately measures the forces generated. Cell morphology was determined in seeded gels fixed in glutaraldehyde, stained with toluidine blue and visualized using a high-definition stereo light microscope. RESULTS: Valve ICs generated an average peak force of 30.9 +/- 10.4 dynes over a 24-h period which, unlike other cell types tested, increased as cell density decreased (R = 0.67, p <0.0001). The temporal pattern of force generation in mitral valve cells was significantly faster than in aortic or tricuspid cells (p <0.05). Microscopic examination revealed the formation of cellular processes establishing a cell/cell and cell/matrix network. When externally induced changes in matrix tension occurred, the valve ICs unlike the other cell types - did not respond to restore the previous level of tension. CONCLUSION: Human cardiac valve ICs produce a specific pattern of force generation that may be related to the individual function of each heart valve. The specialized function of these cells may serve as a guide for the choice of candidate cells for tissue engineering heart valves.
Assuntos
Células do Tecido Conjuntivo/fisiologia , Valvas Cardíacas/citologia , Contração Miocárdica/fisiologia , Adolescente , Adulto , Idoso , Técnicas de Cultura de Células , Criança , Pré-Escolar , Matriz Extracelular/fisiologia , Feminino , Valvas Cardíacas/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Miócitos de Músculo Liso/fisiologia , Pericárdio/citologia , Veia Safena/citologia , Engenharia TecidualRESUMO
The cells that reside within valve cusps play an integral role in the durability and function of heart valves. There are principally two types of cells found in cusp tissue: the endothelial cells that cover the surface of the cusps and the interstitial cells (ICs) that form a network within the extracellular matrix (ECM) within the body of the cusp. Both cell types exhibit unique functions that are unlike those of other endothelial and ICs found throughout the body. The valve ICs express a complex pattern of cell-surface, cytoskeletal and muscle proteins. They are able to bind to, and communicate with, each other and the ECM. The endothelial cells on the outflow and inflow surfaces of the valve differ from one another. Their individual characteristics and functions reflect the fact that they are exposed to separate patterns of flow and pressure. In addition to providing a structural role in the valve, it is now known that the biological function of valve cells is important in maintaining the integrity of the cusps and the optimum function of the valve. In response to inappropriate stimuli, valve interstitial and endothelial cells may also participate in processes that lead to valve degeneration and calcification. Understanding the complex biology of valve interstitial and endothelial cells is an important requirement in elucidating the mechanisms that regulate valve function in health and disease, as well as setting a benchmark for the function of cells that may be used to tissue engineer a heart valve.
Assuntos
Valvas Cardíacas/citologia , Calcinose/metabolismo , Comunicação Celular , Tecido Elástico/citologia , Doenças das Valvas Cardíacas/metabolismo , Valvas Cardíacas/metabolismo , Transdução de SinaisRESUMO
A crucial step towards the goal of tissue engineering a heart valve will be the choice of scaffold onto which an appropriate cell phenotype can be seeded. Successful scaffold materials should be amenable to modification, have a controlled degradation, be compatible with the cells, lack cytotoxicity and not elicit an immune or inflammatory response. In addition, the scaffold should induce appropriate responses from the cells seeded onto it, such as cell attachment, proliferation and remodelling capacity, all of which should promote the formation of a tissue construct that can mimic the structure and function of the native valve. This paper discusses the various biological scaffolds that have been considered and are being studied for use in tissue engineering a heart valve. Also, strategies to enhance the biological communication between the scaffold and the cells seeded onto it as well as the use of bionanotechnology in the manufacture of scaffolds possessing the desired properties will be discussed.
Assuntos
Biotecnologia/métodos , Matriz Extracelular/metabolismo , Nanotecnologia/métodos , Engenharia Tecidual/métodos , Valva Aórtica/anatomia & histologia , Valva Aórtica/fisiologia , Materiais Biocompatíveis , HumanosRESUMO
BACKGROUND AND AIM OF THE STUDY: Human mesenchymal stem cells (MSCs) are a potential cell source for the tissue engineering of biological structures, including cardiac valves. A comprehensive, phenotypic analysis of MSCs and, for the latter, their comparison with valve interstitial cells (ICs) is therefore essential. METHODS: Isolates of bone marrow-derived human MSCs and human cardiac valve ICs were extensively phenotyped for their expression of membrane proteins involved in adhesion and cell-cell communication, cytoskeletal components, extracellular matrix (ECM) proteins and gene expression of WNT/FZD/SFRP/DKK/LRP family members. RESULTS: MSCs and valve ICs (>80%) expressed fibroblast surface antigen, smooth muscle alpha-actin, vimentin and CD44; expression of MHC class I and II and calponin was inconsistent, and a small proportion expressed desmin and smooth muscle myosin. CD105 was weakly expressed by a low percentage of valve ICs (<10%) compared to MSCs (>90%). ECM components made by both cell types demonstrated similar levels and patterns of staining, although expression of elastin was not detected by both cell types. Adhesion molecule expression was highly variable among the MSC isolates and between the two cell types, with the predominant integrins being alphal, alpha3, alpha5, and beta1 by both cell types. PCR analysis of WNT/FZD/SFRP/LRP family members revealed a greater range of the WNT family of genes being expressed in MSCs compared to ICs. CONCLUSION: The study results provided an extensive fingerprint of valve ICs and of MSCs for the tissue engineering of biological structures and for the manipulation of their desired phenotype. MSCs represent a promising cell type for valve tissue engineering, and will require extensive phenotyping after differentiation.
Assuntos
Valvas Cardíacas/citologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Células-Tronco Mesenquimais/fisiologia , Adulto , Idoso , Diferenciação Celular , Expressão Gênica , Humanos , Pessoa de Meia-Idade , Fenótipo , Engenharia TecidualRESUMO
To generate an ''off the shelf'' tissue-engineered heart valve, the cells would need to be of allogeneic origin. Here, we report the possibility of using human bone marrow-derived mesenchymal stem cells (MSCs) as a suitable allogeneic cell source for tissue-engineered heart valves. Proliferative responses of primary and primed CD4+ T cells to allogeneic MSCs were examined. A protein microarray system was used to detect soluble factors from supernatants collected from the T cell assays. MSCs are poor stimulators of primary and primed CD4+ T cell proliferation, despite provision of B7-1 trans-co-stimulation. MSCs not only directly inhibited primary and primed T cell responses to allogeneic peripheral blood mononuclear cells (PBMCs), but 24-h pre-culture of T cells with MSCs suppressed subsequent T cell proliferative responses to allogeneic PBMCs in a contact-dependent manner. Analysis of supernatants revealed a distinctly different cytokine profile after co-culture of T cells with MSCs than with PBMCs or endothelial cells. Pro-inflammatory Th1 cytokines interleukin (IL)-1alpha and beta, interferon (IFN)gamma, and tumor necrosis factor (TNF)alpha were downregulated, whereas, anti-inflammatory Th2 cytokines IL-3, IL-5, IL-10, and IL-13 and the Th2 chemokine I-309, a chemoattractant for regulatory T cells, were upregulated. Further analysis revealed that after co-culture with MSCs, the T cells exhibited a regulatory phenotype (CD4+ CD25(lo) CD69(lo) FoxP3+). MSCs downregulate T cell responses through direct contact and secretion of anti-inflammatory and tolerogenic cytokines, which may involve the recruitment of regulatory T cells. This implies that allogeneic MSCs could be a suitable cell source for tissue engineering a heart valve.
Assuntos
Anergia Clonal/imunologia , Valvas Cardíacas/imunologia , Isoanticorpos/biossíntese , Células-Tronco Mesenquimais/fisiologia , Células Th2/imunologia , Engenharia Tecidual , Células Cultivadas , Humanos , Células Th2/metabolismoRESUMO
Matrix remodeling, which involves proteolytic enzymes, such as the matrix metalloproteinases (MMPs), is of significant importance with respect to tissue engineering a heart valve construct. The ability of valve interstitial cells (ICs) to release these enzymes in biological scaffolds and to synthesize their own matrix has not been adequately studied, and this has important implications for tissue engineering. Cultured human aortic valve ICs were seeded onto a 3-dimensional type I collagen matrix for 28 days, whereby the presence of the remodeling enzymes, MMPs, were determined using immunohistochemistry, and detection of extracellular matrix (ECM) gene expression was performed using in situ hybridization. The collagenases, stromelysins, and membrane-type MMPs were expressed in 1%, 2%, and 5% collagen scaffolds after 28 days, whereas gelatinase expression was not observed. In situ hybridization revealed the presence of the ECM messenger ribonucleic acid (mRNA) in cells cultured in collagen scaffolds however, an increase in all three mRNAs was only detected in the 1% collagen scaffolds. The presence of collagenases, stromelysins, and membrane-type MMPs indicate that human valve ICs have the capacity to remodel type I collagen scaffold and that the genes necessary for synthesizing matrix have been turned on within the cells themselves. Scaffold composition also demonstrated differential effects onMMPexpression. These observations are of relevance with respect to the development of tissue-engineered heart valves.
Assuntos
Materiais Biocompatíveis , Bioprótese , Células do Tecido Conjuntivo/enzimologia , Proteínas da Matriz Extracelular/biossíntese , Matriz Extracelular , Metaloproteinases da Matriz/biossíntese , Valva Aórtica/citologia , Valva Aórtica/enzimologia , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Células do Tecido Conjuntivo/citologia , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Regulação Enzimológica da Expressão Gênica , Próteses Valvulares Cardíacas , Humanos , Engenharia Tecidual/métodosRESUMO
OBJECTIVE: The synthesis of appropriate extracellular matrix by cells in tissue engineered heart valve constructs will be important for the maintenance of valve cusp integrity and function. We have examined and compared the capacity of mesenchymal stem cells to synthesise collagen in response to stretch in comparison with native aortic valve interstitial cells. METHODS: Cells were stretched on a Flexercell FX4000 apparatus and total collagen synthesis was measured by the incorporation of [3H]-proline. The effect of stretch on gene expression of different collagen types was assessed by RT-PCR. RESULTS: There was a significant (p<0.01) increase in [3H]-proline incorporation into stretched valve cells at 10%, 14% and 20% stretch. The response of mesenchymal stem cells at 14% stretch was similar to that seen in the valve cells. Incorporation of [3H]-proline into soluble proteins in the cell media was significantly higher (p<0.01) only at 14% and 20% stretch in valve interstitial cells. These effects were shared with mesenchymal stem cells at 14% stretch. RT-PCR experiments demonstrated that 14% stretch up-regulated levels of mRNA for COL3A1 gene (type III collagen) but did not increase the expression of COL1A1 gene (type I collagen) in valve interstitial cells. However, both collagen genes could be detected in non-stretched and stretched mesenchymal stem cells. There was no evidence that the mesenchymal stem cells had started to adopt an osteoblastic cell phenotype in response to stretch. CONCLUSIONS: Collagen synthesis by valve interstitial cells is dependent upon the degree and duration of stretch. This response can be mimicked closely by exposure of mesenchymal stem cells to the same stretching profile. These properties could have important implications for the choice of cells and programme of conditioning with which to tissue engineer heart valves.
Assuntos
Valva Aórtica/metabolismo , Colágeno/biossíntese , Mecanotransdução Celular/fisiologia , Células-Tronco Mesenquimais/metabolismo , Adolescente , Adulto , Fosfatase Alcalina/metabolismo , Animais , Valva Aórtica/citologia , Células Cultivadas , Criança , Colágeno/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Pessoa de Meia-Idade , Fenótipo , Prolina/farmacocinética , RNA Mensageiro/genética , Estresse Mecânico , Suínos , Engenharia TecidualRESUMO
Rapid prototyping is a novel process for the production of scaffolds of predetermined size and three-dimensional shape. The aim of the study was to determine the feasibility of this technology for producing scaffolds for tissue engineering an aortic valve and the optimal concentration of collagen processed in this manner that would maintain viability and promote proliferation of human valve interstitial cells. Scaffolds of 1%, 2% and 5% w/v bovine type-I collagen were manufactured using rapid prototyping. Valve interstitial cells isolated from three human aortic valves were seeded on the scaffolds and cultured for up to 4 weeks. Cell viability was assessed using the CellTiter 96 Aq(ueous) One Solution Cell Proliferation Assay and cell death by lactate dehydrogenase (LDH) measurement. Valve interstitial cells remained viable and proliferated within the collagen scaffolds. Cells consistently proliferated to a greater extent on 1% collagen scaffolds rather than either 2% or 5% collagen and after 4 weeks reached 212+/-33.1%, 139+/-25.9% and 129+/-38.3% (mean+/-SD) of their initial seeding density on 1%, 2% and 5% collagen scaffolds, respectively. LDH analysis demonstrated that there was minimal cell death indicating that the collagen scaffold was not toxic to human valve interstitial cells. Rapid prototyping provides a route to optimize biological scaffold designs for tissue engineering cardiac valves. This technology has the versatility to create scaffolds that are compatible with the specific needs of the valve interstitial cells and should enhance cell viability, proliferation and function.
Assuntos
Valva Aórtica/citologia , Valva Aórtica/fisiologia , Materiais Biocompatíveis/química , Bioprótese , Colágeno Tipo I/química , Próteses Valvulares Cardíacas , Engenharia Tecidual/métodos , Animais , Apoptose , Bovinos , Técnicas de Cultura de Células/métodos , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Manufaturas , Teste de Materiais , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Projetos PilotoRESUMO
Mesenchymal stem cells (MSCs) are a promising candidate cell for tissue engineering. Magnetic resonance imaging (MRI) has been proven effective in visualizing iron-labeled stem cells; however, the efficiency of this approach for visualization of cells seeded on scaffolds intended for use as tissue-engineered heart valves has not been assessed. MSCs were labeled by incubating for 48 h with ferumoxide and poly-L-lysine as transfecting agent. Any detrimental effect of iron labeling on cell viability, proliferation, and differentiation was examined using appropriate functional assays. Change in the nuclear magnetic relaxation properties of labeled cells was determined using in vitro relaxometry of cells seeded in 3-dimensional collagen gels. Images of labeled and non-labeled cells seeded onto 1% type I bovine collagen scaffolds were obtained using MRI. The presence of intracellular iron in labeled cells was demonstrated using Prussian blue staining, confocal microscopy, and electron microscopy. Cell viability, proliferation, and differentiation were comparable in labeled and non-labeled cells. The T2 relaxation time was 40% to 50% shorter in ferumoxide-labeled cells. Labeled cells seeded on scaffolds appeared as areas of reduced signal intensity in T2 weighted images. Ferumoxide labeling persisted and remained effective even on scans performed 4 weeks after the labeling procedure. Ferumoxide labeling of human MSCs seeded on collagen scaffolds is an effective, non-toxic technique for visualization of these cells using MRI. This technique appears promising for cell tracking in future tissue-engineering applications.
Assuntos
Colágeno/química , Imageamento Tridimensional/métodos , Ferro , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Óxidos , Engenharia Tecidual/métodos , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Meios de Contraste , Dextranos , Óxido Ferroso-Férrico , Humanos , Imageamento por Ressonância Magnética , Nanopartículas de MagnetitaRESUMO
BACKGROUND AND AIM OF THE STUDY: The pattern of expression and distribution of extracellular matrix (ECM) components in human cardiac leaflets was analyzed. Additionally, interstitial cells (ICs) from the four different leaflets were isolated and studied. METHODS: Immunohistochemistry and immunocytochemistry were used for localization, and flow cytometric analysis to quantify the expression of specific markers on these ICs; the synthesis and expression of ECM components was assessed. RESULTS: Elastin was found predominantly on the inflow layer, but fine fibers were also present in the central and outflow layers. Collagen I was predominantly on the outflow layer but permeated throughout the leaflets. Collagen III was expressed ubiquitously. Proteoglycan expression was throughout the leaflet, but was predominant in the central layer. Fibronectin and vitronectin were expressed strongly in the inflow layer, moderately in the central layer, and weakly in the outflow layer. Biglycan expression was ubiquitous, with strong filamentous strands in the central layer. Keratan sulfate and decorin were ubiquitous. Chondroitin-4-sulfate and chondroitin-6-sulfate were strongly expressed in the outer layers, and laminin was restricted to the basal lamina of the endothelial cells. Cultured ICs showed synthesis and expression of various ECM components. CONCLUSION: This study of the pattern of expression of ECM components may provide a basis for a fingerprint on which to base future valve alternatives. The results provide useful information for valve tissue engineering and an understanding of the structural basis of some sophisticated functions of the valves.
Assuntos
Proteínas da Matriz Extracelular/biossíntese , Matriz Extracelular , Valvas Cardíacas/citologia , Valvas Cardíacas/metabolismo , Humanos , Imuno-Histoquímica , Microscopia Eletrônica , Pessoa de Meia-IdadeRESUMO
Cardiac valve interstitial cells (ICs) are a heterogeneous and dynamic population of specific cell types that have many unique characteristics. They are responsible for maintaining the extracellular scaffold that provides the mechanical characteristics vital for sustaining the unique dynamic behaviour of the valve. A number of cellular phenotypes can be distinguished: some are sparsely arranged throughout the valve leaflets, whilst others are arranged in thin bundles. These cells express molecular markers similar to those of skeletal, cardiac and smooth muscle cells (SMCs) and in particular, many ICs express smooth muscle (SM) alpha-actin, a marker of myofibroblasts. In this respect, these cells exhibit a profile unlike skin fibroblasts, which may allude to their role in valve function.