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Differentiation of mesenchymal stem cells on fibrin assemblies supported by immobilized growth factors FGF2 and VEGF
Musílková, Jana ; Filová, Elena ; Kaplan, Ondřej ; Bačáková, Lucie
Bioartificial heart valves and vascular grafts prepared from decellularized tissues could be recellularized with bone marrow-derived mesenchymal stem cells (MSCs) that are able to differentiate into both smooth muscle cells and endothelial cells. MSCs differentiation is facilitated by sustained release of growth factors. In our study assemblies based on fibrin, fibrin with heparin, fibrin with adsorbed or covalently-immobilized vascular endothelial growth factor A165 (VEGF) or basic fibroblast growth factor (FGF-2) via binding to heparin attached to fibrin have been prepared and were evaluated for their stimulation of MSCs differentiation. We estimated the mRNA expression of endothelial marker CD31 (PECAM1), smooth muscle marker α-actin (ACTA2), osteoblast markers osteocalcin (BGLAP) and alkaline phosphatase (ALP). The gene expression was estimated using RT-PCR on days 1, 7 and 21 after seeding. The cell morphology and viability was evaluated by LIVE/DEAD staining. VEGF, both adsorbed and covalently bound, increased significantly the expression of smooth muscle marker α-actin. The mRNA expression of ACTA2 on day 7 and 21 raised more than 200 times in comparison to control samples (undifferentiated cells before seeding). The ACTA2 gene expression significantly exceeded the expression of all other evaluated genes at all time intervals. Moreover, on day 21, the late smooth muscle marker desmin (DES) was steeply rising in cells cultivated on assemblies containing heparin and covalently bound VEGF. The expression of osteocalcin was minimal. We conclude that fibrin assembly containing covalently bound VEGF is the most convenient for MSCs differentiation towards smooth muscle cells.
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Application of nanofiber scaffolds for vesel regeneration
Bezděková, Dagmar ; Amler, Evžen (advisor) ; Holzerová, Kristýna (referee)
Although plenty of systems for vessel regeneration have been developed, no system is successful in small diameter (under 6 mm) vessel replacement yet. Synthetic materials, such as Dacron and ePTFE, have good results in large vessels replacement, but they cause thrombosis in small vessels. In addition, they are not degradable and do not allow a natural remodeling of the vessel system. Furthemore, endothelial cells, which are essential for creating natural antithrombogenic endothelium, do not adhere on these materials, as well as smooth muscle cells. Decellularized xenogenic material is the non-synthetic alternative for vessel regeneration. Appropriate detergent removes donor's cells and only extracellular matrix remains, which is able to host acceptor's cells. The main disadvantages of this system are difficulties with animal's nurture and structure violations after detergent is used. It appears that electrospun materials are the best alternative. The relatively simple process can be modified in many ways and provides then a scaffold, which mimics extracellular matrix. A big advantage of this process is the possibility to incorporate bioactive substances into a fiber. The substances serve there as an attractant for blood cells or as an anticoagulation factor. In combination with the progenitor cells seems...
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Application of nanofiber scaffolds for vesel regeneration
Bezděková, Dagmar ; Amler, Evžen (advisor) ; Holzerová, Kristýna (referee)
Although plenty of systems for vessel regeneration have been developed, no system is successful in small diameter (under 6 mm) vessel replacement yet. Synthetic materials, such as Dacron and ePTFE, have good results in large vessels replacement, but they cause thrombosis in small vessels. In addition, they are not degradable and do not allow a natural remodeling of the vessel system. Furthemore, endothelial cells, which are essential for creating natural antithrombogenic endothelium, do not adhere on these materials, as well as smooth muscle cells. Decellularized xenogenic material is the non-synthetic alternative for vessel regeneration. Appropriate detergent removes donor's cells and only extracellular matrix remains, which is able to host acceptor's cells. The main disadvantages of this system are difficulties with animal's nurture and structure violations after detergent is used. It appears that electrospun materials are the best alternative. The relatively simple process can be modified in many ways and provides then a scaffold, which mimics extracellular matrix. A big advantage of this process is the possibility to incorporate bioactive substances into a fiber. The substances serve there as an attractant for blood cells or as an anticoagulation factor. In combination with the progenitor cells seems...
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