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Mineralised polylactide and polycaprolactone soft foams with hierarchical micro-macro porous structure for tissue engineering
Beran, M. ; Berková, E. ; Musílková, Jana ; Sedlář, Antonín ; Slepička, P. ; Fajstavr, D.
The purpose of the study was development of soft foams from resorbable polymers with unique micro-macro porous interconnected hierarchical structure specially designed as scaffold for engineering of soft tissues. The foams have been prepared by freeze-drying of solutions of polylactide (PLA) and polycaprolactone (PCL) in 1,4-dioxane. The foams prepared by freeze-drying had interconnected porous aerogel characteristics. The hierarchical structure with bimodal micro-macro pore size distribution were obtained after addition of sucrose or NaCl porogens with defined crystal size distributions to the solutions before freeze-drying and leaching the porogen crystals from the freeze-dried foams with demineralised water. Polyethyleneimine was chemically conjugated to the alkali-treated foams followed by conjugation of citric acid using carbodiimide chemistry. Finally, they were mineralised by immersing and incubating in a simulated body fluid with ionic concentration similar to that of human blood plasma, to obtain tissue engineering scaffolds. To verify their biocompatibility, the scaffolds were seeded with adipose-derived stem cells (ASC) and sarcoma osteogenic-2 (SaOs-2) human osteoblast-like cells. Morphology of the cells attached to the scaffolds was evaluated and their viability was verified by a metabolic test. Biocompatibility and usability of the scaffolds was successfully verified by incubation with adipose-derived stem cells and SaOs-2 human osteosarcoma cell line. Mineralised scaffolds are more suitable growth supports for both the cell types than unmineralized collagen scaffolds. The scaffolds have been specially designed for engineering of soft tissues, but they can be used in other categories of tissue engineering, too.
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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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