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Fibrin as scaffold for endothelial progenitor cells: influence of fibrinogen and thrombin ratio
DI STEFANO ROSSELLA
BARSOTTI MARIA CHIARA
- tipo prodotto
- Abstract su Index Medicus e/o Science Citation Index
- GIORNALE ITALIANO DI CARDIOLOGIA
- numero pagine
- pagina iniziale
- volume/numero rivista
- Suppl. 1 al n. 12),
- Aims: Human peripheral blood endothelial progenitor cells (EPC) are promising current therapies for irreversible myocardial damage, heart failure and peripheral ischemia disease. Natural biopolymers such as human fibrin are appealing in tissue engineering, because fibrin is biocompatible, bioresorbable and essential in normal wound healing. In vitro studies indicate that fibrin can support the growth migration and proliferation of several cells types, including endothelial cells, fibroblasts and mesenchymal stem cells. Up to date numerous studies have proved the potential of fibrin based injectable cell delivery systems. No studies are available with fibrin as scaffold for EPC.
The goal of this study was to investigate whether
1) fibrin is a suitable matrix for EPC culture as compared with fibronectin
2) different concentrations of fibrinogen (Fb) and thrombin (Th) can influence the fibrin scaffold structure and the behaviour of EPC.
Methods: Fibrin scaffolds (Kedrion S.p.a. Lucca, Italy) were prepared mixing an aqueous solution of Fb (final 4.5-9-18-36 mg/ml) and an aqueous solution of Th (final 6-12.5-25-50 U/ml) containing CaCl2. All gels were visually examined to assess turbidity and shrinkage after polymerization as well as degradation and/or dissolution over time. Polymerization rate was measured. The scaffolds were maintained for 1 hour at 37°C, 5 CO2 before cell seeding. The ultrastructure of fibrin was investigated by scanning electron microscopy (SEM), cryogenic SEM (CRYO-SEM) and atomic force microscopy (AFM) that allow the hydratating analysis of the sample, to evaluate fibre diameter and density. EPC were obtained from peripheral blood of healthy donors and cultured for 1 week on fibrin scaffold at the concentration of 1x106 cell/ml in endothelial growth medium containing 5 FBS and specific growth factors. EPC seeded on fibronectin were used as control. Metabolic cell activity on the different scaffolds was assessed after 7 and 14 days by using a tetrazolium salt reduction (WST1) while cell viability by confocal microscopy (Calcein AM incorporation).
Results: Fibrin polymerization rate ranged between 17 and 68 seconds and increased at higher Fb or Th concentrations. Both AFM and SEM analysis revealed a nanometric fibrous structure, with a decrease in fiber diameter with higher fibrinogen concentrations (4.5 mg/ml: 166±4 nm. vs. 36 mg/ml: 119±3 nm, p