Fibrin gel: a new scaffold for cardiovascular applications

Abstract

Aims: Peripheral blood endothelial progenitor cells (EPC) are promising therapies for irreversible myocardial damage, heart failure and peripheral ischemia disease. Natural biopolymers as fibrin are appealing in tissue engineering, because fibrin is biocompatible and bioresorbable. In vitro studies indicate that fibrin can support the growth migration and proliferation of several cells types. 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 if fibrin is a suitable matrix for EPC culture as compared with fibronectin and if different concentrations of fibrinogen (Fb) and thrombin (Th) can influence fibrin structure and EPC behaviour. Methods: Fibrin (Kedrion S.p.a. Lucca, Italy) was prepared mixing Fb (final 4.5-9-18-36 mg/ml) and Th (final 6-12.5-25-50 U/ml). 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 at the concentration of 1x106 cell/ml in endothelial growth medium. EPC seeded on fibronectin were used as control. Metabolic cell activity on the different scaffolds was assessed after 7 and 14 days by 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<0.005, n=5). Different concentrations of Th didn\u27t affect fibre diameter and density. CRYO-SEM suggested a reticulate structure with mesh-size up to 10?m. WST1 assay showed that EPC metabolic activity was better with lower fibrinogen concentrations (4.5 mg/ml: 0.890?0.134 a.u. vs. 36 mg/ml 0.234?0.046 a.u., p<0.05, n=5), while Th had no significant effect. Calcein staining demonstrated that EPC were viable at 14 days and even organised in cluster. Conclusions: Fibrin combines important properties of an ideal biological scaffold, like the nanometric structure, important for the growth and migration of cells. Fibrin is also an ideal scaffold for EPC but the ratio between fibrinogen and thrombin is important for cell viability