Fibrinogen concentration influences fibrin microarchitecture and endothelial progenitor cells viability

Purpose: Biodegradable scaffolds for cell release are a key issue in cardiovascular tissue engineering. An ideal scaffold should have a nallometric fiber srrucrure to mimic the extracellu lar matrix and a suitable elasticity 10 direct cell lineage specification. Fibrin promotes the adhesion, proliferation and migration of several cells types. The variation of fibrin compo nents modulates the matrix elasticity and structure. Endothelial progeniror cells (EPC) are bone marrow cel ls contributing to neovascularization. No studies are ava ilable on different concentratio ns of fibrin components as scaffold for early EPC. Our aim was m study the effect of d ifferent concelll rations of fibrinogen (Pb) and thrombin (Th) on EPC viability. Methods: Fibrin was prepared mixing Fb (final 4.5 ~9· 18 ~36 mg/m\) and Th (final 6-12.5·25- 50 U/ml). Polymeri'l:uion r.ue was measured. Scaffolds were maintained for 1 hour at 3rC, 5% CO2 before cell seeding. EPC were obtained from peripheral blood of healthy donors and cu ltured for I week on fibrin (I x 1 If cell/cm2) in endOthelial growth medium containing 5% FBS and specific growth factors. Fibroncctin was used as control. Fibrin microarchirccture was investigated by scanning dectron microscopy (SEM); while. mechan ical properties (stiffness and elasticity) by atomic force microscopy (AFM). The effect of different fibrin scaffolds on cell metabolism was assessed after 7 days by a tetrazolium salr reduction (WST·I) assay. Results: Fibrin polymerization rate ranged between 17 and 68 s, in creasing at higher Th concentrations. SEM revealed a nanometric fib rous structure, with a decrease in fi ber diameter with higher Fb concentrations (4.5 mglml: 166±4 nm vs. 36 mg/ml: lI9±3 nm, p<O.005, n'"-5). Different Th concentrations didn't affect fibre diameter and density. Stiffness measures (4.5 mg/lll l: 0.0115 N/m vs 36/25 mg/ml: 0.2880 N/m, p<0.005. n= 5) revealed an elasticity decrease with higher Fb concentration. WSTI assay showed that EPe metabolic activity was higher with lower Fb conccntmions (4.5 mg/ml: 0.S90±0.134 a.u. vs. 36 mg/ml 0.234±0.046 3.u., p<0.05, n",5), corresponding to a scaffold with higher diameter fib rin prolOfibrils and reduced stiffness. Th had no signi fi cant effect. Conclusions: Fb concentration affects fibrin structural properties, where fibrin co ntaining lower Fb co ncentration has a more elastic structure. The di fferent EPe metabolic activity based on scaffold mechanical properties might be taken in account. Indeed the app ropri ate in vitro matrix conditions might partially overcome an inappropriate ill vivo micro environment during cellbased injection therapies.