Quantification of Cell Oxygenation in 2D Constructs of Metallized Electrospun Polycaprolactone Fibers Encapsulating Human Valvular Interstitial Cells

The present study brings developments in the area of tissue engineering of heart valves, with the design and application of a new 2D model for human heart valve. This model contains valvular interstitial cells (VIC) encapsulated in gold covered electrospun polycaprolactone (PCL/Au) fibers, the latter serving also as an electrochemical sensor that enables the monitoring of oxygen levels of cellular media. The biocompatibility of PCL and PCL/Au with VIC was evaluated after the encapsulation of VIC in both scaffolds using the lactate dehydrogenase assay. Fluorescence microscopy of the encapsulated cells was used for the investigation of cell morphology, by employing phalloidin labelled F-actin (red) and DAPI nuclear staining (blue). The electrochemical techniques allowed to investigate the effect of cell encapsulation within the PCL/Au 2D constructs and to quantify the dissolved oxygen levels in cell culture media at both PCL/Au and PCL/Au with encapsulated cells (PCL/ Au/cells). Experiments were performed in a hypoxistation, which allowed a fine control on the cellular media oxygenation. Electrochemical impedance spectroscopy (EIS) was used to evaluate the electrochemical behavior of the 2D constructs, PCL/Au and PCL/Au/cells, in cellular media at different O-2 concentration. Results were analyzed and both impedance values at a fixed frequency and values of the equivalent circuit elements were used to construct calibration curves for dissolved O-2 determination. The second electrochemical technique used to monitor the levels of O-2 at PCL/Au and PCL/Au/cells in the cellular media was square wave voltammetry with sensitivities of 1.5 and 2.5 mu A cm(-2) mu M-1, for PCL/Au and PCL/Au/cells, respectively.