Vascular endothelial and smooth muscle cell culture on NaOH-treated poly(epsilon-caprolactone) films: a preliminary study for vascular graft development.

Tissue engineering offers the potential of providing vessels that can be used to replace diseased and damaged native blood vessels. The endothelization of a synthetic vascular graft minimizes the failures associated with blood clotting and platelet activation. The aim of this study was to culture vascular-derived endothelial and smooth muscle cells on both untreated and NaOH-treated poly(epsilon-caprolactone) (PCL) films, a biocompatible and bio-resorbable polymer, and to evaluate the behavior of both cell types as a preliminary study for vascular graft development. PCL films were prepared by hot pressing; characterized by DSC, IR, SEM, and scanning force microscopy; and treated with NaOH to increase the surface hydrophilicity before cell culture. Endothelial and smooth muscle cells, isolated from pig cava vein, were characterized by immunofluorescence and confocal microscopy studies of endothelial nitric oxide synthase and alpha-smooth muscle actin. Good adhesion, growth, viability and morphology of both the endothelial and smooth muscle cells on PCL films were obtained, but a light stimulation of mitochondrial activity was observed during short culture times. NaOH treatment improved the adhesion and enhanced the proliferation in both cell types. This verified the possible use of this modified polymer as a support in the preparation of a synthetic vascular graft. [Diagram: see text] SEM micrograph of smooth muscle cells cultured on NaOH-treated PCL film. (Original magnification: 1000x).

Progenitor-derived endothelial cell response, platelet reactivity and haemocompatibility parameters indicate the potential of NaOH-treated polycaprolactone for vascular tissue engineering.

The haemocompatibility of NaOH-treated poly(ε-caprolactone) (PCL) has been evaluated in vitro by analysing several parameters, including plasma recalcification time, whole blood clotting time and platelet adhesion/activation. NaOH-treated PCL films showed a significant decrease in the clot formation speed and a reduced number of adhered platelets, which mainly exhibited non-activated morphologies. Furthermore, mature endothelial cells derived from peripheral endothelial progenitor cells were cultured on the polymer to investigate the effects of the endothelial lining on polymer haemocompatibility. Interestingly, cells cultured on NaOH-treated PCL films showed a significant stimulation of NO production. Although further research is required, NaOH treatment could be an interesting and simple strategy to modify PCL-based materials in order to enhance endothelial NO production, where compromised, and provide a better interaction of the scaffold with the blood components. In conclusion, these results reinforce the use of NaOH-treated PCL as a haemocompatible polymer for vascular tissue-engineering applications.

Nitric oxide production by endothelial cells derived from blood progenitors cultured on NaOH-treated polycaprolactone films: A biofunctionality study.

Poly(epsilon-caprolactone) (PCL) is a biodegradable polyester whose biocompatibility has been widely demonstrated both in vivo and in vitro. In the last few years, our group has confirmed that NaOH-treated PCL films can serve as a suitable biomaterial for vascular tissue engineering by supporting the culture of primary vascular cells and, more recently, endothelial-like EC(2) cells derived from endothelial progenitor cells (EPC). In the present study, NO production in basal conditions and after stimulation with different agents has been evaluated and related to the reactive oxygen species (ROS) content and the intracellular calcium levels on EC(2) cells cultured on NaOH-treated PCL films. The results obtained demonstrate that EC(2) seeded on NaOH-treated PCL films enhance the basal NO levels and show a faster, more intense response to physiological stimuli such as VEGF, bradykinin and thrombin than vein endothelial cells (ECv). This result could be indicative of a better capacity of EC(2) cells to maintain their endothelial functionality when seeded on polymers. On the other hand, the culture of both EC(2) and ECv cells on NaOH-treated PCL films induces a significant increase in both ROS content and intracellular calcium that is balanced out through the stimulation of NO production in these cells. In conclusion, these results demonstrate the ability of NaOH-treated PCL films to support endothelial cell production of nitric oxide and reinforce the idea of considering the endothelial-like EC(2) cells derived from blood progenitors as an adequate source of endothelial cells to functionalize vascular grafts. Furthermore, NaOH-treated PCL films could be considered as a promising cellular NO production-inducing biomaterial for vascular tissue engineering applications.