Proliferation and β-tubulin for human aortic endothelial cells within gas-plasma scaffolds

Purpose: We determined if human aortic endothelial cells (HAEC) enhanced proliferative and angiogenic phenotypes within gas-plasma treated bioresorbable D,L-polylactic acid (D,L-PLA) three-dimensional scaffolds. Method: 6 × 10³ HAEC (N=120) were incubated for 6, 12 or 18 days within either non-treated control or treated scaffolds. Before removing media, unstained wells were observed for apparent cell densities. Quantitative colorimetric WST-1 mitochondrial assays were determined for pooled conditioned media from both HAEC attached to wells and their respective HAEC-containing scaffolds. Fixed HAEC in scaffolds were examined using non-quantitative laser confocal microcopy with FITC-conjugated consensus, Types-I/II or Type-III β-tubulin. Results: WST-1 indicated that significantly (p<0.05) less mitochondria were on cell culture plates than inside scaffolds but for different reasons. For example, a 12-18 days comparison between WST-1 and β-tubulin indicated that wells decreased because of overgrowth apotosis; whereas, mitochondrial activity inside treated scaffolds decreased with increased tubulogenesis. Observed with consensus and Type-I/II β-tubulin, HAEC-treated scaffolds exhibited increased cell-cell interconnections and angiogenic cords undergoing tubulogenesis to form vessels with central lumens as well as increased Type-III β-tubulin, predominantly in cells of smaller surface areas. Moreover, β-tubulin inside HAEC-treated scaffolds appeared in discrete cytoskeletal and podial regions; yet, β-tubulin for HAEC-control scaffolds was located in more diffuse cytoplasmic regions especially at 18 days. Conclusions: HAEC-treated scaffolds undergo increased migration, proliferation, β-tubulin expression and quiescent cord formation. HAEC in scaffolds represent a potential model to study mechanisms for vascular cord progression into tubes. WST-1 does not represent accurate cell densities in three-dimensional scaffold matrices.