Experimental study of affinity of endothelial outgrowth cells cultured on aligned plla nanofibrous scaffolds

Abstract

Objective Tissue engineering holds great promise in providing vascular grafts as substitutes for damaged small-di-ameter blood vessels. Two of the key factors in vascular tissue engineering are biocompatible scaffolds that mimic the effects of extra-cellular matrix and the source of seeding cells. To study the cellular affinity and adhesion and proliferation of endothelial outgrowth cells (EOCs) obtained from rabbit peripheral blood cultured with the aligned synthetic poly-L-lactic acid(PLLA) nanofibers in vitro so as to provide a solid foundation for future vascular tissue engineering involving both PLLA and EOCs. Methods The random, aligned and super-aligned PLLA nanofibers were prepared by an insulated high-speed roller collector covered with parallel steel sticks rotating at 0 rpm, 1000 rpm and 2500 rpm, respectively. Finally, the electorspun PLLA nanofibers were modified by hypothermy plasma and type Ⅰ collagen was coated onto the materials physically. The resulting rabbit peripheral blood mononuclear cell (PBMC) were collect-ed and resuspended in endothelial basal medium (EBM-2) supplemented with EBM-2-SingleQuots, which were continually cultured with complete EBM-2 medium for 3 weeks. To confirm EOC generation during culturing, morphological changes of adherent cells were visualized with Olympus phase-contrest microscopy. The attached Cells were either incubated with Ac-LDL that was labeled with the fluorochrome DiI or incubated with FTTC- conjugated UEA-I. Incorporation of DiI-labelled Ac-LDL and binding of FTTC-UEA-I was determined by fluorescent microscopy. First passage EOCs were seeded onto sterilized PLLA nanofiber scaffolds in 96-well tissue cul-ture plates. To determine the growth curves, cells were taken from 4 randomly chosen wells for growth each condition every other day from day 3 until day 17. Cells were incubated in MTT (Thiazolyl blue) and subsequently resuspended in DMSO(Dimethyl sulfoxide) with shaking. The absorbance A representing cell density in each well was measured with a ELISA immune detector at a wavelength of 490 nm. Third passage cell adherency and proliferation were also investigated directly on the PLLA scaffolds. After incubation for 4 hours, 12 hours, 24 hours, 3 days, and 7 days, respectively, PLLA nanofiber scaffolds were randomly taken from 3 wells for each condition. Cells attached to the PLLA scaffolds were then treated with 25% trypsin-EDTA mixture and counted. After incubation for 48h, the third passage, cells adherent to PLLA scaffolds labeled with CM- DiI were observed with fluorescent microscopy. After incuba-tion for 72 hours,cells were fixed with 2.5% glutaraldehyde and 30% - 100% gradient ethanol dehydration. Samples were critical point dried, sputtered with gold, and observed with scanning electron microscope, Results Anisotropic electrospinning is an excel-lent technique for fabricating oriented fibers with a high length/diameter ratio and well-defined configuration. The diameters of these fi-bers ranged from 300 nm to 400 nm. Their porosities were more than 90%. Colony-forming cells with cobblestone-like morphology were designated as EOCs according to their endothelial morphology and their ability to uptake of ac-LDL and to bind UEA-I (Dual pos-itive). Although random PLLA scaffolds did not have detectable effect on the growth EOCs, both aligned and super-aligned PLLA nanofibrous scaffolds significantly enhanced their growth (P < 0.05). These results indicate that not only the PLLA scaffolds are bio-logically compatible with the EOCs, the aligned nanofibers actually promote growth of EOCs. This conclusion was further corroborated by the observation with EOCs grown on and attached to PLLA scaffolds. Adherency of EOCs was completed during 12 hours incuba-tion. Both aligned and super-aligned PLLA nanofibrous scaffolds was significantly higher than the rate of adherency and proliferation of random PLLA scaffolds after 12 hours incubation (P <0.01). The rate of proliferation after 24 hours incubation between aligned and super-aligned PLLA nanofibrous scaffolds was also significant (P<0.05). When grown in composite cultures, we found that EOCs maintained the spindle-shaped morphology in the presence of all types of PLLA scaffolds. Importantly, cell orientations correlated with the structures of the scaffolds-they were randomly oriented in the presence of random scaffolds and aligned reasonably well along the aligned and super-aligned scaffolds EOCs. We also observed intimate intercellular contacts among adjacent cells, suggesting the forma-tion of tight endothelial cell layers surrounding the fibers of the scaffolding material. Conclusion EOCs can be adhere well to aligned and super-aligned scaffolds of PLLA and proliferate, keep well on cell morphology. EOCs are ideal seeding ceils for vascular tissue en-gineering. We found that PLLA nanofibers are not only biocompatible with EOCs, the aligned PLLA fibers actually promoted and guid-ed their sustained proliferation. These results suggest that aligned PLLA could be excellent both as the scaffolds and as a promoter of cell growth during vascular tissue engineering. Key words: Tissue engineering; Blood vessel prosthesis; Stents; Super-aligned nanofibers; Endothelial out-growth cells