Preparation Of Bilayer Small-diameter Nanofibrous Vascular Scaffolds By Phase Separation And Their Biological Evaluation

Thermally induced phase separation (TIPS) technology is a facile way to fabricate three-dimension (3D) nanofibrous scaffold for tissue engineering. In TIPS, a homogeneous polymer solution with the initial high temperature was driven to separate into a multi-phase system (such as polymer-rich phase and polymer-lean phase) by fast-freezing the solution. Then a nanofibrous scaffold was obtained after solvent exchanging and freeze-drying. TIPS is considered to be a promising approach to fabricate the3D nanofibrous scaffold for tissue engingeering, because the obtained scaffold possesses nanofibers ranging from50to500nm in diameter and has a porosity of exceed90%in combination with porogen leaching method. In this paper, a bilayer tubular scaffold was prepared by using TIPS. The main contents are as follows:Part1:The PLLA/PCL, PLLA/PS, PLLA/PLCL, PLLA/PLGA composite scaffold were prepared by using TIPS, and the effects of different polymer mixture system on the pore-forming were studied to explore the mechanism of "self-porogen" method. The SEM results suggested that PLLA/PCL, PLLA/PS composite scaffolds had the macroporous nanofibrous structure, while the PLLA/PLCL, PLLA/PLGA composite scaffolds had no macroporous structure. Further studies demonstrated that all of the PLLA/PCL, PLLA/PS mixture solutions with the concentration of10%(w/v) were immiscible at60℃, while PLLA/PLCL, PLLA/PLGA mixture solutions were miscible at the same condition. This result indicated that the pore-forming of "self-porogen" method was related to the immiscible nature of polymer mixture system. Besides, the concentration of polymer mixture solution affected its miscibility. For example, the PLLA/PCL mixture solution of6%was miscible, however, it became immiscible when the concentration increased (e.g.,8%,10%and12%). Furthermore, ternary PLLA/PLCL/PCL, PLLA/PLCL/PS and PLLA/PLGA/PCL composite scaffolds also possessed the macroporous nanofibrous structure. Taken together, this "self-porogen" method based on TIPS holds the great potential to fabricate3D macroporous nanofibrous scaffold for tissue engineering application.Part2:A bilayer small-diameter tubular scaffold, which inner layer of its tubular wall was PLLA/PLCL composite material and outer layer was PLLA/PCL composite material, was fabricated by using a two-step TIPS. The SEM images showed the hierarchical distribution of macroporosity on the tubular wall of bilayer scaffold, which possessed dense nanofibrous structure in the inner layer and macroporous nanofibrous structure in the outer layer. Also, we studied the effect of the introduction of PLCL on the PLLA/PLCL composite scaffold. When PLCL content increased to60%, the average fiber diameters of scaffolds decreased from317to205nm, which closely mimic the nano-structure of native extracellularmatrix (ECM). However, a sharp increase to695nm in average fiber diameter and severe shrink in wall thickness when PLCL content reached to70%. In addition, the elastic property of PLLA/PLCL scaffolds was the most optimal when the PLCL content was60%, so PLLA/PLCL40:60composite material was chosen as the inner layer of bilayer scaffold. While PLLA/PCL70:30composite material was selected for the outer layer, because such scaffold possessed macroporous structure with average pore size in38.4±19.3μm, a maximum strain of12.93±1.94%and nanofibers with average diameter in288.1±94.3nm. Furthermore, the tensile mechanical tests indicated that the bilayer scaffolds had the better mechanical properties than the single PLLA/PLCL scaffolds and PLLA/PCL scaffolds. The tensile strength and ultimate strain were1.310±0.153MPa and117.5±50.1%respectively, which was comparable to the coronary artery of human being.Part3:In vitro tests suggested that PLLA/PLCL scaffolds can facilitate the adhesion, proliferation and growth of pig iliac endothelial cells (PIECs). The heparinized modification had no significant effect on the behavior of PIECs. Besides, PLLA/PCL scaffolds can also promote the adhesion of human vascular smooth muscle cells (HVSMCs), and their macropores were large enough for permeation of HVSMCs. In vivo tests showed that the cells infiltrated into the PLLA/PCL70:30scaffolds after implanted subcutaneously in Sorague-Dawley (SD) rats for eight weeks. Furthermore, a bilayer tubular scaffold with heparinized inner structure of wall was implanted in the jugular vein of New Zealand white rabbit. The result showed that the scaffold was patent after four weeks post-implantation. Therefore, such bilayer tubular scaffolds have the great potential for vascular tissue engineering application.