Construction And Research Of Vascular Chip Based On Microfluidic Technology

At present,most of the endothelialization of vascular tissue engineering scaffolds is achieved by static culture of endothelial cells in vitro.However,there is little report on whether the endothelial layer of vascular scaffolds formed under static culture will be damaged under the shear effect of blood flow when they are transplanted into the body.Moreover,according to previous studies,the arrangement of endothelial cells on the surface of vascular tissue engineering scaffolds is different from those on corresponding transplant site.In order to study the integrity and orientation of the endothelial layer on the surface of endothelialized tissue-engineered vascular polycaprolactone(PCL)fibrous scaffolds in human arterial blood flow in vitro,a bionic removable blood vessel chip and its perfusion system were designed and constructed based on microfluidic technology.Further,the constructed vascular chip was applied to the preliminary evaluation of a novel paclitaxel(PTX)/L-polylactic acid(PLLA)nanoparticle sustained-release drug.The 3D modeling and numerical simulation technology were used to optimize the early design of the vascular chip structure,and the various modular components of the chip were prepared through a variety of processing techniques such as computer numerical control(CNC)engraving,electrostatic spinning,and thermosetting molding.After that,the all parts of the chip were fastened by screws to realize the repeated disassembly of the chip.The chip and its perfusion system were assembled as a whole.According to the numerical simulation calculation,when the chip inlet velocity was 0.75 m/s,the width of single grid channel of vascular chip was 0.8 mm,and the channel depth was 0.5 mm,the fluid above the PCL fibrous scaffold met the blood flow parameters of human artery,which indicated that the internal fluid environment of the vascular chip designed in this thesis was close to the human artery microenvironment.The results showed that the machining accuracy of CNC engraving,the diameter and the arrangement of the fibers in the electro-spun PCL fibrous scaffold met the expected requirements;the chip packaging film met the requirements for chip sealing and biocompatibility;and the perfusion system with good sealing performance met the requirements of the next experiment.The endothelialized PCL fibrous scaffold and other components were aseptically assembled and operated to realize the construction of the vascular chip and its perfusion system.Then,immunofluorescence staining,CCK-8 detection and other analysis were applied to investigate whether statically cultured endothelialized the aligned PCL fibrous scaffold and the random PCL fibrous scaffold would have local endothelial damage under arterial blood flow conditions.And the orientation of endothelial cells on the two PCL fibrous scaffolds was explored.The experimental results showed that when statically cultured endothelialized aligned PCL fibrous scaffold and random PCL fibrous scaffolds were directly exposed to the arterial blood flow environment(Pressure=70 mm Hg,Velocity=0.5 m/s,Shear stress=50dynes/cm~2)in vitro,local endothelial damage occurred on the surface of both scaffolds.Furthermore,the method of slowly upgrading from low-shear perfusion(20 dynes/cm~2)culture to high-shear perfusion(50 dynes/cm~2)culture was used to achieve the integrity and stability of the aligned PCL fibrous scaffold in static culture endothelialization in the arterial-like blood flow in vitro.In addition,it was demonstrated that the arrangement of endothelial cells on the surface of the fibrous scaffold was closer to the oriented arrangement of endothelial cells in normal arteries under the combined effect of the contact guidance effect and the flow-mediated mechanotransduction.According to the conditions of drug release in vitro,some parts of the chip were replaced and the operation conditions of the device were optimized under the condition of numerical simulation.PTX/PLLA nanoparticles were initially evaluated by the updated chip.The main parameters,such as dispersion,drug loading rate and drug cumulative release in vitro,were characterized by optical microscope and high performance liquid chromatography(HPLC).The cumulative release curve of paclitaxel was fitted with various release models to clarify the release mechanism of nanoparticles.The experimental results showed that the nanoparticles formed by coating the PTX with PLLA enabled the water-insoluble PTX to be stably dispersed in an aqueous environment at a relatively large concentration.Compared with PTX,it showed a sustained release time and a gentle drug release in PTX/PLLA nanoparticles.It achieved a sustained low-dose release of PTX,which was a cell cycle specific anticancer drug.The release model fitting results showed that the release of PTX/PLLA nanoparticles was a synergistic effect of the diffusion of PTX and the degradation of PLLA,and the diffusion mechanism was the main part in the early stage of drug release.