Construction Of The Microvascular System Based On The Sacrificial Materials Alginate And Its Application In Liver Tissue Engineering Research

With the development of tissue engineering technology,liver-like tissue fabricated by the hepatocytes and biomaterial in vitro,provide a promising therapy for the hepatopathy.However,there are still two major problems in the present study of liver tissue engineering.One is the hepatocytes polarity losing cultured in vitro,and the other is cell necrosis caused by material transfer restrictions.According to the structure of the liver sinus,this research constructed a hepatoid based on the hepatocytes,vascular endothelial cells and gel matrix materials in order to explore and solve the core problem in liver tissue engineering.Specifically,the alginate was used as sacrificial material,through microfabrication,surface modification,embedding,gelation and other operations,to construct a microfluidic channel system based on gel matrix.The human umbilical vein endothelial cells?HUVECs?was cultivated in the microfluidic channel and incubated in 3D pattern forming an endothelium layer,which was similar to the vascular wall in vivo.And on this basis,an orderly co-culture system was formed by loading liver parenchyma cells into the external gel matrix for studying the effect of the system on material delivery and the function of the hepatocytes.The specific research content are as follows:?1?The construction of a microchannel system based on the sacrificial materials alginate.At first,the alginate micro-column gel was fabricated by fluid spinning,and the effects of divalent cation,needle inner diameter and fluid velocity on the morphology and physical properties of alginate gel were investigated.The study found a positive correlation between the diameter of the cylindrical gel and the inner diameter of the printing needle by using Ca²⁺as cross-linking factor and controlling the fluid velocity in the range of 60-180 L/s.Then the micro-column gel was embedded in the external matrix,and the influence of the composition on the mechanical stability and the biological of the matrix were investigated.Finally,the microchannel was formed by liquefaction of alginate gel in the external matrix system with sodium citrate solution as liquefacient.By investigating the structure and performance of microchannel in the system,we selected a fast effective and biocompatible method for gelation removing.?2?The construction of microvascular system.Firstly,the calcium alginate?Ca-Alg?was modified with chitosan?CS?to form a composite membrane structure on its surface.The effects of reaction time of chitosan and Ca-Alg on composite membrane structure and biological properties were investigated.A chitosan membrane with a thickness of 5?m-15?m can be obtained in 3-7 min.Then,the HUVECs was cultured on the surface of modified Ca-Alg.After culturing 9 days,a dense cell monolayer was formed.Cell proliferation and apoptosis analyses revealed that the calcium alginate gel with composite membrane?CS/Ca-Alg?can promote cell proliferation and inhibit cell apoptosis.After that,the CS/Ca-Alg gel covered with HUVECs was encapsulated within a three-dimensional?3D?composite gel and dissolved by sodium citrate solution to form the microvascular structure.We investigated the effect of sodium citrate solution concentration on completely dissolution time and cell activity.The results showed that the Ca-Alg with a diameter of 500-1000 mM can be completely liquefied within 20-40 min in 15 mM sodium citrate,and the cell activity was remain above 80%.Finally,we evaluated the transmission of matter and found that microfluidic channels with membrane structure and microvascular system can selectively penetrate molecules.?3?The construction of liver-like tissue with microvascular system in vitro.On the basis of the above research,the human hepatoma cell line?HepG2?used as the model combined the microvascular system to form an orderly co-cultured system.On the one hand,the microvascular system as nutrients transport system provided adequate nutrition for hepatocyte metabolism in 3D system,thus the problem of material transfer constraints formed only by diffusing effect was solved.On the other hand,the microvascular system as framework of orderly co-culture system provided a microenvironment for the polarity reconstruction and functional repair of hepatocytes.The study found that the activity of hepatocytes in the microvascular system was significantly enhanced and the liver-special functions of hepatic cells,including the albumin secretion and the expression levels of hepatocyte-specific genes,are significantly improved.The gene expression levels associated with the xenobiotic metabolism CYP1A1?CYP3A4?UGT1A1 and GSTA1 were noticeably increased3.5?11.1?7.9?5.6 fold.In addition,the results of synthesis-associated transcription factors NDUFA3 and GCLM were also markedly increased2.0 fold and 4.2 fold.After culturing for 5 days,the albumin secretion level of Hep G2 cells was significantly improved by 2.8 time under the influence of the HUVECs on the surface of microvascular system.In conclusion,this study proposed a method for constructing microchannel and microvascular system.The microvascular system as a material delivery system can provide rich nutrients for cells in the tissues.More importantly,it can serve as an orderly co-culture system,providing a favorable microenvironment for the polarity reconstruction and functional repair of hepatocytes.This study provides a new idea for the research of liver tissue engineering.