Preparation And Propertiies Of Polycaprolactone/collagen/silk Fibroin Composite Micro-nanofibrous Scaffolds

The development of hepatocyte culture in vitro has great significance for the study of liver tissue engineering.Three-dimensional scaffold culture has been paid more and more attention by researchers as an effective method for hepatocyte culture in vitro.At present,electrospun micro-nanofibrous scaffolds which were made of natural and synthetic polymer are widely used in tissue engineering.However,there have been few studies on the application of fibrous scaffolds in hepatocyte culture in vitro.The electrospun collagen/silk fibroin micro-nanofibrous scaffolds were studied for hepatocyte culture,but the practical application is still limited by some shortcomings: electrospun collagen/silk fibroin fibrous membranes are brittle,easy to shrink and deformation,and the stability of fibers is insufficient;the traditional fibrous membranes have a dense structure and small pore size,which would hinder hepatocytes infiltration into the internal structure of scaffolds.In order to solve these problems,electrospun PCL/COL/SF composite micro-nanofibrous scaffolds were fabricated,the mass ratio of compositions was regulated,probes array collectors and the method of preparing nanofiber aerogels were used to create large pores fibrous scaffolds,which could be used for hepatocytes culture in vitro.The main work of this thesis is as follows:?1?PCL/COL/SF composite micro-nanofibrous membranes were prepared by electrospinning.The composite fibrous membranes could maintain the morphology of fibers after glutaraldehyde?GTA?vapor crosslinking.With the increase of PCL content,the fiber diameter decreased,and the mechanical properties of composite fibrous membrane were enhanced.The ultimate tensile strength and elongation were highest of 11.07 MPa and 56.3% when the mass ratio was 30:70.The results indicated that the addition of polycaprolactone could improve the brittleness and stability of composite fibrous membranes.The HepG₂ cell culture results showed that PCL/COL/SF composite fibrous membranes had good biocompatibility,and hepatocyte adhesion and proliferation were best when the mass ratio was 30:70 and 50:50.These results showed that electrospun PCL/COL/SF fibrous membrane with mass ratio of 30:70 had a good performance and could as a good carrier in hepatocyte culture.?2?PCL/COL/SF composite micro-nanofibrous scaffolds with large pores were prepared by using the probe array collectors.Compared with flat-plate scaffold,the fibrous scaffolds obtained by using the probe collectors showed ordered arrangement,large pore size and porosity.Among these array scaffolds,pore size and porosity of 6 × 6 array scaffold was largest of 10-25 ?m and 91.1%.The contact angle of scaffolds decreased from 75.3 ° to 40 °,and the hydrophilicity was higher than the flat-plate scaffold.The Hep G2 cell culture results showed that the array scaffolds had good biocompatibility,the 6 × 6 array scaffold showed good hepatocyte adhesion and proliferation ability,cell morphology and three-dimensional growth tendency,indicating that it has the potential as a three-dimensional scaffold for hepatocyte culture in vitro.?3?According to the method of preparing nanofiber aerogels,PCL/COL/SF composite micro-nanofibrous scaffolds with large pore size and three-dimensional structure were prepared by crushing,dispersing,freeze-drying and crosslinking the prepared fibrous scaffolds.The structure of three-dimensional composite fibrous scaffold was stable after GTA vapor crosslinking.The fibers were adhered together to form porous structure with large pores?5-35 ?m?and small pores?1-3 ?m?,the average size of large pores was 19.29 ?m,scaffolds have lamellar structure.The Hep G2 cell culture results shown that this structure could support hepatocyte migration and growth,promote the cell deep into the scaffolds and cell function,which indicated that the three-dimensional composite fibrous scaffolds had the potential as a three-dimensional medium for hepatocytes three-dimensional culture in vitro.