Scale Fabrication Of Three Dimensional Scaffolds With Large Pores Based On Electrospun Fiber/Yarn And The Application In Tissue Engineering

The defects of tissue and organs are of great harm to the health of human beings,which is the most serious problem in clinic.The common defects of tissue include bone tissue defect,peripheral nerve injury,vascular injury,skin injury,and so on.In order to repair the defects,several common methods are applied in clinic,such as autologous transplantation,allogeneic transplantation and xenotransplantation.Autologous transplantation is known as the gold standard.However,it is also facing a lot of problems,including the limited source of transplantation and secondary damage to the patients.Considerable endeavour has been conducted by researchers to prepare artificial scaffold which can replace autologous transplantation.In 1980 s,a new way to solve this problem was provided by the emergence of tissue engineering.To repair the defects,the corresponding cells were cultured on the artificial scaffold to regenerate new tissue or organs.Seed cells,scaffolds,and growth factor are the three elements of tissue engineering.Among which,the key is tissue engineering scaffold.It was indicated that in the human tissue,extracellular matrix(ECM)is a key role in regulating cell behaviour in tissue cells.The cell adhesion,proliferation,and differentiation all occur in the environment constructed by ECM.To provide the most suitable living environment for cells,it is quite necessary for the scaffold to mimic the ECM.The ECM of human is a three dimensional nanofibrous net constructed by nanofibers,which contains collagen and glycosaminoglycan,with a diameter of 50~500 nm.It is generally believed that mimicking the nanofiber structure of ECM is a shortcut for the preparation of tissue engineering scaffolds.Thus,a variety of methods to prepare nanofiber materials were applied in the preparation of tissue engineering scaffolds.The most widely used method is electrospinning due to its simplicity,low cost.Polymer fibers of tens nanometre to several micrometre could be produced via electrospinning,with high specific surface area,high porosity.The nanofiber scaffold could biomimetic ECM in nanoscale,promoting cell adhesion,proliferation,differentiation,and migration.Currently,two problems limited the use of electrospun nanofiber tissue engineering scaffold.One is the low production rate of electrospinning,and the other is tightly packed structure.In the electrospun scaffold,pores between nanofibers are too small for cell infiltration in to the scaffold.Generally,cells are inhibited on the surface of the scaffold.No tissue with a certain thickness could be formed.The purpose of this study is to prepare three-dimensional scaffold via electrospinning.In order to improve the yield of electrospun nanofibers,three dimensional scaffolds were fabricated by needleless electrospinning technique.At the same time,three-dimensional scaffolds with high orientation and controllable structure were prepared with electrospun nanofiber yarn.1.In the study of needleless electrospinning,a disc-needleless electropsinning setup was built and modulated for electrospinning.The key devices of this setup include disc-spinneret,collecting cylinder,solution tank,and high voltage generator.With this instrument,the basic conditions of the preparation of PCL fiber scaffolds without needle electrospinning were studied.For the TFE solution system,when the concentration of PCL is low(2-7%),the fiber with nest structure could be obtained at the appropriate voltage.The beads formed the "wall" of the bird’s nest,dividing the nanofibers into polygonal structures.Nanofiber film collected at different time were observed and analysed.It could be concluded that the formation of nest structure was related the charge concentration at the surface of the collector caused by the polymer beads.The nest structure disappeared while increasing the concentration of PCL.For the single solvent system,the spinnability of the solution is poor(TFE and DCM),which is prone to the formation of beads and inter-fiber adhesion.The introduction of double solvent system could significantly improve the spinnability and get clear and single nanofiber.When using DCM/DMF(9/1)as the solvent,fiber with porous surface could be collected,which is related to the phase separation caused by the rapid evaporation of DCM.2.Based on the preparation of PCL scaffold via needleless electrospinning,a three dimensional PCL scaffold with porous surface structure were fabricated with DCM/DMF as solvent,which was applied in tissue engineering.The fiber morphology,pore size,and biocompatibility of the scaffold were tested.Large pores could be observed in the three dimensional scaffold with an average pore diameter of 24.75 μm and the largest pore size of 55.85 μm.As the needle electrospun scaffold possessed a smaller pore diameter(4.83 μm).The results of protein adsorption test showed that the porous PCL scaffold could adsorb 55% more protein compared with the same material.According to the experiments in vitro,more cells were adhered on the needleless electrospun PCL scaffold in 4 hours.During 14 days culture period,cell proliferation on the PCL scaffold was significantly higher than the control group.It is indicated that the three dimensional scaffold could benefit the cell adhesion and proliferation.With the help of confocal fluorescence microscope,fibroblasts could be observed in the depth of 800 μm.Z-stack imaging also indicated cells were densely distributed in the scaffold at a thickness of 0~350 μm.All these results indicated the great potential of discelectrospun 3D PCL scaffolds in soft tissue engineering.3.Synthetic materials possessed good physical and mechanical properties while natural materials are of good biocompatibility.In tissue engineering,synthetic materials and natural materials were combined to prepare scaffold with the merits of both materials.Herein,PCL and gelatin were mixed and electropsun into three dimensional component scaffold via needleless electrospinning.A three dimensional macro-/nanofiber scaffold was obtained due to the phase separation of these two solute in TFE solution.In this scaffold,the scaffold was supported by the skeleton of microfiber,forming large pores.Nanofiber were inserted in the scaffold and bridged between microfibers,forming nanofiber webs in the large pores.The average pore diameter of the macro-porous scaffold was 13.39 μm,which is much higher than that of the control.The macro-porous structure of the scaffold could promote the infiltration and migration of the cells in the scaffold,and the presence of the nanofibers could provide more adhesion sites for cells,thereby promoting cell adhesion,spreading,and proliferation.With the support of the nanofiber web,cells can grow in the three dimensional space of the microfibers.Such a combination of nanofiber and microfiber,may solve the problems facing electrospun scaffolds for tissue engineering.Not only could the requirements of the pore size be met,but also biomimetic the nanoscale structure of ECM.The scaffold could,promote cell adhesion and proliferation,and allow cells to grow in three-dimensional space and eventually form a certain thickness of tissue.4.In the study of the preparation of three dimensional tissue engineering scaffolds by electrospinning technology,the scaffolds are mostly non-woven structure and the fibers are randomly ranged.For the electrospun scaffold,it quite hard to re-regulate the structure after electrospinning.Thus,electrospun nanofiber yarn were applied in the fabrication of tissue engineering scaffolds.Nanofiber yarn is constructed by nanofiber,with highly aligned topology.A wide variety of fabric structure could be prepared by traditional textile methods,which could expand the scope of application of scaffold materials.In the trial and error,PLLA nanofiber yarn with highly aligned PLLA nanofibers was prepared via dual-spinneret electrospinning technology.The application of PLLA yarn in tissue engineering was preliminarily explored.Three dimensional fabric with a certain thickness was manufactured by Noobing,which could support the cell growth in a three dimensional space.After culturing for 7 days,mouse myocardial cells could be observed in scaffold at different positions.And the alignment of cells was same to the yarn direction.At the same time,PLLA nanofiber yarn was inserted into the P(LLA-CL)tube to form a novel nerve conduit scaffold.The highly aligned PLLA nanofiber in the surface could induce nerve cells migrate unidirectionally.The results indicated that after 7 days of culture,Schwann cells could penetrated into the whole nerve conduit scaffold.All these studies shown the potential of nanofiber yarn in the fabrication of controllable three dimensional tissue engineering scaffolds.