Preparing Collagen-Chitosan Nanoscale Fibrous Biomimetic Extracellar Matrices By Electrospinning

Tissue engineering is a newly emerging biomedical technique at the convergence ofcell biology and material science. The objective of tissue engineering is to regeneratenatural tissues from living cells to replace defective or lost tissues and organs. Itstypical method is to produce synthetic tissues by incorporating isolated living cellsinto porous scaffolds and create conditions for cells to proliferate, organize anddevelop into the desired tissues or organs. As a regenerative therapy, tissueengineering avoids the problems associated with organ transplantation, such as donorshortages and permanent immunosuppresive medication, and it does not require theimplantation of artificial biomaterials, which might have poor biocompatibility. Withthese advantages, its medical significance will undoubtedly increase in the 21stcentury. The key and challenge for tissue engineering is how to create excellentartificial ECMs. Here the manual ECMs are three-dimensional biomaterial scaffoldswith excellent biocompatibility and porosity. The functions of biomaterial scaffoldsact as analogues of the natural ECMs found in tissues, which provide information forcells expressing their functions, e.g. adhesion, proliferation, differentiation. Therefore,the aim for biomaterial scaffolds design is to mimic the natural ECMs on both thecomponents and the microstructure.The native extracellular matrix is a molecular complex made up of proteins and polysaccharides and comprises 3-dimensional hierarchical fibrous structures of nanometer scale dimensions. From the point of view of mimic, electrospinning of biocompatible collagen and chitosan blends was studied to biomimic the natural ECMs on both the components and the microstructure for the first time in this paper. At first, the history, the method, the theories, the influencing factors and the applications of electrospinning were introduced. In the experiments, the mixture of 1,1,1,3,3,3 hexafluoro-2-propanol (HFP) and trifluoroacetic acid (TFA) (v/v, 90/10) was found as an appropriate solvent for electrospinning of chitosan and collagen blends, and the collagen-chitosan nanofibers were obtained by electrospinning for the first time. Moreover, the dependence of the fibers diameter on the electrospinning parameters (voltage, feed rate and distance), the concentration of solution and the ratio of chitosan to collagen in the electrospun blends was studied. It was found that the diameter of spun fibers became thick with the increase of the feed rate, the distance and the concentration of solution and became fine with the increase of the voltage and the ratio of chitosan to collagen. The electrospun collagen-chitosan single fibers and fibrous bundles were also collected, which would be benefit to the investigation of fibers properties further.The properties of the electrospun collagen-chitosan nanofibres have also been investigated. Fourier transform infrared spectroscopy (FTIR) of fibers has proved that intermolecular interactions exist between collagen and chitosan. The X-ray diffraction (XRD) has shown collagen, chitosan and their complex fibers give typical amorphous after electrospinning. Thermal properties of fibers have been analyzed by thermogravimetry (TG) and differential scanning calorimentry (DSC) and DSC measurement has also proved the existence of intermolecular interactions between collagen and chitosan. The electrospun collagen-chitosan single fiber and fibrous membrane show different mechanical properties with the difference of chitosan content in the electrospun fibers. The mechanical properties of the single fiber have also proved the existence of intermolecular interactions between collagen and chitosan further. Both the porosity and the surface hydrophilic property of fibrous membrane increase with the increase of chitosan content in electrospun fibers.The electrospun collagen-chitosan nanofibers are so hydrophilic that they can be soluble in water and not keep the fiber form, which can limit its applications. In order to improve both water-resistant ability and mechanical properties of nanofibers, the fibrous membrane was crosslinked by glutaraldehyde (GTA) vapor and time of crosslinking was 2 days. The properties of the crosslinked fibrous membrane were also investigated further. The fibrous form of the membrane has been grossly preserved even after 4 days soaked in 37℃deionized water. FTIR shows that the characteristic absorption bands of the fibers before and after crosslinking do not have obvious difference owing to the same main functional groups of both collagen and chitosan. XRD analyses shows the crosslinked fibers still are amorphous. The thermal properties of the fibers are improved after crosslinking. The average ultimate tensile strength of the collagen-chitosan fibrous membrane has been enhanced, but the average ultimate tensile elongation of the fibrous membrane decreases except for the increase of pure collagen and chitosan fibers. Comparing with the dry crosslinked fibrous membrane, the soaked crosslinked fibrous membrane has better tensile elongation but worse tensile strength.The interaction between cells and artificial ECMs is an important field in tissue engineering research. In this paper, the biocompatibility of the electrospun collagen-chitosan nanofibres was tested by cell culture on the fibrous membrane. The porcine iliac artery endothelial cells and the myocardial artery smooth muscle cells of the mouse were seeded on the electrospun fibrous membrane, the tissue culture plates (TCPs) and the cover slips as controls. Cellular adhesion, cellular proliferation and cellular shape on the fibers were investigated and compared with those on the TCPs and the cover slips. The conclusions can be drawn as follows.(1) MTT measurement shows that the endothelial cells adhesion on the fibers is better than that on both the TCPs and the cover slips when chitosan content is 100%, 20% and 50%, but it is worse at chitosan content of 80% and 0%.(2) MTT measurement indicates that both endothelial cells proliferation and smooth muscle cells proliferation on fibrous membrane are better than that on both the TCPs and the cover slips when chitosan content is 20% 100% and 50%, however, it still is worse at chitosan content of 80% and 0%.(3) SEM measurement shows that cells can migration into the fibrous membrane and grow in the three-dimensional space.Therefore, the ratio of chitosan to collagen will affect cell growth on the electrospun collagen-chitosan fibrous membrane, moreover, the porocity is also an important factor. The effect of other factors on cells growth has been analyzed, such as, the fibers morphologies, the surface free energy, the surface hydrophilic/hydrophobic properties, the surface charge and the surface bioactivity, which means the complexity of the interactions between cells and biomaterials.As a result, electrospun collagen-chitosan fibrous membrane is a promising biomedical material. The research will provide the data and the base for electrospun collagen-chitosan fibrous membrane to be selected as tissue engineering scaffold in clinic.