| With the improvement of social conditions and people's living standards,cardiovascular diseases which are caused by irregular eating habits and low-frequency exercise have become the number one killer threatenting human health.Although artificial blood vessels can alleviate the severe pressure of donor shortage in the vascular transplanation operation to a certain extent and provide a new method for the treatment of cardiovascular diseases,the thrombosis and restenosis problems caused by artificial blood vessels in the late stage of implanation can not be totally avioded.After learning about importance of the endothelial layer in inhibiting thrombosis and maintaining the long-term patency of artificial blood vessels,scientists begin to study the rapid endothelialization of tissue-engineered blood vessels.The process of endothelialization on the surface of tissue engineering blood vessels mainly includes three important steps: 1,Stem cells or endothelial cells(ECs)in the blood stick to the surface through interactions with artificial blood vessels and migrate over large areas.2,Stem cells complete the directed differentiation towards ECs.3,The differentiated cells retain the function of ECs and secrete corresponding extracellular matrix and growth factors.Therefore,how to effectively complete the above three steps and improve their efficiency are the research hotspots and difficulty in the field of vascular tissue engineering by far.To solve the specific problems mentioned above,our research employs synthetic polymer PCL as a raw material.The investigation uses electrospinning technology to fabricate three-dimentional porous scaffolds,and then modify these scaffolds based on the knowledge and theories in engineering science,thus modulate cell behaviors,such as proliferation,migration and differentiation,on scaffolds.The main research work of this paper is as follows:First part.Fabrication of PCL/Gelatin composite electrospun scaffold and its effects on the behaviors of mesenchymal stem cells(MSCs).At present,tissue engineering scaffolds made of synthetic polymeric materials have been widely used in clinical experiments and scientific research.However,the insufficiency of biocompatibility and biochemical function is still a chock point limiting their further applications.Therefore,the combination with natural materials to improve the biocompatibility and biochemical properties of synthetic polymers is considered as one of the most effective solutions.In this part of study,the author mixed PCL and gelatin with different weight ratio,and produced composite nanofibrous scaffolds via electrospinning method.The biocompatibility of composite scaffolds were enhanced with the introduction of peptides sequence,which are provided by gelatin molecules.Apart from that,with the change of gelatin weight ratio,the surface wettability,crystallization and mechanical properties of composite scaffolds were also developed.This research proved that the interactions between MSCs and PCL/gelatin scaffolds could be modulated with gelatin addition,suggesting the potential application of PCL/gelatin scaffolds in vascular tissue engineering.Second part.Endogenous biological factors modulated by substrate stiffness regulate endothelial differentiation of MSCs.In addition to growth factors,the differentiation of MSCs to adult cells is also affected by the extracellular matrix environment.Therefore,the efficiency of directional differentiation can be enhanced by synergistic stimulation combining the inherent characteristics of tissue engineering scaffolds with exogenous growth factors.In this part of investigation,the author adopted a simple and convenient annealing method to treat electrospun PCL/PLA composite scaffolds for different time,so as to adjust the surface stiffness of the composite scaffolds.The MSCs were then seeded on the scaffolds with different surface stiffness and induced to differentiate to ECs.The experimental data showed that the differentiated MSCs could produce more amount of EC-related genes and proteins,including platelet endothelial cell adhesion factor(CD31)and von willebrand factor(vWF),proving that modulating on EC differentiation of MSCs by scaffold stiffness.This research provides experimental guidance on the design and optimization of scaffolds.Third part.Explore the potential signal channel by which the scaffold stiffness modulates MSC differentiation to ECs.By using the inherent characteristics of tissue engineering scaffolds,such as physical and chemical properties,to influence and regulate the directional differentiation of pluripotent stem cells to adult cells has important research significance in the field of tissue engineering and stem cell therapy.However,the underlying mechanism and complex signal pathway related with the differentiation of stem cells have not been fully and clearly revealed.Based on the conclusion made in the second part of study,i.e.,the increase of scaffold stiffness can enhance the efficiency of MSC differentiation to ECs,the author introduced a small interfering RNA technology(siRNA)to explore the invloved signal pathway in this part of study.The MIF-mRNA and VEGF-mRNA in the MSCs were transfected via siRNA technology respectively,and then MSCs without and with teansfection were seeded on scaffolds with different surface stiffness and induced with endothelial differention medium.The results showed that when MIF-mRNA in MSCs were transfected,there were no significant difference in the gene and protein amounts of VEGF,CD31 and vWF between differentiated MSCs on two kind of scaffolds.However,when VEGF-mRNA in MCSs were transfected,the gene and protein amounts of CD31 and vWF were too less to detect,while the different gene and protein amounts of MIF were observed in differentiated MSCs on two kind of scaffolds.The conclusions above demonstrat that the directional differentiation of MSCs to ECs can be regulated by scaffold stiffness via a MIF-VEGF-CD31/vWF signal pathway,providing a new reference in tissue engineering and stem cell therapy.Forth part.PCL nanofibers containing VEGF-encapsulated gelatin particles enhances MSC differentiation and angiogenesis of ECs.Growth factors paly vital roles in both the self-proliferation of cells and the interactions between cells and tissue engineering scaffolds.However,during the experimental operation,growth factors are usually added into the culture medium by the experimenter.With the rapid degradation,their influence on cells will be weakened or even disappeared as time goes on.Therefore,the fabrication of functional scaffolds modified with growth factors and with the ability to release growth factors are attracting more and more attentions.In this part,the author integrated the first three parts of study and encapsulated VEGF into nanoparticles made of gelatin,then assemble these nanoparticles into electrospun PCL nanofibers.After experiments,the author found that the PCL nanofibrous scaffold containing VEGF-encapsulated gelatin nanoparticles could induce the differentiation of MSC to ECs,meanwhile extended the tubular structure formed by ECs for a long period.This part of study demonstrates this kind of composite scaffolds can not only stimulate MSC differentiation to ECs in early stage,but also maintain the stability of specific function,providing a new experimental scheme for the design and optimization of vascular tissue engineering scaffolds. |
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