Fabrication And Performance Evaluation Of Biological Scaffolds With Three-dimensional Vascular Networks By Using Composite Process

In the clinical field,due to the limited number of organ donations,the demand for various regenerative tissues and organs caused by accidents and diseases is increasing.The manufacturing of biocompatible regenerative tissues and organs in vitro becomes an important area that has attracted the attention and the intensive investment from the current society.Since the publication of tissue engineering in 1993,some significant progress has been achieved in the manufacturing of in vitro tissues and organs.For example,the reconstructed tissues and organs with simple geometry and internal structure have entered the clinic or clinical experimental phase,such as skin,osteochondral,bladder,and so on.However,the in vitro tissues and organs with large thickness or volume has not yet entered the clinical trial stage,such as heart,kidney,and large segmental bone.This is mainly due to the complex structure of the large tissues and organs.The internal entangled vascular network is the basis for maintaining cell viability and successful tissue regeneration.Therefore,how to build an in vitro natural vascular network in engineered tissues and organs,using the method in tissue engineering to create a biological scaffold with a complex three-dimensional vascular network,promote the culture of in vitro tissues,enhance the efficiency of tissue regeneration,has become a major problem for the domestic and foreign scholars to be solved.The research on the manufacturing technology of biological scaffolds with complex three-dimensional vascular networks and the key technologies of the manufacturing equipment also have extremely important research significance and application value.This dissertation aims at the key technical issues in the regeneration of thick tissue defect,focuses on the research of the fabrication process and the key equipment technology of biological scaffolds with three-dimensional vascular networks.The basis of this study includes the controllable process,the hemodynamic flow model,and simulation analysis.The target of this study is that the fabricated biological scaffolds have biocompatibility both in vitro and in vivo.A fabrication method combines additive manufacturing and sacrificial process for producing vascular networks was proposed.The fabrication of three-dimensional sacrificial templates by using melting extrusion printing and three-dimensional vascular networks was systematically illustrated.This dissertation focuses on the process parameters of the fabrication process,the wall shear environment generated by blood flow,and the in vitro and in vivo biocompatibility of the prepared biological scaffolds to conduct the theoretical and experimental research.It provides reliable theoretical evidence and experimental basis for the material of the biological scaffolds,the structural optimization of the three-dimensional vascular networks,and the validation of the effectiveness of the biological scaffolds.The research content and results of the thesis mainly include the following aspects:1)According to the basic principle of mimicking the native vasculatures,this dissertation proposed a multi-process method for fabricating biological scaffolds with three-dimensional vascular networks.Sacrificial material was printed on a rotary receiving device to obtain a sacrificial template with a three-dimensional geometric shape by using melting extrusion printing.Thus,the fabricated vascular network has a corresponding three-dimensional structure.Moreover,the fabricated vascular networks can provide a nutrient transport channel for the loaded cells.The printing principle of the three-dimensional sacrificial templates and the fabrication process of the biological scaffolds are elaborated in this dissertation.The framework,composition,and function of the three-dimensional sacrificial templates printing system are designed and built.2)Based on the native vasculatures of the human body,two receiving devices of the drum device and the stepped device were used to obtain a sacrificial template which has a three-dimensional shape in both the axial direction and the radial direction.The effect of the inclination angle from the stepped device on the shape of sacrificial templates was studied.Drum vascular networks and stepped vascular networks were successfully fabricated in the biological scaffolds.3)The comparative test and analysis of Pluronic F-127,calcium alginate,and PVA,which are commonly used currently,were conducted.The advantages of PVA was studied from three aspects of mechanical properties,printability,and dissolution swelling ratio,which verified the effectiveness of using PVA as the sacrificial material to print three-dimensional sacrificial templates and fabricate biological scaffolds with three-dimensional vascular networks.4)The wall shear force generated from the human blood flow was set as the research object in this dissertation.The numerical simulation method was used to simulate the flow of blood in the three-dimensional vascular networks.A mathematical model of blood flow was established.The effect of the vascular network structures on the wall shear effect produced during blood flow was investigated to verify the rationality of the structure of the three-dimensional vascular networks theoretically.5)For the cytocompatibility of the fabricated biological scaffolds,the endothelial cells were implanted into the three-dimensional vascular networks.Fluorescent labeling methods were used to study the cell attachment,the cell viability,the cell morphology,and the ability of cells to form new blood vessels.The reaction of reduction was used to study the proliferative ability of cells in the biological scaffolds.The diffusion behavior of macromolecules was utilized to study cell function.All these experiments were conducted to verify the ccytocompatibility of the biological scaffolds and the formation of endothelial cell monolayer required for the formation of blood vessels.For tissue engineering and clinical medical applications,the biological scaffolds with threedimensional vascular networks were implanted into animals for co-culturing.The histological comparison was performed to observe the in vivo compatibility of the biological scaffolds,the function of the vascular networks,and the degradation phenomenon of the biological scaffolds,which could further verify the degradation performance,tissue regeneration function,and vascularization of the biological scaffolds three-dimensional vascular networks.