| The cellular environment in human tissues is a typical un-homogeneous environment,which includes various types of cells and extracellular matrix,and it is highly heterogeneous.Efficient and rapid construction of the in vivo environment has always been a hot topic.Due to the simple and symmetry stuctre,hydrogel microspheres have been wildly used in cell carriers,drug delivery agents,and identifiers for biological assays.Hence,the construction of in vitro cell microenvironments as well as complex micro-tissue structures by using heterogeneous microspheres deserves further investigation.And lastly,in area of hydrogel microsphere fabrication,researches usually use microfluidic methods to achieve heterogeneous inner structures inside the microspheres,and finally to arrange the multi-components spatially.Among the common heterogeneity patterns are partitioned,core-shell,and non-spherical shapes.However,we still unable to make more complex microspheres.Besides,the tiny size of the micro-channel greatly limits the applicable viscosity range and the flow quantity of biomaterials,which will makes it hard to achieve mass production.In this article,according to the application demands of heterogeneous microspheres in tissue printing and in vitro cell culture,basic cell printing studies were carried out in four specific research directions:gradient manufacturing,rapid fabrication,micro-size fabrication,and multi-scale fabrication of heterogeneous microspheres.We proposed the printing process of multi-scale heterogeneous distribution of spiral hydrogel microspheres for the first time.On this basis,the applications of different cell density hydrogel microsphere printing,single cell carriers,and spherical spiral distribution of cell microspheres were respectively performed.At last,cell-laden hydrogel microspheres were cultured in vitro and also in vivo using vascular endothelial cells,and initially achieved vascularization.The main research and work of this article are summarized as follows:(1)The concentration distribution of cells in a three-dimensional environment in vitro has a great influence on cell viability and function expression.According to the turbulent mixing characteristics of sodium alginate bioink,a fabrication scheme of cell-laden heterogeneous hydrogel microsphere with adjustable cell concentration was proposed.A multi-channel dynamic micromixer was invented as a cell printing nozzle that uses active mechanical mixing to achieve rapid mixing with multi-component materials.By studing the mixing efficiency of the micromixer,its size and process parameters were optimized.Besides,fabrication of hydrogel microspheres with adjustable cell concentration was achieved using dynamic gradient printing.At last,the effects of cell concentration on cell viability and proliferation ability under three-dimensional culture conditions were studied,providing experimental basis for cell culture in three-dimensional micro spheres in vitro.(2)Mass production and single cell encapsulation of heterogeneous hydrogel microspheres have high research value in clinical applications and basic biomedical studies.A multi-component symmetrical coaxial nozzle was invented using coaxial jet technology.This nozzle consists of a multi-component mixing front end and a symmetric coaxial nozzle,which improves the stability of the coaxial air flow during spraying.Systematic experiments were performed on the satellite hydrogel droplets which was found during the printing process,and its feasibility for single cell encapsulation was investigated.By studying the experimental influence of injection parameters,optimal solution was proposed for the encapsulation of single cell.Besides,the heterostructures of microspheres produced by coaxial jetting were characterized using bimaceral hydrogels.Finally,cell viability and proliferation ability of the co-axially jet printed microspheres were verified.(3)The controllable adjustment of heterogeneous hydrogel microspheres size can greatly extend their applications,while few technologies were reported so far.Here,a printing process of partitioned heterogeneous microspheres is proposed by applying electrostatic jet technology.This method can be universally integrated into the existing bioprinting system.The electrostatic field enables the printing of hydrogel microspheres with adjustable diameters.The bioprinting system for hydrogel microspheres based on this method was built,then the fluid properties and force conditions of the hydrogel microspheres were analyzed in the electrostatic field.Through series of experiments on parameters such as electric field strength,extrusion pressure,and nozzle diameter,controllable fabrication of hydrogel microspheres based on electrostatic spraying was realized.Finally,cell carriers and proliferation of hydrogel microspheres with different diameters were successed using electrostatic spraying.(4)Microspheres with complex three-dimensional heterostructures can be used to build complex artificial micro-organizations,but it is still a big problem in the field of microsphere manufacturing.In this article,the manufacturing method of airflow-assisted rotation is proposed,which fills in the gap of the current manufacturing ability of producing heterogeneous hydrogel microspheres with three-dimensional controllable internal structures.An experimental platform for the manufacture of spherical heterogeneous hydrogel microspheres was built.In order to reveal the impact of various process parameters on the feature sizes of formed microspheres,theoretical analysis and systematic experimental studies were carried out,and controlled fabrication of spiral microspheres was achieved.Finally,spiral distributed cell-laden hydrogel microspheres were fabricated by cell printing,and the ability of cells to survive and proliferate in the spiral hydrogel microspheres was successfully achieved.(5)In vitro vascularization is the premise of tissue functionalization.Two typical applications of in vitro organ production and in vivo cell therapy were operated using heterogeneous microsphere.Firstly,complex cartilage microtissues were created with endothelial cells and bone marrow mesenchymal stem cells,and the endothelial cells are formed into spatial spiral shape inside the microspheres.Then,the vascularization of endothelial cells and the osteogenesis of bone marrow mesenchymal stem cells were both achieved in vitro environment,thus a complex cartilage model was establishment.For in vivo therapy,293T cells that can stably release angiogenic growth factors were encapsulated inside the hydrogel microspheres and implanted into mice with organ ischemia.Eventually,blood vessel regeneration in the ischemic limbs of mice was achieved. |
