Regulation Of Stem Cells Fate Through Different Physical Cues Mediated By Micro/Nanostructures

Tissue engineering is a combination of cell biology and material science,which contains three main contents,including cells,scaffold,and growth information.Expanding cells especially stem cells in vitro,designing and fabricatng suitable cell culture scaffold,and providing efficient growth information to direct stem cell's proliferation and differentiation are three main aims in tissue engineering,which have great significance for the regeneration of damaged tissue and organs.Biochemical molecules are usually used to regulate stem cell fate even though these molecules are expensive and easy to spread.Physical cues are believed to be efficient and safe and have attracted people's attention.In this dissertation,based on the needs of physical cues,various micro/nano-structured materials were prepared for stem cell amplification and differentiation.The effects of micro/nanomaterials mediated physical cues including structural information,plasmonic effect,and piezoelectric signal on stem cell fate were investigated,which will lay the theological and practical foundations for the design and construction of materials for tissue engineering applications.This dissertation mainly includes the following aspects:1.Fabricating composite microspheres for stem cell expansion and phenotype maintenance.Stem cell is the basic element in tissue engineering.However,the insufficiency of stem cell sources is still a problem that limits the further development of tissue engineering and clinical medicine.For traditional two-dimensional(2D)culture,the obtained number of cells is limited,and the cultivation environment is quite different from that in vivo.Three-dimensional(3D)cell culture can provide a microenvironment similar to that in vivo,which is conducive to the growth and proliferation of cells and the maintenance of biological functions of stem cells.In this dissertation,a simple,low-cost and productive method for preparing genipin cross-linked chitosan/graphene oxide composite microspheres as cell culture scaffold was proposed.The composite microspheres have good biocompatibility and physical properties,which will maintain the long-term stability of cell culture environment.Mesenchymal stem cell phenotype can be maintained while spreading and expanding on the surface.In addition,due to the spontaneous fluorescence generated by the crosslinking of genipin,it is convenient to observe the behavior of stem cells on microspheres.This dissertation provides a method for in vitro 3D stem cell amplification via rapid-and mass-produced chitosan/graphene oxide composite microspheres,which will provide an abundant stem cell source for our following researches of stem cell differentiation.2.Construction of ordered nanoarrays and their regulation of osteogenic differentiation.Based on the effective amplification of stem cells,the differentiation of stem cells is also an important content of tissue engineering.Nanostructures are believed to have better biological stability and safety than biochemical factors.Nanopillar array has been widely used in tissue engineering,and a large number of studies have proved the promotion effect of nanopillar array on osteogenic differentiation.However,the effect of arrays with different nanopillar diameters on stem cell fate regulation has not been reported.In this dissertation,polylactic acid(PLA)nanoarrays with different nanopillar diameters were fabricated by a nano-imprinting method using porous anodic aluminum oxide(AAO)as a template.By culturing hADSCs without the assistance of growth factors,the differentiation tendencies of hADSCs on nanopillar arrays were assessed at gene and protein levels.Moreover,the ectopic osteogenic ability of the nanopillar array was also assessed via an in vivo animal model.Therefore,this dissertation provides a practical foundation for the application of nanopillar array-induced stem cell differentiation in tissue regeneration.3.Regulating neural differentiation by NIR-driven electromagnetic signals of nanostructured plasmonic materials.After proving the efficacy of nanostructures for regulating osteogenic differentiation,other physical signals were also used to direct stem cell differentiation.Compared to osteogenic differentiation,neural differentiation of MSCs is faced with more challenges.Because electrical stimulation has been proved to have a positive effect on nerve regeneration,in this part,electric stimulation was introduced to improve the neural differentiation efficiency.We used near-infrared(NIR)light to provide non-contacting electric stimulation to cells through a plasmonic nanostructure.In this dissertation,the effect of plasmonic nanostructures mediated by near-infrared light on neural differentiation of hADSCs was investigated.hADSCs were cultured on the copper sulfide nanostructure and stimulated with near-infrared light(808 nm)for 3 min per day.The differentiation of hADSCs was detected by q-PCR and immunofluorescent staining.The results showed that the nanostructure,plasmonic thermal effect,and intense local electromagnetic field of copper sulfide nanostructures had synergistic effects on the neural differentiation of hADSCs.Therefore,plasmonic nanostructure with near-infrared light absorbance is used to realize the remote near-infrared light-mediated non-invasive stem cell fate regulation,which further verified the regulation of stem cell fate through physical cues driven by nanostructured materials.4.Regulating neural differentiation by ultrasonic mediated piezoelectric signal.In order to develop more approaches of providing untethered electric stimulations and improving neural differentiation efficiency of hADSCs,besides NIR,the ultrasonic-mediated electric signal of piezoelectric materials was also introduced.Based on previous studies,a soft matrix is conducive for neural differentiation of stem cells.Therefore,in this dissertation,ultrasound-assisted piezoelectric signal was used to apply non-contacting electrical stimulation to stem cells.In the absence of growth factor and differentiation medium component,hADSCs cultured on nano-cellulose(NC)hydrogel have shown neuron-like morphology.Ultrasonic wave is introduced as the source of untethered mechanical force to generate electrical signals.The degree of neural differentiation of hADSCs was assessed at gene and protein levels.The results showed that the hADSCs cultured on nano-cellulose hydrogel without ultrasound treatment had a tendency to differentiate into astrocytes,while the hADSCs treated with ultrasound showed a tendency to differentiate into both astrocytes and neurons,which has far-reaching significance and important application prospect for nerve tissue repair with autologous stem cells.In this section,the regulation of neural differentiation by materials-mediated physical cues,including nanostructure,matrix stiffness,and electric signal,is further confirmed.In summary,this dissertation is devoted to studying the regulation of stem cell fate by microenvironment provided by composite materials,nanostructures,light-and ultrasonic-mediated physical cues of nanostructured materials,which confirmed the regulation effect on stem cell fate by different physical cues mediated by micro/nano-structured materials.This dissertation will further accelerate the application of physical cues mediated by biomaterials in tissue engineering and biomedical researches,and provide new insights for stem cell therapy.