Electrical Signals Mediated By Functional Materials Driven By An External Field To Regulate Stem Cell Differentiation

After the human body is damaged,the damage of tissues and organs will seriously affect people’s life and health.The development and application of tissue engineering technology provide a new way to study this problem.As an important element of tissue engineering,seed cells possess strong regeneration and differentiation potential,and play a vital role in tissue repair.However,it is still a difficult problem to regulate the fate of stem cells,induce their targeted differentiation,and repair damaged tissues precisely.Factors regulating stem cell differentiation include biological factors,chemical factors and physical factors.The biological and chemical factors have many problems,such as high cost and toxic side effects,which restrict the development of clinical stem cell therapy.However,physical factors have attracted wide attention because of their advantages of simple operation,low cost and small toxic and side effects.All cells in the body are in a three-dimensional dynamic microenvironment.Physical signals provided by extracellular matrix and mechanical stimulation in the microenvironment affect the migration,self-renewal and differentiation of cells and other behaviors and functions.Therefore,the construction of scaffold materials that simulate natural cell microenvironment and physical signals in vivo can regulate cell fate,which has become a research hotspot in tissue repair and regenerative medicine.Nanomaterials have many advantages in regulating the fate of stem cells.First,the extracellular matrix has a specific microenvironment composed of active substances such as collagen.Using nanomaterials to simulate the cell microenvironment can affect the behavior and function of stem cells.Second,nanomaterials can achieve targeted contact effect on the damaged tissue,with little impact on other tissues and cells,achieving precise targeted repair;Third,functional nanomaterials with specific morphologies,such as piezoelectric materials and magnetoelectric materials,can mediate physical signals in the external field and regulate the fate of stem cells more efficiently.In addition,electrical stimulation can significantly regulate cell growth,differentiation and tissue regeneration.Therefore,electroactive biomaterials are considered as the most ideal scaffolds for tissue engineering.However,delivering electrical signals to damaged tissue in the body remains challenging.The use of external wires can cause infection during surgical implantation and affect movement and behavior.In addition,the conductive paste or glue required to connect the conductor and the stent will also bring inconvenience to the implantation in vivo,which greatly restricts its practical clinical application.The breakthrough to solve the above problems is to effectively coordinate the relationship between seed cells,inducing factors and scaffold materials,and make scaffold materials serve as the carrier of cell support and internal electrical signals,and regulate the directional differentiation of stem cells under the local electrical signals generated by the scaffold itself.Functional materials under certain conditions will generate charge redistribution,so as to achieve the generation of current without external power supply.Physical signals(electrical signals)generated by scaffold materials can be used to regulate the differentiation of stem cells,thus laying a foundation for tissue repair.Therefore,the purpose of this study is to explore the role of scaffold material morphology and its mediated radio signal to regulate the directed differentiation of stem cells.Mesenchymal stem cells(MSCs)were used as seed cells to prepare PLLA and PLLA/Gr nanofiber membranes by electrostatic spinning technology.The regulation of piezoelectric nanofiber membranes on the differentiation of stem cells toward osteogenesis was systematically studied,providing theories and new ideas for the research and treatment of bone injury.With the help of rotating magnetic field,non-contact induced current generated on graphene paper(GP)is realized to systematically study the regulation effect of electrical signals on the differentiation of stem cells to neurons,providing theories and new ideas for the research and treatment of neurological diseases.The specific work and conclusions are as follows:1.Piezoelectric PLLA/Gr nanofiber membrane regulates the osteogenic differentiation of MSCsPiezoelectric materials have been shown to generate piezoelectric potentials mediated by internal or external mechanical forces.L-polylactic acid(PLLA)is an FDA approved material with good biocompatibility,biodegradability and piezoelectric properties,which is widely used in tissue repair.Graphene(Gr)has attracted extensive attention in the medical field in recent years due to its excellent electrical conductivity and biocompatibility.In this paper,PLLA/Gr nanofiber material was prepared by electrostatic spinning technology.The fiber morphology can simulate the natural cell microenvironment,and Gr can effectively improve the piezoelectric properties of the material.The resulting piezoelectric potential is significantly maximized through cell adhesion and migration.The effects of piezoelectric and nano-morphology on proliferation and osteogenic differentiation of MSCs cells were studied comprehensively.CCK-8,cell viability and nuclear skeleton staining showed that PLLA and PLLA/Gr nanofiber membranes had good biocompatibility,which was conducive to cell adhesion and proliferation.By real-time quantitative polymerase chain reaction(q PCR)and immunohistochemistry,the effects of nanofiber membrane on osteogenic differentiation of MSCs were analyzed at gene and protein levels,respectively.The results showed that the osteogenic differentiation related genes OPN,OCN and Runx2 were up-regulated by 5.4,16.2and 6.5 times,respectively.Moreover,osteogenic differentiation related proteins OPN,OCN,Runx2 and COL-1 were highly expressed in the cells.Alkaline phosphatase and alizarine red staining were used to further verify the osteogenic differentiation effect,and the results showed that there were more stained cells on the PLLA/Gr nanofibers.In summary,the results fully indicate that PLLA/Gr nanofiber membrane can effectively promote the osteogenic differentiation of mesenchymal stem cells.This discovery provides new ideas for the rational design of bone tissue engineering scaffolds.2.The rotating magnetic field drives the conductive graphene paper to generate local electrical signals to regulate the neural differentiation of MSCsIn view of the principle of electromagnetic induction,this paper proposes to use graphene paper(GP)with high conductivity and good biocompatibility as the conductive scaffold,and realize the electrical signal generation without wire contact by means of external rotating magnetic field drive,to explore its regulation on the neural differentiation of MSCs.GP is a thin film prepared from graphene with excellent mechanical properties and electrical conductivity.A permanent magnet and an electric stirrer were used to construct an external rotating magnetic field to detect the induced current generated by GP at different speed magnetic fields.The results show that the induced current is proportional to the rotation speed of the magnetic field,and the current up to 6 μA can be generated at the rotation speed of400 rmp.MSCs were used as seed cells,and GP was mediated to generate induced current at a rotation speed of 400 rmp to explore the regulation of the generated electrical stimulation on the differentiation of MSCs into neurons.CCK-8,cell viability and nuclear skeleton staining showed that GP had good biocompatibility and was conducive to cell adhesion and proliferation.The electrical signals generated do not affect cell activity.The regulation of GP-induced electrical stimulation on neural differentiation of MSCs was analyzed at gene and protein levels by real-time quantitative polymerase chain reaction(q PCR)and immunohistochemistry,respectively.The results showed that neuronal differentiation related genes Nestin,Tuj1 and MAP2 were up-regulated by 2.8,2 and 2.9 times under 400 rmp rotating magnetic field,respectively.Neural differentiation-related proteins Nestin,Tuj1,GFAP and MAP2 were highly expressed.At the same time,vivo differentiation test was carried out in rats.Hematoxylin-eosin(HE)staining showed that GP had good histocompatibility and no obvious inflammatory reaction.In addition,immunofluorescence staining showed that the implanted GP could regulate the neuronal differentiation of MSCs assisted by rotating magnetic field.This technique of regulating neural differentiation of stem cells by in situ radio signals based on electromagnetic induction may lay a preliminary foundation for the development of non-invasive field-driven neural repair techniques.