The Preparation Of PVDF-based Piezoelectric Film And Its Application In Regulating Neural Differentiation Of Stem Cells

Ageing of population,cardiovascular and cerebrovascular disease,and traffic accidents causes much more neurodegenerative diseases and nervous system damage which becomes a social problem.The nervous system which mobilizes the body to respond to various signal stimuli can regulate various functional activities in the human body.Once damaged,it will cause pathological changes and cause the body to produce functional disorders.Loss of functional neurons is the major pathogenesis of neural dysfunction.Although the medical scientists and clinicians have paid great efforts and take great progress,neural repair and regeneration still face huge challenges in clinical.One promising method is to generate sufficient new neurons through neural stem cells differentiation to recover the function of nerve systems.Neural stem cells are thought to be the best seed cells for therapy of neurodegenerative diseases due to their great differentiative potential to neurons and neuroglia cells.However,the lack of resources of autologous neural stem cells prohibits their clinic applications.Bone marrow-derived mesenchymal stem cells(BMSCs),as a type of pluripotent adult stem cells,are widely present in umbilical cord blood,bone marrow and other tissues,with good proliferation ability and multiple differentiation potentials.Because of its extensive self-source,simple extraction,and no ethical issues,BMSCs are considered to be the most ideal cell source for neural tissue engineering.Nervous system repair needs to be controlled within a short treatment cycle to reduce irreversible damage caused by the damage.Therefore,it is necessary to develop a faster and more effective treatment to repair the nervous system damage.Tissue engineering is a cross-discipline developed from the integration of cell biology,materials science,medicine and other disciplines since the 1990s,which aims to repair damaged tissues or organs through co-cultivation of cells and scaffold materials.Neural tissue engineering which includes three parts:seed cells,scaffold materials,and growth factors is a part of tissue engineering.It mainly uses tissue engineering methods to repair the damaged nervous system.Among them,the scaffold material is the core element of nerve tissue engineering.The unique physical,chemical or biological signals of the scaffold can regulate cells'attachment,arrangement,migration,proliferation,cell polarization,differentiation and other behaviors.At present,using scaffold materials to regulate the neural differentiation of BMSCs has some problems,such as loss of neural function and low differentiation efficiency.Therefore,in nerve tissue engineering,the development of scaffold material that can accelerate nerve regeneration and improve the quality of nerve regeneration is of great significance for nerve injury repair.Nerve cells are electrically active cells,and their function is closely related to the biological electrical activity in organisms.Therefore,electrical stimulation has become a commonly used means of regulating the neural differentiation of stem cells in neural tissue engineering.Electrical stimulation is a non-drug treatment method,with advantages such as less damage,economy and safety,convenient operation,and controllable parameters.However,most studies have used conductive scaffold to connect an external electric field to induce stem cell differentiation.When these wire conductive scaffolds are used to repair nerve damage,there will be problems such as infection and inconvenience,and they are not suitable for clinical application.The ideal approach is to use wireless electrical stimulation to induce stem cells to undergo neural differentiation.Therefore,as a typical power conversion material related to electricity,piezoelectric material which can convert various mechanical stimuli into electrical signals has become a suitable scaffold material.PVDF is an organic macromolecular polymer with excellent piezoelectric properties.When subjected to mechanical stress,polarization will occur inside the material,and local electric potential will be generated on the surface of the material.Meanwhile,due to its light density,stable physical and chemical properties,flexibility,and good biocompatibility,PVDF has become an ideal scaffold material.In this study,PVDF-based piezoelectric films prepared in different ways were utilized to induce the neural differentiation of rat bone marrow-derived mesenchymal stem cells(rBMSCs)under the action of non-contact ultrasonic waves.The main contents are as follows:1.PVDF piezoelectric films were utilized to induce neural differentiation of rBMSCs.In this study,PVDF piezoelectric films with different piezoelectric responses were used as the research object.rBMSCs were selected as seed cells.Ultrasonic force was used to deform PVDF to produce electrical stimulation,which was used to induce neural differentiation of rBMSCs.First,one of the PVDF piezoelectric film was prepared by casting method and uniaxial mechanical stretching technology,and its d₃₃coefficient was 0.6 p C/N.Then,rBMSCs were seeded on the PVDF piezoelectric film to test its cell compatibility.After5-day ultrasonic treatment,the number of cells cultured on the film accounts for about 70%compared with the cells cultured on the tissue culture plates(TCPs)of the 5 days,showing a good proliferation trend.The results of live/death experiment,cytoskeleton experiment and cell dehydration experiment confirmed that the cells seeded on the PVDF film have good adhesion and spreading effects.In order to verify whether the stem cells undergo neural differentiation,real-time quantitative polymerase chain reaction(q-PCR)detection was performed after 21-day ultrasonic treatment.The results show that the expression of neural-related genes Nestin,Tuj1,MAP2 and GFAP is relatively weak.However,related studies have found that a certain intensity of electrical stimulation signals can induce neural differentiation of mesenchymal stem cells.Considering the poor piezoelectricity of the PVDF film prepared by the first method,the electrospinning technique was used to prepare another PVDF film with high voltage electrical response.Similarly,a certain number of rBMSCs were seeded on the film to explore the effect of local electrical stimulation signals generated by the deformation of the PVDF film on the differentiation of stem cells under the action of ultrasonic wave.The d₃₃coefficient of the PVDF piezoelectric film prepared by the electrospinning technology can reach 26.8 p C/N,which is two orders of magnitude higher than the piezoelectric response of the above-mentioned PVDF film.CCK-8 experiments show that PVDF piezoelectric films have good cell compatibility.After 2-day ultrasonic treatment,the live/death experiment,cytoskeleton staining and cell dehydration experiments confirmed that the PVDF piezoelectric film is beneficial to the attachment and spreading of rBMSCs.The q-PCR test results show that compared with the cells on the TCPs without ultrasonic treatment,the expression of Nestin,Tuj1,MAP2 and GFAP increased by about 40,10,30 and1.5 times,respectively,after 21-day ultrasonic treatment.This indicates that the PVDF piezoelectric film prepared by electrospinning technology is beneficial to induce the neural differentiation of rBMSCs.Immunofluorescence staining experiments also confirm this point at the protein level.In order to verify whether the differentiated nerve cells have neural function,calcium spark experiment were further carried out.In the experiment,no changes in the intracellular calcium ion concentration are observed,indicating that the differentiated nerve cells are immature and do not have nerve function.From the above results,it can be concluded that the PVDF film with high-voltage electrical response can induce the neuron-like differentiation of rBMSCs,that is,the differentiated nerve cells are nerve cells at the gene and protein level,but do not have the function of nerve cells.Although this work is inadequate,it confirms the potential of electrical stimulation signals in regulating the neural differentiation of stem cells,laying the foundation for future work.2.FeOOH/PVDF piezoelectric films were utilized to induce neural differentiation of rBMSCs.The previous work confirms that the electrical stimulation signal provided by the PVDF piezoelectric film can regulate the differentiation of rBMSCs into immature nerve cells.However,if there is no neurological function,it does not have the potential to repair the damage of the nervous system.Some studies have pointed out that iron ions can promote the development and maturation of neurons and play an important role in the development of the human nervous system.In view of this,the high-voltage electrical response PVDF fiber film prepared by electrospinning technology has been modified to obtain a FeOOH/PVDF piezoelectric film with both high-voltage electrical response and uniform surface morphology.The piezoelectric film was used as the research object,the PVDF piezoelectric film was used as the control group,and rBMSCs were selected as the seed cells.Ultrasound can deform the piezoelectric film to produce electrical stimulation and release iron ions into the solution.The application of the dual effects of electrical stimulation signals and iron ions in regulating the neural differentiation of stem cells has been studied.Because PVDF film is a hydrophobic material,it is not conducive to the growth of semiconductor oxide.Therefore,this chapter uses the complexation reaction and hydrothermal reaction to load a layer of rod-shaped FeOOH on the PVDF film.Preliminary work has confirmed that the electrospinning technology can prepare high-voltage electrical response PVDF film.In order to verify whether the load of FeOOH affects the piezoelectricity of the composite film,XRD,PFM,and d33 experiments were used to characterize the piezoelectric properties of the film.The results prove that the FeOOH/PVDF piezoelectric film still has a good piezoelectric response,and its d33 piezoelectric coefficient can reach 27.2 p C/N.The CCK-8 experiment shows that the cells on the FeOOH/PVDF piezoelectric film account for about 80%of the cells on the FeOOH/PVDF piezoelectric film after 5-day ultrasonic treatment,compared with the cells cultured on TCPs on the same day,indicating that the cells on the film have a good proliferation trend.When the cells cultured on the film were subjected to ultrasonic treatment for two days,the live/death staining experiment results show that the piezoelectric film has good cytocompatibility.And the cytoskeleton staining and cell dehydration results of the same days also prove that the cells have good spreading and attachment effects on the piezoelectric film.Cytoskeleton staining experiments on different days show that the cells cultured on the FeOOH/PVDF piezoelectric film have a tendency of neural differentiation,and the cell shape gradually changes from a long spindle to a spherical shape.After 21-day ultrasonic treatment,the q-PCR test results show that the expression levels of Nestin,Tuj1,MAP2 and GFAP increased by about 80,130,200,and 1.25 times,respectively,compared with the cells on the TCPs without ultrasonic treatment.This shows that,compared with PVDF piezoelectric film,FeOOH/PVDF piezoelectric film is more conducive to regulating the neural differentiation of rBMSCs.The results of immunofluorescence staining can also confirm that the rBMSCs cultured on the FeOOH/PVDF piezoelectric film differentiate into nerve cells.Further calcium spark experiments confirm that the nerve cells differentiated by rBMSCs have the functions of aminobutyric and aminergic neurons.Therefore,the FeOOH/PVDF piezoelectric film can induce the neural differentiation of rBMSCs through the dual effects of iron ions and electrical stimulation without any biological growth factor.This research provides a new strategy for nerve tissue engineering,which is of great significance for repairing nervous system damage.This paper aims to explore a method of wireless electrical stimulation to induce neural differentiation of stem cells.The electrical stimulation generated by the piezoelectric effect of the PVDF-based piezoelectric material is used to induce the neural differentiation of stem cells,it is found that the effect of simple electrical stimulation on the neural differentiation of stem cells is limited,and it needs to work with other factors to induce rBMSCs to differentiate into mature nerve cells.This work is hoped that it can provide some ideas or theoretical basis for neural tissue engineering.