Biological Effects Of Electrical Stimulation On MBMSCs And RAW264.7 Cells

As an effective mean to regulate cell behaviors,electrical stimulation has attracted widespread attention in the field of tissue engineering.Tissue engineering mainly seeds cells on conductive biological scaffolds,regulating and controlling the behaviors of cell with electrical stimulation,so as to achieve the purpose of treating tissue damage.In this study,to study the effect of electrical stimulation combined with conductive polymers on specific behaviors of cell,a composite hydrogel PDA-GO-PAAM with good conductivity and mechanical properties was prepared by polydopamine partially reduced graphene oxide(pr GO).The physicochemical properties and biological activity of PDA-GO-PAAM hydrogels were characterized,and it was used in combination with electrical stimulation to study the effects of electrical signals on the behavior of mBMSCs and RAW264.7macrophages.1.Fabrication and characterization of composite hydrogels:PDA-GO-PAAM composite hydrogels with good conductivity and mechanical properties can be obtained by PDA partially reduced GO and mixed with acrylamide monomer,and the PAAM and PDA-PAAM hydrogels were used as control.Morphological observation was used to observe the morphology.And the Raman spectrum,the conductivity test,and the dynamic thermomechanical tensioner were performed to analyze the physicochemical properties of the hydrogels.Morphological observation shows that all hydrogels showed similar pore structures in terms of porosity,indicating that the doping of PDA and pr GO had little interference on the pore structure.The Raman spectrum suggested that the I_D/I_Gof pr GO was 1.100,which was higher than that of GO(1.008)and lower than r GO(1.330),indicating that GO was partially reduced.The conductivity results showed that the doping of pr GO endows the composite hydrogel with good electrical conductivity(8.23×10^-4S/cm),almost 25 times higher than the conductivity of 3.34×10^-5S/cm of pure hydrogels.In addition,the tensile strength of the composite hydrogels can reach 9.1 KPa,which can due to the combination of nanocomposite effect of nanoparticles and self-generated covalent cross-linking of the hydrogels to enhance the mechanical properties of the hydrogels.2.Biological properties of composite hydrogels:mBMSCs and RAW264.7macrophages were used to determine the cytotoxicity and differentiation/polarization ability of different hydrogels.Cell proliferation on different hydrogels was measured by cell counting kit 8(CCK8).Real-time quantitative fluorescence PCR,alkaline phosphatase activity,immunofluorescent staining and flow cytometry were used to evaluate the biological function of different hydrogels.The results showed that,compared with the control group,the composite hydrogel had good biocompatibility and could promote the proliferation of cells on its surface.Meanwhile,the adhesion factors(NCAM,integrin)secreted by mBMSCs cells on the surface of the composite hydrogel was higher,which could significantly promote cell adhesion.In addition,PCR,alkaline phosphatase activity and flow cytometry results showed that the composite hydrogel could significantly promote the osteogenesis and chondroblast differentiation of mBMSCs,and regulated the immune polarization response of RAW264.7.3.Effect of electrical stimulation with composite hydrogels on mBMSCs cells:By applying different electrical stimulation parameters,cell proliferation was measured by cell counting kit 8,real-time quantitative fluorescence PCR,alkaline phosphatase activity and immunofluorescent staining was used to evaluate the effect of electrical stimulation on the behavior of mBMSCs.The results showed that ES posed a lesser impact on the cell proliferation,but significantly promoted the expression of cell adhesion molecules compared with the unstimulated group.Electrical stimulation increased the diffusion area of MBMSCs on the surface of the conductive hydrogel,and the expression of osteoblast genotype and phenotype was stronger,especially under the parameter of 1V/cm and 1 hour stimulation every day.In addition,electrical stimulation also significantly promoted chondroblast differentiation.4.Effect of electrical stimulation with composite hydrogels on RAW264.7 cells:By applying different electrical stimulation parameters,cell proliferation was measured by cell counting kit 8,real-time quantitative fluorescence PCR and flow cytometry was used to the effect of electrical stimulation on the behavior of RAW264.7 cells.Compared with the unstimulated group,the appropriate electrical stimulation parameters significantly promoted the cell proliferation,while the excessive voltage or long stimulation time inhibited the cell proliferation.In addition,under the effect of electrical stimulation,at the early stage of culture,the expressions of pro-inflammatory factors and anti-inflammatory factors secreted on the surface of the composite hydrogel increased significantly.With the growth of culture time,the expression levels of the two immune factors will decrease,to prevent excessive inflammation and excessive pro-healing adverse effects.The effect of electrical stimulation further enhanced the immune response of the composite hydrogel.5.Effect of electrical stimulation with composite hydrogels on the co-culture of mBMSCs and RAW264.7 cells:By applying different electrical stimulation parameters,real-time quantitative fluorescence PCR and flow cytometry was used to evaluate the effect of electrical stimulation on osteogenic differentiation of mBMSCs and immune polarization of RAW264.7.The results showed that the expression trend of pro-inflammatory factors and anti-inflammatory factors in macrophages on the surface of electrostimulation hydrogel was the same under indirect co-culture and direct co-culture.More pro-inflammatory factors were secreted in the early stage of culture,while more anti-inflammatory factors were expressed in the late stage of culture,which could cause appropriate foreign body reaction,but also could effectively promote inflammation healing.Proinflammatory factors and anti-inflammatory factors could promote the osteogenic differentiation of mBMSCs.Electrical stimulation further increased the expression of osteogenic gene.In conclusion,under the co-culture condition,electric field intensity and the expression of immune-related genes regulated by electrical stimulation are the main factor affecting osteoblastic differentiation.