The Effect And Mechanism Of Polarized Poly(Vinylidene Fluoridetrifluoroethylene) Biofilm On Osteogenic Differentiation In Rat Bone Marrow Mesenchymal Stem Cells

Background and ObjectiveAlveolar bone defect caused by tumors,trauma,and periodontal disease creates difficulties for dental implant.Thus,improved bone condition by bone augmentation surgery is required in clinic.Guided bone regeneration is one of the most widely used methods,in which the barrier membrane plays a key role.Most of the available membranes merely act as mechanical barrier and lack of the ability of osteogenic induction,which compromised bone regeneration efficacy in massive alveolar bone defect.Therefore,it’s urgent to improve the bone induction ability of the barrier membranes to accelerate bone regeneration.Poly(vinylidene fluoridetrifluoroethylene)membrane is known as a biomimetic electroactive material because of its good biocompatibility.In addition,this membrane is also designed to effectively prevent infection induced by exogenous electrical signal devices as well as to facilitate the biological effect and biosecurity.Recent studies have shown that polarized membrane can promote the biological activity of osteoblasts,but it is still controversial with regard to the cellular response on different charge properties.Few reports have investigated the effects of different charge properties on osteogenic activity.Therefore,the purpose of this study is to apply electroactive materials onto barrier membranes,and investigate the regulation efficiency of different charge properties of barrier membrane on the osteogenic differentiation of rat bone marrow mesenchymal stem cells,and to provide theoretical basis and practical reference for future application of biomimetic electroactive materials in oral implants.Methods1.P(VDF-TrFE)was prepared in a casting manner.The experiment was set up with negative group,positive group and non-polarization group which is the control group.The polarization was achieved by charging the membrane with 6kV/cm electricity in an oil bath polarizing device at 120℃ for 1 hour.2.The morphology and crystal form of the material were analyzed using scanning electron microscope,Fourier transform infrared spectroscopy and X-ray diffraction spectroscopy.The physical and electrical properties after polarization were tested by wide range d33-meter.3.rBMSCs were co-cultured with the membrane.The biocompatibility of the membrane was detected by cck-8 and LDH.Cell morphologies on the membrane were observed through fluorescent microscope,and the ability of different electrical properties on osteogenic differentiation is detected by alkaline phosphatase activity,alizarin red staining,PCR,and Western blot.4.Mitochondrial Membrane Potential Assay,dead/live staining and mitophagy related assays were performed to investigate the relevant mechanisms between different electrical properties and osteogenesis.Results1.β phase of P(VDF-TrFE)were significantly increased after polarization,and the piezoelectric constant(d33)reached±10pC/N.The surface morphology、Water contact angle and elastic Modulus of P(VDF-TrFE)had no obvious changes after polarization.2.LDH results showed Polarization did not cause cytotoxicity.cck-8 results showed the cell proliferation rate in negative P(VDF-TrFE)group was significantly higher than that in positive group and control group(p<0.05).ALP,Alizarin red results showed that negative P(VDF-TrFE)group had the highest osteogenesis efficiency,followed by positive group(p<0.05).PCR and Western blot experiment had the same results.3.MMP results showed the mitochondrial membrane potential of the negative P(VDF-TrFE)group was depolarized(p<0.05).Dead/live staining results shown the living cells reach 80%of the total on three groups.PCR and Western blot results showed mitophagy was activated in negative P(VDF-TrFE)group,Which indicating that negative P(VDF-TrFE)promoted osteogenic differentiation through mitophagy.Conclusions1.P(VDF-TrFE)were prepared by casting method,and the micro-electric environment was successfully constructed through polarization.2.Polarized P(VDF-TrFE)membrane had good biocompatibility.Both positive and negative polarization promoted the proliferation and differentiation of rBMSCs.The negative group had higher biological activity,and better osteogenic differentiation efficiency.3.Mitophagy might play a key role in electrical stimulated-osteogenesis.This study provided new ideas and theoretical basis for the further application of electroactive biomaterials.