Promotive Role Of P(VDF-TrFE)Membranes In Osteogenic Differentiation Via The Immunomodulatory Effects On Macrophage Phenotype Transition

Background and ObjectiveWith the development of bone immunology,the positive effect of immune cells on bone defect repair has attracted widespread attention in the academic community.Abandoning the previous concept of exploiting "inert" biomaterials,more scholars are committed to designing a biomaterial with osteoimmunomodulatory properties.Specifically,materials can adjust the behavior of immune cells to create an immune micro-environment which facilitates bone regeneration,and ultimately achieve the purpose of biomaterials to promote bone regeneration.Macrophages are the main participants in the body response mediated by biomaterials.The transition of pro-inflammatory M1 and anti-inflammatory M2 types regulates the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)and osteoblasts/osteoclasts homeostasis balance.In the inflammatory state after implantation,the transition from M1 to M2 phenotype of macrophages is a critical role in bone defect repair.Based on the high plasticity of macrophages,previous studies demonstrated that negative charges can inhibit the inflammatory response of macrophages,while positive charges stimulate the secretion of inflammatory factors.To our knowledge’s,only a few research involved in the regulation of surface charges on macrophages in the field of bone regeneration.Poly(vinylidene fluoride trifluoroethylene)(P(VDF-TrFE))membrane is an electroactive thin membrane suitable for tissue regeneration and has the ferroelectric properties of surface charging after high-voltage polarization.Considering the biological effects of macrophages on the surface charges,the aim of this study is to develop a bio-membrane with osteoimmunomodulatory properties.A double-sided heteroelectric P(VDF-TrFE)membrane was fabricated.We further investigated the regulation of the M1/M2 phenotypic transition of macrophages with positive and negative charges via in vivo and in vitro tests.This study provides new insight for the development of biomaterials with osteoimmunomodulatory properties and experimental foundation for the clinical application of electroactive material.Methods1.P(VDF-TrFE)membrane preparation and high-voltage polarization,and physical properties and crystal phase composition were characterized.Also,materials electrical properties and surface charges were evaluated by piezoelectric constant D33 detector,scanning probe microscope,Zeta potential analyzer,respectively.2.Cell proliferation and cytotoxicity were evaluated by CCK-8 assay and lactate dehydrogenase kit.RAW264.7 macrophages were seeded on the P(VDF-TrFE)membrane,followed by LPS induction of M1 polarization.After the successful induction was verified,the macrophages were cultured for 24 hours using this model,and skeletal staining,ELISA,PCR,WB techniques detected M1/M2 polarization of macrophages to reveal the regulatory effects of different charged P(VDF-TrFE)membranes on the conversion of M1 phenotype macrophages to M2 phenotype.3.Macrophage-P(VDF-TrFE)membrane CM were prepared to stimulate rBMSCs.The osteogenic differentiation of rBMSCs was evaluated by fluorescence staining,ALP staining,alizarin Red Staining,PCR,WB techniques to evaluate the effect of charged P(VDF-TrFE)membrane-macrophage microenvironment on the osteogenic differentiation of rBMSCs.4.An in vivo model of the critical bone defect in the mandibular angle of rats was established for the implantation of the membrane.The osteogenesis efficiency of different charged P(VDF-TrFE)membranes and the phenotype distribution of macrophages were observed by using HE staining and immunohistochemical staining.Results1.After high-voltage polarization,the Zeta potentials of both sides of the P(VDF-TrFE)membranes were(59.67±5.03)mV and(-74.00±5.57)mV,respectively.Based on the property of the charges carried on the surface,the polarized membranes were divided into negatively charged(N-C)and positively charged(P-C)P(VDF-TrFE)membranes.The unpolarized uncharged(U-C)P(VDF-TrFE)membrane was used as a control.2.Charged P(VDF-TrFE)membranes had no apparent toxic effects on macrophages.Regarding cell proliferative rates,the P-C groups was significantly higher than those of the N-C and U-C group(p<0.05).The results of ELISA and PCR tests indicated that negatively charged P(VDF-TrFE)membrane could effectively up-regulated anti-inflammatory factors Arg-1,IL-10 expression of macrophages.Additionally,the secretion of LPS mediated inflammatory factors iNOS,TNF-α can be inhibited.Also,the expression levels of macrophages autophagy-related protein LC3II were up-regulated(p<0.05).3.The results of ALP and alizarin red staining of rBMSCs showed that CM of N-C group stimulated mineralized nodules as well as the ALP production of rBMSCs.The results of WB and PCR demonstrated significantly higher expression levels of osteogenic related genes and proteins,namely,COL-I,OPN and ALP(p<0.05).4.More new bone tissues were observed in N-C group.Moreover,the immunohistochemical images exhibited that more Arg-1 positive cells around the bone defect can be found in the N-C group(p<0.05).Conclusions1.The P(VDF-TrFE)membrane was successfully fabricated,which obtained stable piezoelectric properties and surface electrical activity through high-voltage polarization.2.Negatively charged P(VDF-TrFE)membrane promoted the transition from M1 to M2 phenotype of macrophages,and its induction of macrophage M1 to M2 phenotype conversion has a certain promotion effect on the osteogenic differentiation of rBMSCs.3.Negatively charged P(VDF-TrFE)membrane still has a good osteogenic effect and the activation function of M2 polarization in vivo.