Osteogenesis Of BMSCs-GelMA/MS-BMP9 In Inflammatory Microenviroment And Its Effect On The Biological Behavior Of Marophage

Background:trauma,inflammation and tumor are the main causes of bone defect in oral and maxillofacial region.Bone regeneration is a major problem to be solved in clinic.With the development of immunology,the importance of bone immune microenvironment in the repair of bone defect has been paid more and more attention.The treatment of bone defect is mainly bone transplantation with bone substitute material,and the existing bone graft material is mainly through operation flap to expose the bone defect for bone transplantation,it has some disadvantages,such as great trauma,much bleeding,slow repair,and the immune inflammatory reaction may occur after the implantation of this kind of material,which affects the formation of new bone.The aim of this study was to find an injectable and minimally invasive bone graft substitute that can regulate the bone immune microenvironment.Objective:(1)To prepare injectable gel microspheres with good Biocompatibility by microfluidic technology,and to prepare engineered microspheres capable of continuously secreting BMP9 protein by gene transfection and three-dimensional cell culture,as a scaffold material in bone tissue engineering.(2)To verify the osteogenic effect of engineering microspheres in vivo and in vitro.(3)To explore the effect of engineering microspheres on polarization of macrophages in vivo and on osteogenesis under the control of immune microenvironment through rat inflammation model.(4)To investigate the biological changes and mechanism of macrophages under the regulation of inflammation by engineered microspheres in vitro.Methods:(1)In this study,BMSCs,the ideal cells in bone tissue engineering,were selected by using the principle of tissue engineering and microfluidic technology,the engineered gel microspheres,which can continuously express and secrete BMP9 protein,were prepared.The Biocompatibility of the gel microspheres were investigated by CCK-8,vivisection and phalloidin staining.(2)ALP staining,ALP activity and alizarin red staining were used to observe the effect of the engineered microspheres on the osteogenic differentiation of BMSCs in vitro.(3)The rat model of inflammation was established to prepare the bone defect of distal femur.After 4,8 weeks,the new bone formation was observed by Micro-CT,HE and Masson,the changes of biological behavior of macrophages under the change of immune microenvironment in vivo were observed by immunofluorescence.(4)In vitro,gene sequencing,immunofluorescence,Flow cytometry and RT-q PCR were performed by preparing conditioned medium,to investigate the effect of engineered microspheres secreting BMP9 protein on the biological behavior of macrophages.Results:(1)The injectable GelMA gel microspheres with a diameter of about153μm were successfully prepared by using two-phase microfluidic technology.The GelMA gel microspheres had good physical properties and biocompatibility.(2)The engineered microspheres were successfully constructed,which can secrete BMP9 protein continuously,promote the osteogenesis of BMSCs to differentiate in vitro,and form tissue engineered bone in vivo.(3)The engineered microspheres were injected into the osteomyelitis bone defect animal model,which proved that the engineered microspheres could promote the bone tissue regeneration under the inflammatory condition.(4)The engineered microspheres promoted the change of biological behavior of macrophages from M1 to M2 in vitro,and inhibited the expression of pro-inflammatory factors and promoted the secretion of anti-inflammatory factors.(5)Engineering microspheres secreting BMP9 protein were confirmed by gene sequencing and WB to inhibit ROS expression in macrophages under inflammatory state and down-regulate JAK-STATs signaling pathway.Conclusion:GelMA/MS prepared by microfluidic control in this study has good Biocompatibility,and the engineered BMP9-secreting microspheres can promote osteogenesis in vitro and in vivo,under the condition of inflammation in vitro,it can change the biological behavior of macrophages,regulate the immune microenvironment,regulate the phenotype of macrophages,promote the polarization of M1 macrophages to M2 macrophages,and inhibit the expression of inflammatory factors,promotes the production of anti-inflammatory factors,and constructs tissue-engineered bone in vivo,modulates the proportion of macrophage-polarized phenotypes,regulates the bone immune microenvironment,and achieves bone tissue regenerative repair in an inflammatory environment.