The Effect Of Mineralized Collagen On RAW264.7 Macrophage Polarization:an Experimental Study

Objective:With the development of tissue engineering and regenerative medicine,an increasing number people begin to realize that biomaterials can be used to regulate the inflammatory.Understanding the interaction between biomaterials and immune system has become more and more important.Macrophages play a key role in this process,the M1/M2 polarization state and inflammatory secretion affects the prognosis of the organization.Mineralized collagen(MC)which is consist of hydroxyapatite(HA)and Collagen(COL)has the same chemical components and microstructures to the nature bone tissue,and which is considered as a better biomaterial for bone prostheses compared to HA.However,after biomaterials implanted in to the body,the earliest state is surgical trauma and inflammation and the repair process is not only related to osteoblasts.However,there is little information about how MC affects inflammatory responses.Therefore,it is necessary to investigate the influence of MC to macrophage polarization and inflammation,and explain the outstanding performance of MC comprehensively and lay the foundation for MC in clinical application in the future.Methods:RAW264.7 macrophages were cultured on the surfaces of culture glasses(control grouop,LPS+IFN-γinduced group and IL-4 induced group),MC,HA and COL in the corresponding time points:1.Immunofluorescence method is applied to observe the cell morphology,cell spreading area was calculated by the NIS-Elements D software.2.Immunofluorescence method is applied to detect the M1/M2macrophage cell membrane surface specific markers CD86/CD206 expression.3.The application of ELISA method to detect the secretion of inflammatory cytokine:IL-10and TNF-αlevels in the macrophage culture supernatant.Results:1.The morphology of macrophages cultured on MC,HA and COL were different with control group and there are also differences among three groups.LPS+IFN-γinduced group cell spreading area(522.964±113.0528μm~2)is the largest,is significantly higher than the other 5 groups(P<0.0001);HA surface cells spreading area(415.695±98.681μm~2)is 1.7 times than that cultured on MC surface(242.004±31.759μm~2)and have significant difference(P<0.0001)between them.2.MC increases the number of CD206+staining macrophages and HA increases the number of CD86+staining macrophages,whereas in 3d,COL increases the number of CD86+/CD206staining macrophages.3.MC promote the secretion of anti-inflammatory cytokines IL-10,and HA promote the secretion of proinflammatory cytokines TNF-α.Conclusion:The mineralized collagen(MC)can induce macrophage RAW264.7 to M2polarization,and has the better property in the aspect of the body’s inflammatory response than that of hydroxyapatite(HA)and collagen(COL),and MC is good for tissue reconstruction.