Immunomodulatory Effects Of Calcium And Strontium Co-Doped Titanium Oxides On Osteogenesis

As an implantable medical biomaterial,titanium(Ti)and its alloys have a wide range of application in the fields of orthopedics and dentistry due to their good corrosion resistance,low modulus,good anti-fatigue performance and excellent biocompatibility.However,the immune response will be activated when these biomaterials are implanted in host,which can cause the foreign body reaction,the undesirable inflammation,the fibrous tissue formation and so on.It could impair the integration between the biomaterial and its adjacent tissue,and cause the loosening or even failure of the biomaterial.Macrophages play an important role in the immune response,and polarize into M1 macrophages to cause inflammation or M2 ones to repair tissue under certain conditions.It has been proved that M2 macrophages contribute to the use of biomaterials in clinic.Calcium(Ca)and strontium(Sr),as two elements the body needs,concurrently appear around the implant in clinic.Though many studies find that Ca or Sr plays a role in osteogenesis or immunomodulation,their potential synergistic effects on osteogenesis and the immune response have not been explored to date.In this work,based on Ti substrate,the coating with different ratios of Ca and Sr was fabricated through micro-arc oxidized(MAO)andelectrochemical treatment(ECT),and further evaluate the immunomodulatory effects of Ca and Sr co-doped titanium oxides on osteogenesis.In term of the characteristics of samples,Sample surface morphology was visualized by scanning electron microscopy(SEM),and the chemical states of Ca and Sr were determined by X-ray photoelectron spectroscopy(XPS).Ca and Sr release kinetics were assessed by inductively coupled plasma atomic emission spectrometry(ICP-AES).Sample wettability was determined by measuring the contact angles.Then the in vitro experiments were carried out.After RAW264.7 cells were seeded onto the surfaces of samples,the cell viability was assessed by live/dead staining,the cell proliferation by CCK-8,the cell phenotypes by flow cytometry and immunofluorescence staining,the cytokines by enzyme-linked immunosorbent assay(ELISA),the genes and proteins of cell polarization by real-time polymerase chain reaction(RT-PCR)and western blotting,respectively.In addition,after the co-culture of BMSCs and RAW264.7,extracellular matrix(ECM)mineralization assay,alkaline phosphatase activity(ALP)assay,expression of osteogenic and adipogenic genes were carried out to assess the effect of macrophage polarization on the osteogenic differentiation of BMSCs.Finally,the mouse air pouch model was established in order to evaluate the effect of samples on macrophage polarization.Flow cytometry and ELISA were respectively used to analyze macrophage phenotypes and cytokines in the air pouch exudate.HE staining,Masson trichrome staining and immunofluorescence staining were respectively used to analyze the formation of fibrous tissue,the infiltration of inflammatory cells and the phenotypes ofmacrophage polarization.According to the in vitro results,the Ca and Sr co-doped samples induced more M2 macrophages when compared to other samples without Ca and Sr or with one of Ca and Sr.And the coating with a Ca/Sr ratio of 2:1 was more favorable to induce macrophage polarization toward M2,up-regulate anti-inflammatory cytokine(IL-10),growth factors(BMP2 and VEGF),and down-regulate pro-inflammatory cytokine(TNF-?).The micro-environment created by M2 macrophages significantly induced the osteogenic differentiation of BMSCs.Being consistent with the in vitro results,the in vivo results further demonstrated that the coating with a Ca/Sr ratio of 2:1 showed a similar effect on macrophage polarization and the production of cytokines.Moreover,the analyses of histology showed that the coating significantly inhibited the formation of fibrous tissue and reduced the infiltration of inflammatory cells.In conclusion,the in vitro and in vivo results make it clear that the osteoimmunomodulatory effect of a titanium-oxide coating can be enhanced by modulating the concentration ratio of its components such as Ca and Sr.It also providing new method and insight into the development of novel medical biomaterials in clinic.