Immunological Effects Of Tantalum Particles And Their Application In Tissue Engineering Periosteum

Background:(1)The inflammatory microenvironment created by macrophages has been proven critical for bone regeneration.Both tantalum nanoparticles and micron particles have been applied to bone tissue engineering and have achieved good efficacy,but their effects on immune microenvironment remain unclear(2)The periosteum is rich in vascular networks,osteoprogenitor cells,and stem cells and plays an important role in bone defect repair.Previous studies on bone defect repair mainly focused on the optimization of bone graft materials,structural design,and drug delivery to promote bone repair,while ignoring the importance of periosteum in bone defect repair.Objectives:(1)To clarify the different effects of tantalum nanoparticles and tantalum microparticles on macrophage innate immunity in vitro and in vivo and the specific mechanisms.(2)Construct polycaprolactone composite tissue engineered periosteum doped with tantalum nanoparticles and zinc oxide nanoparticles and verify its biological properties.Method:(1)The morphological characteristics of tantalum nanoparticles and tantalum microparticles were observed and recorded by transmission electron microscope(TEM)and scanning electron microscope(SEM).The biocompatibility of the two tantalum particles was evaluated by CCK-8 kit and Live/Dead staining.The morphological changes of RAW264.7 cells were observed by confocal microscopy.The effects of the two tantalum particles on the expression of inflammation-related genes in macrophages with or without LPS stimulation were detected by real-time fluorescence quantitative PCR.The polarization typing of macrophages was detected by flow cytometry and immunofluorescence.The content of reactive oxygen species in macrophages was detected by reactive oxygen species detection kit.The Proteome Profiler Mouse XL Cytokine Array was used to detect the changes of secretory cytokines in macrophages.The effects of two tantalum particles on peri-tissue inflammation in vivo were investigated by the mouse air pouch model.(2)The construction of tantalum nanoparticles,zinc oxide nanoparticles and PCL composite tissue engineering periosteum was completed by electrospinning method.Scanning electron microscope(SEM)and laser confocal microscope were used to observe the surface morphology of composite tissue engineering periosteum.The physical properties of tissue engineered periosteum were tested by tensile test,ion release test and X-ray photoelectron spectroscopy(XPS).Rat mesenchymal stem cells(BMSC)and rat bone marrow endothelial progenitor cells(EPC)were co-cultured with the constructed tissue engineered periosteum to verify their biocompatibility.The in vitro osteogenic properties of tissue engineered periosteum were tested by ALP staining,Sirius red staining,alizarin red staining and real-time fluorescence quantitative PCR.The in vitro canalization performance of tissue engineered periosteum was tested by Transwell chamber,real-time fluorescence quantitative PCR and tubularization test.The subcutaneous angiogenesis,immune regulation and antibacterial properties of tissue engineered periosteum were tested by subcutaneous implantation in rats.The ability of tissue engineering periosteal to promote bone integration in vivo was examined by rat model of large skull bone defect.Results:(1)Tantalum nanoparticles and tantalum micron particles were characterized and found to have good biocompatibility.Under the activation of lipopolysaccharide,the morphology of macrophages co-cultured with tantalum nanoparticles was more similar to M2 phenotype.At the same time,tantalum nanoparticles also decreased the gene expressions of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and inducable nitric oxide synthase(i NOS),and increased the expressions of transforming growth factor-β1(TGF-β1)and interleukin-10(IL-10).In addition,under the intervention of tantalum nanoparticles,the infiltration of M2 macrophages increased in the tissues surrounding the mouse model of subcutaneous balloon inflammation.(2)In vitro results showed that PCL/Ta/ZnO tissue engineered periosteum has good biocompatibility,excellent in vitro osteogenic properties,antibacterial properties and promoting angiogenesis.In addition,the results of in vivo experiments show that PCL/Ta/ZnO tissue engineered periosteum still has good angiogenic and antibacterial properties in rats,and can create an immune microenvironment to promote tissue repair through the release of zinc ions.Finally,PCL/Ta/ZnO tissue engineered periosteum showed a good ability to promote bone tissue repair in the rat model of large skull defect.Conclusion:(1)Compared with tantalum nanoparticles,tantalum nanoparticles can effectively regulate the immune response of macrophages and improve the immune microenvironment of local tissues.ROS may be the key factor for tantalum nanoparticles to regulate the immune microenvironment.(2)The successfully prepared PCL/Ta/ZnO tissue engineered periosteum can effectively antibacterial,promote angiogenesis,and promote bone defect repair.