Research On The Construction Of Micro/Nano Structures On Titanium Surface And Their Immune Response And Osteogenic Differentiation

Chapter 1 The effect of micro-nano structured titanium surface on immunological behaviorObjective:To construct different micro/nano morphologies on Ti surface,providing study models for subsequent cell experiments.Methods:The Mg-Al-LDH film was directly formed on Ti surface by hydrothermal method after acid pickling of Ti disc,and different morphologies were constructed on the surface of samples by adjusting the concentration of Mg（NO₃）₂,Al（NO₃）₃ and urea.A scanning electron microscope（SEM）was used to observe the morphology of samples,a X-ray diffractometer was used to detect the phase composition of the film,and a X-ray photoelectron spectroscopy was used to test the chemical components and chemical state of samples.A optical contact angle measuring device was used to detect the wettability of material surface,and inductively coupled plasma atomic emission spectrometry was used to detect ion release.Results:By observing the surface of samples with SEM,three different morphologies,i.e.nano foliation structure（Nano）,micro lamellar structure（Micro）and micro-nano hybrid structure（MN）appeared on the surface of samples.The three groups of samples had the same elemental composition.The water contact angle of Nano sample group was significantly reduced,while that of Micro sample group was significantly increased.Ion release detection found that the release of Mg²⁺and Al³⁺in Nano sample group was the lowest,while that in Micro sample group was the highest.Conclusion:Nano samples have better hydrophilicity,with the lowest release of Mg²⁺and Al³⁺,while Micro samples have strong hydrophobicity,with the highest release of Mg²⁺and Al³⁺.Chapter 2 The effect of micro-nano structured titanium surface on osteogenic differentiationObjective: To explore the effect of different micro-nano morphologies on Ti surface on the polarization and inflammatory secretion of mouse monocyte-macrophages（RAW264.7）M1/M2.Methods: Mouse mononuclear-macrophages were inoculated on the surface of different samples for culture.The alamarBlue^TM kit was used to detect the proliferation of macrophages on the surface of each sample.SEM was used to observe the morphological changes of macrophages on the surface of different samples.Real-time quantitative PCR was used to detect the expression of inflammatory genes in macrophages inoculated on the surface of different samples.ELISA was used to detect the release of inflammatory factors from macrophages.Immunofluorescence was used to detect the secretion of inflammatory factors in macrophages.Flow cytometry was used to detect the surface markers CCR7 and CD206 of macrophage membrane to determine the polarization status of macrophages M1/M2.Results: Samples with different morphologies had no obvious cytotoxicity to macrophages,and macrophages proliferated well on the surface of each group of samples.The macrophages on the surface of Nano samples were fusiform with few pseudopods,while the morphology of macrophages on the surface of Micro,MN and Ti samples were similar,spherical and pseudopodia.The results of immunofluorescence staining and flow cytometry showed that compared with Ti sample,Nano sample could significantly promote M2 macrophage polarization,MN sample could promote M1 / M2 macrophage polarization,and Micro could promote M1 macrophage polarization.The results of ELISA and PCR showed that compared with Ti samples,Nano samples could promote the high expression of macrophage antiinflammatory genes,inhibit the expression of anti-inflammatory genes and promote the synthesis and release of anti-inflammatory cytokines,followed by MN samples,while Micro could promote the expression of macrophage inflammatory genes and inhibit the release of antiinflammatory genes and anti-inflammatory factors.Conclusion: The micro-nano morphology of Ti surface can regulate the polarization direction of macrophages.The surface morphology of Nano samples is beneficial to promote the polarization of M2 macrophages.The surface morphology of Micro samples promotes the polarization of M1 macrophages.MN samples can simultaneously promote the polarization of M1/M2 macrophages due to their special morphology.The surface morphology of samples has a greater effect on the polarization of macrophages than the release of Mg²⁺ and Al³⁺.Chapter 3 The effect of micro-nano structured titanium surface on the osteogenic differentiation of BMSCs by regulating the polarization of macrophagesObjective: To study the effect of the secreted products of macrophages regulated by different morphologies on the osteogenic differentiation of BMSCs.Methods: The conditioned medium of macrophages was collected by establishing an indirect co-culture model.PCR was used to detect the expression of osteogenesis genes BMP-2,OPN and OCN.ALP staining and ALP activity detection were used to observe the differentiation of BMSCs into osteoblasts.Results: Compared with Ti samples,Micro samples could promote the expression of osteogenic related genes in BMSCs,while Nano and MN samples did not significantly promote bone differentiation.There was no significant difference in the ALP expression level of BMSCs on the surface of samples.The secreted products of macrophages on the surface of Nano sample group could significantly promote the osteogenic differentiation of BMSCs,and the expression of osteogenesis genes（BMP-2,OPN,OCN）was significantly up-regulated.The results of ALP staining and ALP activity showed that the conditioned medium of macrophages in Nano sample group could promote the osteogenic activity of BMSCs,the ALP staining was deepened,and the activity of ALP was significantly higher than that of other groups.Micro structure also had a certain effect on promoting osteogenic differentiation of BMSCs,which was manifested as a significant increase in the expression of osteogenesis genes.Conclusion: Micro of the sample surface can promote the osteogenic differentiation of BMSCs.M2 macrophages play an important role in promoting the osteogenic differentiation of BMSCs.