Detection Of Corrosion Resistance Of Titanium Modified By Antimicrobial Peptide GL13K And Regulation Of Macrophage Behavior To Promote Osteogenic Differentiation And Angiogenic Differentiation

Objective:?To explore the corrosion resistance of pure titanium modified with antimicrobial peptide GL13 K and how to regulate the behavior of macrophages to promote osteogenic differentiation and angiogenic differentiation.Methods: 1.The change of corrosion resistance of pure titanium surface modified by antimicrobial peptide GL13K:(1)the phase structure difference among pure titanium,silanized titanium and antimicrobial peptide GL13 K modified titanium was analyzed by X-ray diffractometer(X-Ray diffraction,XRD).(2)the ratio of AC potential to current signal of three groups of titanium materials in the electrochemical system was detected by electrochemical impedance spectroscopy(Electrochemical impedance spectroscopy,EIS).According to the Nyquist and Bodephase and amplitude diagrams of impedance data,the corresponding curves were drawn and the differences of corrosion resistance of the three groups of titanium materials were analyzed.2.The proliferation of bone marrow mesenchymal stem cells modified by antimicrobial peptide GL13 K on the surface of pure titanium was detected:(1)the proliferation of bone marrow mesenchymal stem cells cultured directly on the surface of three groups of titanium was detected by CCK-8 kit;(2)the proliferation and aggregation of bone marrow mesenchymal stem cells on the surface of three groups of titanium were directly observed by immunofluorescence staining.(3)the adhesion and proliferation of bone marrow mesenchymal stem cells on the surface of three groups of titanium materials were quantitatively analyzed by RT-q PCR to detect the cytoskeleton proteins such as FN,VCL,ITGA3 and so on.3.Detection of RAW264.7 proliferation and M1 / M2 polarization of macrophages modified by antimicrobial peptide GL13 K on the surface of pure titanium:(1)the changes of RAW264.7 proliferation of macrophages cultured directly on the surface of three groups of titanium materials were detected by CCK-8 kit.(2)the high expression of CD11 c in M1 macrophages and the high expression of CD206 in M2 macrophages were used as markers for the identification of macrophage polarization.The macrophage markers were detected at cellular level by flow cytometry to determine the M1/M2 polarization state of macrophages.4.Effects of cultured macrophages on bone healing after antimicrobial peptide GL13 K coating on pure titanium: to observe the osteogenic differentiation potential of bone marrow mesenchymal stem cells on GL13 K coated titanium surface and the expression of related bone forming gene proteins,osteoblast differentiation and related gene expression of macrophages under the stimulation of macrophages.5.To explore the effect of antimicrobial peptide GL13 K coating on angiogenesis of pure titanium:(1)to detect the proliferation of human umbilical vein endothelial cell HUVECs on GL13 K coated titanium surface under the stimulation of macrophages by CCK-8 kit,and(2)to detect the effect of three groups of titanium on the angiogenic differentiation of human umbilical vein endothelial cell HUVECs by Tanswell migration test.(3)the difference of HUVECs angiogenesis related gene protein expression in human umbilical vein endothelial cells on the surface of the three groups was quantitatively detected by RT-q PCR at the molecular level.Results :1.The corrosion resistance tests of three groups of titanium materials were carried out:(1)XRD results show that the phase structure of silanized coating titanium and antimicrobial peptide GL13 K coating titanium is basically consistent with that of pure titanium,that is,silanized coating and GL13 K coating do not destroy the internal structure of titanium material,but still maintain the internal structure stability of titanium material.(2)the data of Nyquist curve in the results of EIS show that pure titanium and antimicrobial peptide GL13 K coating titanium show a larger impedance radius,while the titanium radius of silanized coating is the smallest,that is,antimicrobial peptide GL13 K coating has good electrochemical corrosion performance,and the impedance modulus of Bode amplitude diagram shows the same trend in the low frequency range.2.The proliferation of bone marrow mesenchymal stem cells on the surface of three groups of titanium materials:(1)the results of CCK-8 kit showed that the proliferation of bone marrow mesenchymal stem cells cultured directly on the surface of the three groups increased with the extension of culture time,and there was no significant difference among the three groups.(2)the results of immunofluorescence detection showed that bone marrow mesenchymal stem cells proliferated with the prolongation of culture time after being directly cultured on the surface of the three groups of materials,and the cells on the surface of titanium coated with antimicrobial peptide GL13 K showed obvious mutual aggregation.(3)RT-q PCR results showed that the expression of FN,VCL,ITGA3 and other cytoskeleton genes of bone marrow mesenchymal stem cells cultured on the surface of three groups of titanium increased with the extension of culture time,and there was no significant difference among the three groups.3.The proliferation of macrophages on the surface of three groups of titanium materials:(1)the results of CCK-8 kit showed that macrophages proliferated with the prolongation of culture time after directly cultured on the surface of the three groups,and there was no significant difference in proliferation among the three groups.(2)GL13K-coated titanium could activate a higher proportion of pro-inflammatory M1 macrophages at the initial stage of culture,but after 7 days of culture,the plan changed,mainly anti-inflammatory M2 macrophages.4.The results of osteogenic differentiation experiment showed that the titanium surface coated with GL13 K was beneficial to the osteogenic differentiation of BMSCs.There was no significant difference between ALP staining and ALP,OC and RUNX2 gene expression.Alizarin red staining and COL-1 α1 gene expression increased on the surface of GL13 K coated titanium.5.In this experiment,the model of indirect co-culture of macrophages and HUVECs on titanium surface was constructed.CCK-8 and Transwell experiments showed that the surface of titanium group coated with GL13 K created a good local microenvironment for the proliferation and migration of HUVECs.The results of this study also showed that GL13 K coated titanium group stimulated the expression of vascular related genes(ICAM-1,angs,VEGF-A and KDR)at different time points,and increased the secretion of VEGF-A cytokines.Conclusion : 1.Titanium materials modified by antimicrobial peptides GL13 K can maintain good internal structural stability,and the corrosion resistance is not changed.2.Titanium modified by antimicrobial peptide GL13 K shows good biocompatibility on bone marrow mesenchymal cells and macrophages,and has a tendency to promote the mutual aggregation of bone marrow mesenchymal stem cells and properly regulate the polarization of macrophages to M1 / M2,thus promoting the occurrence of a series of cell biological behaviors.3.GL13 K modified titanium surface in vitro is beneficial to the formation of local microenvironment for macrophages to promote tissue repair and promote osteogenic differentiation and angiogenic differentiation.4.Osteogenic differentiation of bone marrow mesenchymal stem cells can be regulated by BMP-2/M-CSF pathway.