The Preparation Of 3D Printing β-TCP Scaffold With Dual Bioactivities Of Osteogenesis And Antibacterial

Objective:Osteogenesis and antibacterial are two major difficulties in bone repair.It is meaningful to design bone tissue engineering scaffolds with excellent osteogenic activity and antibacterial properties.Bone morphogenetic protein2-mimetic peptide(BMP2-MP)is a peptide screened by BMP-2 receptor in peptide library.It has the properties of BMP-2,small molecular weight,low cost and it is easy to modify.Ornithodoros savignyi(OS)is a new type of antibacterial peptide derived from insect tick defensins,which has excellent bactericidal effect on gram negative and gram positive bacteria.It can meet the needs of osteogenesis and antibacterial at the same time by combining BMP2-MP and OS with the scaffold.In recent years,some scholars have developed strategies to simulate the adhesion mechanism of mussels to enhance the binding effect of biomolecules.One of the main reasons why marine mussels can firmly adhere to the surface of materials in the ocean is3,4‐dihydroxyphenylalanine(DOPA).DOPA can adhere to the surface of different substances through multiple functional groups,so it has been widely used in surface modification of biological materials.DOPA can be converted from Tyrosine(Y)under the catalysis of tyrosine hydroxylase.Lysine(K)and DOPA have a synergistic effect on adhesion.Therefore,some scholars designed YKYKY pentameric peptides to simulate mussel adhesion proteins for material modification.In this experiment,we first chose β-tricalcium phosphate(β-TCP)as experimental materials as its good biocompatibility,excellent bone conduction and osteoinductivity,and prepared β-TCP scaffolds through 3D printing technology.Then we used genetic recombination to integrate YKYKY into the ends of BMP2-MP and OS,and converted to DOPA,so that DOPA-BMP2-MP and DOPA-OS were fixed on the scaffold.Finally,a bone tissue engineering scaffold with dual biological activities of osteogenesis and antibacterial activity was obtained.Methods:1.The β-TCP scaffolds were prepared by 3D printing.Scanning electron microscope(SEM)and X-ray diffraction(XRD)were used to analyze the surface morphology and composition of the scaffold.And the biocompatibility of the scaffold was studied by cell staining.2.All proteins were labeled with Fluorescein isothiocyanate(FITC).The YKYKY-BMP2-MP and YKYKY-OS obtained by gene recombination were converted into DOPA-BMP2-MP and DOPA-OS by tyrosine hydroxylase.We took OS,BMP2-MP group as negative control,and DOPA-OS,DOPA-BMP2-MP,DOPA-OS /DOPA-BMP2-MP group as positive control.The 3D printing β-TCP scaffolds were incubated with peptides for 12 hours.The peptides binding and the surface morphology of scaffolds were observed by fluorescence imager and SEM.3.Staphylococcus Aureus and Escherichia Coli were the experimental objects.The plate colony counting method was used to evaluate the antibacterial effect of composite scaffold.4.Mouse preosteoblast cell line MC3T3-E1 was seeded on different composite scaffolds for culture,and the cytocompatibility of the composite scaffolds was tested by in vivo staining and SEM.5.After MC3T3-E1 was co-cultured with the scaffolds for 7 days,the ALP activity of different scaffolds were detected,and the osteogenic related genes Runx2 and ON were detected by PCR.Results:1.The β-TCP scaffold,with good biocompatibility,was obtained by 3D printing.Its fibers were crossed at an oblique angle of 45°and the internal pores were interpenetrated with each other.2.By combining different types of peptides with β-TCP,composite scaffolds were finally obtained: β-TCP scaffold,OS@β-TCP scaffold,DOPA-OS@β-TCP scaffold,BMP2-MP@β-TCP scaffold,DOPA-BMP2-MP@β-TCP scaffold,DOPA-OS/DOPA-BMP2-MP@β-TCP scaffold.Fluorescence imaging and SEM showed that more peptides could be bound to the surface of the scaffold by introducing DOPA.3.The results of antibacterial experiments showed that,compared with the OS@β-TCP group,the DOPA-OS@β-TCP group could exert a better antibacterial effect on Staphylococcus Aureus and Escherichia Coli.4.The cytocompatibility results showed that each group of composite scaffolds had good cytocompatibility.5.ALP activity and PCR results showed that DOPA-BMP2-MP@β-TCP group and DOPA-OS/DOPA-BMP2-MP@β-TCP group constituted higher bone activity.Conclusion:The above results showed that,by simulating the mussel adhesion mechanism and introducing DOPA into the ends of OS and BMP2-MP,more peptides could be bound to the surface of the β-TCP scaffold.Finally,the DOPA-OS/DOPA-BMP2-MP@β-TCP scaffold with good osteogenesis,antibacterial properties and excellent biocompatibility was obtained,which had certain reference significance for the repair of bone defects in the future.