Surface Bioengineering Of Orthopaedic Implants With Optional Functions Via Mussel Bioinspired Molecular Adhesion And Bioorthogonal Conjugations

Part Ⅰ:Synthesis of mussel bio mimetic peptide DOPA4-DBCO and its application in surface modification of various materialsObjective:To synthesize biomimetic peptides from mussel and study their potential applications in surface modification of various solid materials.Methods:Based on the standard solidphase peptide synthesis strategy of fluoromethoxycarbonyl(Fmoc),the biomimetic peptide DOPA4DBCO containing dibenzocyclooctyne(DBCO)functional group was synthesized.DOPA4-DBCO was used to modify the surface of various solid materials,such as cell culture polystyrene sheet,glass sheet,stainless steel sheet,titanium sheet and polyetheretherketone sheet commonly used in orthopedics clinic.The effects of surface modification on the structure and properties of the materials were analyzed.Results:The biomimetic peptide DOPA4-DBCO was successfully synthesized and coated on the surface of various materials by simple soaking,thus,the surface roughness and hydrophilicity of the material are significantly improved.Conclusion:The mussel biomimetic peptide DOPA4-DBCO can be used to improve the biocompatibility of various solid materials by simple,rapid and efficient surface modification.which provides the basis for the functional surface bioengineering of orthopedic implants by Strainpromoted Azide-Alkyne Click Chemistry reactionPart Ⅱ:DOPA4-DBCO functionalized surface-modified orthopedic material based on bio-orthogonal loading of multiple functional peptidesObjective:To investigate the effect of DOPA4-DBCO functionalized surface modified orthopedic materials based on biological orthogonal loading of multiple functional peptides.Methods:Azide-RGD,Azide-OGP and Azide-AMP were synthesized.After DOPA4-DBCO coating was formed on the surface of titanium plates,Azide-RGD was loaded on the surface of titanium plates through bioorthogonal reaction to obtain the DOPA4@RGD Titanium plates.We cultured bone marrow-derived mesenchymal stem cells(rBMSCs)on titanium plates with different surface treatments to observe the ability of the cell adhesion.DOPA4-DBCO coating was formed on the surface of polyetheretherketone(PEEK)plates,and Azide-OGP was loaded on the surface of PEEK to get the DOPA4@OGP PEEK plates.By seeding rBMSCs on the surface of PEEK with different surface treatments,and culturing them in osteogenic induction medium,the effect on the osteogenic differentiation of rBMSCs was observed.After the DOPA4-DBCO coating was formed on the surface of tissue culture polystyrenes(TCPs),Azide-AMP was loaded on the surface of TCPS to form the DOPA4@AMP TCPs.The effect of different surface treatments on the antibacterial properties of TCPs was observed by planting bacteria on the surface of TCPs.Results:Compared with the PBS treated and DOPA4-DBCO loaded titanium plates,the results show that the surface of DOPA4-DBCO coated titanium plates were better than that of PBS treated titanium plates,the adhesion and growth of rBMSCs can be significantly promoted by DOPA4@RGD treated Titanium plates.Compared with PBS treated PEEK and DOPA4-DBCO loaded PEEK,DOPA4@OGP PEEK can significantly promote not only the osteogenic differentiation but also mineralization of rBMSCs.Compared with PBS treated TCPs and DOPA4-DBCO loaded TCPs,DOPA4@AMP loaded TCPs displayed obvious antibacterial effects.DAPI staining was used to observe the bacterial adhesion of different surface modified TCPs,the adhesion of three kinds of bacteria was significantly inhibited in DOPA4@AMP group.Conclusion:The mussel biomimetic peptide DOPA4DBCO can construct functional bioactive coating on the surface of the material by simple immersion.Bioorthogonal loading of different functional peptides can further effectively improve the cell adhesion,osteogenic differentiation,and antibacterial properties of the material.It has great application potential in the field of bone regeneration and surface modification of orthopaedic materialsPart Ⅲ:Bioclickable Mussel-Derived Peptides With BMP2p for Osseointegration of PEEKObjective:To investigate the effect of Azide-DOPA4 surface modified polyetheretherketone(PEEK)material,which were based on biological orthogonal loading of BMP-2 peptide(BMP2p).Methods:Dibenzocyclooctyne(DBCO)functional group containing BMP-2 peptide(BMP2p)and azide group containing mussel biomimetic peptide Azide-DOPA4 were synthesized.After Azide-DOPA4 coating was formed on the surface of PEEK,BMP2p was loaded on the surface of PEEK based on biorthogonal reaction to get the DOPA4@BMP2p surface modified PEEK.We used the scanning electron microscope,atomic mechanics microscope,water contact angle and X-ray positron spectroscopy to characterize the structure and properties of modified PEEK.Azide-DOPA4 was labeled with DBCOCy5 to observe the distribution of Azide-DOPA4 on PEEK.Furthermore,we synthesized BMP2p-FITC with green fluorescence to observe whether BMP2p can be better loaded on the PEEK material pretreated by Azide-DOPA4 through biorthogonal reaction.In vitro,rBMSCs were cultureded on the PEEK materials with different surface modification.After cultured in osteogenic induction medium,we used ALP staining,ALP activity detection and alizarin red S staining to observe the osteogenic differentiation of rBMSCs.The mRNA of cells was extracted,and the level of osteogenic related genes were tested by qRT-PCR.In vivo experiment,we constructed a rat critical calvarial-bone defect(5mm)model and implanted peek materials with different surface treatments.Eight weeks after operation,we collected rat calvarial bone samples and observed the repair effect of bone defect by micro-CT and histology.Results:Azide-DOPA4 and BMP2p were successfully loaded on the surface of PEEK,the surface roughness of PEEK was significantly increased,the hydrophilicity of PEEK was improved.XPS and BMP2p-FITC loading experiments results showed that,the surface of DOPA4@BMP2p modified PEEK contained more BMP2p,suggesting that Azide-DOPA4 modified PEEK could bind more BMP2p.5 days after cultured in osteogenic induction medium,cells which ALP staining were positive and ALP activity were significantly increased in BMP2p and DOPA4@BMP2p group than the cells cultured on the PBS treated PEEK group.After cultured in osteogenic induction medium for 21 days,the positive rate of alizarin red S staining was significantly higher in DOPA4@BMP2p group than the other three groups.qRT-PCR tests showed that after cultured in osteogenic induction medium for 7 days,the gene expression levels of COL1A1,BMP2 and β-Catenin were significantly up-regulated in DOPA4@BMP2p group than other three groups.In vivo experiments,the results of micro CT scanning showed that,compared with the sham and PBS group,the bone mineral density(BMD)and the volume fraction of new bone(BV/TV)of the bone defect area in the BMP2p and DOPA4@BMP2p groups were significantly increased,and the BV/TV rate was higher in DOPA4@BMP2p group than in the BMP2p group.The results of H&E and Masson histochemical staining were consistent with micro-CT,BMP2p and DOPA4@BMP2p group had a good effect on promoting bone formation,and DOPA4@BMP2p group showed better osseointegration effect.Conclusion:The biomimetic peptide Azide-DOPA4 can be used to modify the surface of PEEK materials simply,quickly and efficiently,and further loading BMP2p by biorthogonal reaction can effectively improve the biocompatibility and biological function of PEEK materials,and also enhance the osseointegration of PEEK materials and bone tissue,which provides a basis for further application of PEEK materials in trauma and tissue engineering.