Research On Surface Biofunctionalization Of Medical Titanium Alloy And Its Biological Properties

Titanium (Ti) and its alloy are widely used in the biomedical field owing to theirexcellent corrosion resistance and mechanical properties. The bare inert surface oftitanium (Ti) alloy has poor osteointegration with host bone tissue, and then typicallycauses early failures in implants. The aim of this study was to improve the bioactivityof titanium alloy and shorten the bone healing/integration time. Layer-by-layerself-assembly technique and TiO2nanotube (TNT) fabrication were employed tomodify the surface composition and tomography. Furthermore, silver nanoparticle andmicroRNA (miRNA) were loaded on the surface of titanium alloy to improve theantibacterial and osteogenic activities, respectively.Firstly, the morphology and biocompatibility of dopamine (DOP) modifiedhyaluronic acid and chitosan (CHI) multilayer (DHA/CHI) on Ti-Nb-Zr alloy (TNZ)surface via layer by layer self-assembly method were studied. Infrared spectroscopyand zeta potentials results proved the formation of organic multilayer film on thesurface of TNZ, AFM and water contact angle tests showed that multilayer filmreduced the surface roughness of TNZ and increased the surface hydrophilicity.DHA/CHI multilayer enhanced osteoblast cell adhesion and proliferation. Theosteogenic gene expression significantly increased. The expression levels of ALP andOCN were2and3-fold higher, respectively. The alizarin red staining indicatedenhanced mineralization degree in multilayer coated group. Furthermore, thecorrosion resistance of DHA/CHI multilayer modified titanium alloy was evaluatedby the electrochemical impedance test. DHA/CHI multilayer exhibited capacitanceeffect and showed a charge transfer blocking capability, hence reduced the corrosionrate of titanium alloy.Secondly, after modifying sodium alginate (DAL) with DOP and fabricating theDAL/CHI multilayer via layer-by-layer technology, silver nanoparticles(AgNP)-DAL/CHI multilayer was constructed on pure Ti surface as an antibacterialcoating. The silver particles with diameter of50nm uniformly distributed in theDAL/CHI multilayer. The contact angle of AgNP-DAL/CHI multilayer was about40°,which demonstrated excellent hydrophilicity. In the MTT test, the multilayer showedlow cytotoxicity. AgNP-DAL/CHI multilayer considerably reduced the activities ofEscherichia coli and Staphylococcus aureus. The antibacterial circles diameter were2.85±0.24mm and2.13±0.15mm, respectively, and the inhibitory rate was about 60%higher than that of DAL/CHI coating.Thirdly, by using the layer-by-layer self-assembly technology, miRNA(miRNA-218) mixed with conmecial transfection reagent (RNAiMAX) and anionicpolyelectrolyte DAL were constructed on pure Ti surface. The miRNA maintainedgood biological activity in multilayers and sustainably released from DAL/miRNAcoating in situ. The release rate of miRNA was about1.8pmol/μL after4h, andgradually reached the stable level of4.7pmol/μL after24h. The cytotoxicity testindicated no cell toxicity. The fluorescence microscopy observation showed thatmiRNA was transmitted into the cytoplasm. The expression level of ALP increasedsignificantly when compared to Ti and DAL.Furthermore, fetal bovine serum (FBS) was loaded into TNT on the pure Tisurface. SEM observation showed that the tube wall of TNT became thicker, while thediameter was thinner after FBS loading. The contact angle of TNT/FBS was about30°,which suggested good hydrophilic property. TNT/FBS coating significantly promotedthe early cell adhesion. Cell morphology observation under SEM and fluorescencemicroscope showed that preosteoblast cells appeared large lamellipodia. The sustainedrelease time of FBS from TNT/FBS was longer than that of Ti/FBS with burst releaseperiod up to7d. The total amount of FBS loaded on TNT was about30μg/cm2.Influence of coating on cell proliferation was according to TNT/FBS> Ti/FBS> Ti>TNT sequence. The expression levels of ALP and OCN were in accordance withTNT/FBS> TNT> Ti/FBS> Ti. Alizarin red staining results also showed thatTNT/FBS considerably induced mineral deposition.Finally, miRNA was incorporated into TNT to constructed TNT/miRNA coatingon pure Ti surface. TEM observation showed that miRNA was approximatelyspherical size with diameter of30~40nm, and the diameter of TNT was about150nm. The fluorescence microscopy observation indicated that the miRNA uniformlydistributed in TNT. The water contact angle changed from0°to17±3°after loadingmiRNA into TNT. Nanodrop test showed that the release amount of miRNA increasedlinearly with time, and the total amount of miRNA reached the stable level of19pmol/μL after12h. Cytotoxicity test indicated that the TNT/miRNA coating had nocell toxicity. The fluorescence microscopy showed miRNA was transmitted into thecytoplasm, which induced the significant increase of ALP expression level.In summary, both layer-by-layer self-assembly technique and TNT modificationwith silver nanoparticle and/or miRNA loading can reduce the risk of infection and promote bone regeneration. The results of this study showed that biological surfacemodification on titanium alloys has great potential in orthopedic application forstimulating bone repair.