Study On Cytotoxicity Effect And Its Mechanism Of Different Sized Titanium Dioxide Nanoparticles

Titanium and its alloy were widely used as bone implants in clinics, mainly due to theirgood biocompatibility and mechanical property. Nevertheless, owing to their inherentsurface inertness, titanium based implants could only integrate with surrounding bonetissue via mechanical interlocking. Thus, the fabrication of titanium substrates withdesired topography was one of the efforts to develop high quality titanium implantsduring the past decades. With the rapid development of nanotechnology, surfaceengineering titanium with nanotopography to regulate cell behaviors has attracted muchattention in recent years.However, in long-term clinical application （e. g. titanium-based hip joint implant）, wearparticles at micro-/nano-meter scale would be generated at the implant/bone interfacedue to corrosion, aseptic loosening and prosthesis friction. In this study, to simulate thewear debris on nano scale from total joint replacements, TiO₂nanoparticles withdifferent sizes were synthesized by hydrothermal method. Subsqunetly, we investigatedthe influences of different TiO₂nanoparticles on the growth of osteoblasts andmesenchymal stem cells （MSCs）, which accumulated knowledge for the development ofnovel titanium-based implants. The main contents and conclusion of the study are listedas follows:1．The synthesis of TiO₂nanoparticles with different particle sizesTiO₂nanoparticles with different sizes were synthesized via a hydrothermalmethod. Morphological features of the series of TiO₂nanoparticles werecharacterized by field emission scanning electron microscopy （FE-SEM） andtransmission electron microscopy （TEM）, respectively. The results displayed thatthe synthesized TiO₂nanoparticles had uniform feature and good dispersity, withparticle size distribution of14±3nm,39±8nm,74±15nm,108±23nm,136±34nm and196±50nm, respectively. X-ray diffraction （XRD） measurementconfirmed that the highly crystallized structure phase of TiO₂nanoparticles werein anatase phase （No. JCPDS21-1272）.2．Correlation of the cytotoxicity of TiO₂nanoparticles with different particlesizes on a sub-200nmTo evaluate the effect of nano-scale wear particles generated by titanium-basedtotal joint replacements on the growth of cells, in this study, we firstly investigated the influence of TiO₂nanoparticles with various sizes on the biological functions ofosteoblasts. Cell viability assay and alkaline phosphatase （ALP） assay indicated thatnegative effect was significantly increased with increasing the sizes nanoparticles（p<0.05or p<0.01）. Transmission electron microscopy （TEM）, fluorescent staining oflysosomes and confocal laser scanning microscope （CLSM） were used to investigate theeffect of nanoparticles on morphology of osteoblasts and the distributions ofnanoparticles within osteoblasts. The results manifested that TiO₂nanoparticles wereonly located in cytoplasm, while not penetrating into nuclei of osteoblasts. Furthermore,we investigated the uptake mechanisms of different nanoparticles by using endocytosisinhibitor. The result confirmed that TiO₂nanoparticles with different sizes entereinginto cells by different endocytosis pathways. Finally, we investigated the influence ofdifferent TiO₂nanoparticles on the expression of receptor activator of nuclear factor κBligand （RANKL）, activity of matrix metalloproteinases MMP-9and the apoptosis ofosteoblasts. The results displayed that the adverse impact of TiO₂nanoparticlessignificantly increased with increasing the particle size. In short, all results suggestedthat the negative effects of anatase TiO₂nanoparticles on osteoblasts was significantlyincreased along with increasing the particle size, which provided a basis for the safetyevaluation of titanium-based implants.3．Size-dependent influences of titanium nanoparticles on adhesion, migration,proliferation and differentiation of mesenchymal stem cellsTo investigate the influences of nano-scale wear particles derived from titanium-based artificial joint implants on integration of bone, we investigated the potentialimpacts of TiO₂nanoparticles （as models） on adhesion, migration, proliferation anddifferentiation of mesenchymal stem cells （MSCs） from cellular level to molecular level.TiO₂nanoparticles displayed correlation with cell viability, proliferation and cell cyclesof MSCs in dose-and size-dependent manners （p<0.01）. CLSM was employed toinvestigate the effects of internalized nanoparticles on the adhesion, spreading andmorphologies of MSCs. The results manifested that the integrity of cell membrane,cytoskeleton and vinculin of MSCs were heavily destroyed by large TiO₂nanoparticles.The migration assay with transwell and wound healing model suggested that TiO₂nanoparticles had strong adverse impacts on cell migration with particle sizes increasing（p<0.01）. In addition, alkaline phosphatase, gene expressions of osteocalcin （OC） andosteopontin （OPN） and mineralization measurements indicated that the sizes of TiO₂nanoparticles negatively affected the osteogenic differentiation of MSCs. The accumulated knowledge of this study might provide insight into the design of novelnano-structured titanium implants.4．Study of the effects of the surface features and chemistry of differentnanoparticles on the biological functions of endothelial cellsIn this study, three types of nanoparticles （mesoporous SiO₂, Fe₃O₄and TiO₂nanoparticles） with diameters of around100nm were synthesized and characterized byscanning electron microscopy （SEM）, X-ray diffraction （XRD） and Zeta potentialinstrument to monitor the morphology, crystalline phases and zeta-potential of thenanoparticles, respectively. Furthermore, we investigated the effects of the morphologyand surface charge of different nanoparticles on biological functions of endothelial cells.The results displayed that the dose-dependent cytotoxicities of mesoporous SiO₂andFe₃O₄nanoparticles was not obvious when the concentration of nanoparticles was lowerthan0.20mg/mL. However, the negative effects of nanoparticles significantly increasedalong increasing culture time. While under high-dose circumatance （>0.20mg/m）,different nanoparticles demonstrated cytotoxicity correlation with dose and culture time.Furthermore, the activity of lactate dehydrogenase （LDH） displayed the same trend asabove results. TEM observation suggested that the the endocytosis pathways andcytotoxicity of nanoparticles were related to their features and suface charges. CLSMobservation manifested that different nanoparticles led to the damage of cytoskeletonand integrity of cells. The potential mechanism lies in that different nanoparticlesinduced excessive production of ROS and oxidative stress, in turn leading to thedamage of mitochondria and thus accelerating cells’ apoptosis and death.