The Effect Of Surface Properties Of Bone Implant On Protein Adsorption And Cell Behaviors

Titanium and its alloys have been widely used as orthopedic implants due to their excellent biocompatibility and corrosion resistance.The biocompatibility of titanium is believed to be closely related to the existence of native titanium oxide?TiO₂?thin layer on its surface.However,the poor bioactivity of TiO₂ may lead to unsatisfactory osteogenesis and osseointegration,causing implant instability followed by high risk of surgery failure,thus the titanium surface need to be modified for enhanced bone integration.Previous studies have reported the effect of TiO₂ properties including crystal structure,wettability and topography on osteoblast behaviors,yet conflicting results were reported.Most studies have paid much attention to the osteogenic performance,but the detailed influence mechanism is still obscure.Therefore,additional studies on the protein level as well as the molecular pathways inside the cell need to be undertaken.Besides,osseointegration is a complex process in which many kinds of cells act together.Previous reports have focused on how material surface modifications can promote stem cell differentiation toward osteoblast,but neglected the management of osteoimmunomodulation property and nerve regeneration microenvironment.Based on the above background,in this work,the surface properties of titanium implants,including crystalline phase,wettability and topography,were regulated separately.Biological performance including adsorbed protein conformation,integrin binding specificity,intracellular signaling pathways as well as cell responses were investigated.Meanwhile,the influence of microstructure on the photoelectric performance of TiO₂ and the photocurrent stimulation of neuronal differentiation was studied.The main results were summarized as follows:1.Anatase and rutile TiO₂ thin films with similar surface topography and wettability were prepared by atomic layer deposition?ALD?and post-deposition annealing.In vitro cellular assays demonstrated that the anatase phase led to better osteoblastic behaviors in terms of cell adhesion,proliferation,differentiation,mineralization as well as osteogenesis-related gene expression when compared with the rutile phase.We investigated the difference between the anatase and rutile TiO₂films at the biomolecular level to explain the enhanced osteogenic activity of the anatase film.It was found that the presence of more Ti-OH groups on anatase surface induced more exposed cell-binding sites of adsorbed fibronectin?FN?on its surface,which promoted cell adhesion as well as subsequent osteoblastic behaviors.2.Surface wettability of TiO₂ were modulated by ALD,UV irradiation and micro-contact printing.Enhanced anti-inflammatory and pro-healing performance of macrophage was observed on hydrophilic surfaces compared to hydrophobic ones.In addition,macrophage on hydrophilic surface may contribute to tissue regeneration through the higher expression of osteogenic cytokines,such as BMP-2 and TGF-?1.Further study on the influence mechanism demonstrated that the surface wettability controlled specific proteins?fibronectin and fibrinogen?adsorption and conformation,which activated different signaling pathways?PI3K/Akt and NF-?B?through selective expression of integrin?1 or?2 to influence the behaviors of macrophage.3.Sub-micropatterned surfaces?pillars and pits?were fabricated to elucidate how surface topography altered macrophage responses.The results indicated that sub-micron features could help polarize macrophage toward an anti-inflammatory,pro-healing M2 phenotype compared to flat control.Additionally,different sub-micron features caused diverse macrophage responses.Short pillars?height=150nm?and pits?depth=150 nm?in this work could better enhance macrophage spreading and M2 polarization compared to high pillars?height=400 nm?.Further study on the influence mechanism demonstrated that topographical cues could affect the distribution and conformation of adsorbed proteins.We concluded that sub-micron features could induce better fibronectin conformation with more exposed cell-binding sites compared to flat surface.Moreover,surface with short pillars and pits could facilitate subsequent cell binding to adsorbed fibronectin.Better cell binding to fibronectin was related to the higher expression of integrin?1,which activated intracellular PI3K/Akt signaling pathway,inhibiting NF-?B downstream,and thus resulted in the anti-inflammatory phenotype of macrophage.4.TiO₂ thin films were deposited using different ALD cycles and different temperatures to understand the effect of microstructure on the photoelectric properties.It was found that the photoelectric property of TiO₂ film was promoted with the increasing of crystallinity.The band gap of TiO₂ thin film decreased from 3.13 eV to2.90 eV when the grain size decreased from around 200 nm to 20 nm,indicating that the optimal grain size around 20 nm could help reduce the band gap of TiO₂,resulting in higher charge carrier density.Besides,the columnar grains were conducive to charge carrier transportation.Accordingly,TiO₂ film deposited at 300°C using 1500ALD cycles exhibited columnar nanocrystalline structure,which showed significantly enhanced photoelectric property.The photocurrent around 2?A/cm² facilitated PC12cells spreading and proliferation.Neurite development of PC12 cells was also observed to be largely stimulated by photocurrent,indicating the enhanced neuronal differentiation.