Study On The Regulation Of Biological Fnctions Of Cells By Biomedical Titanium Surface Topography And Molecular Mechanisms

Biomedical titanium（Ti）and Ti alloy materials have been widely used in clinical orthopedics and dentistry as replacement and orthodontic applications of hard tissues,mainly owing to their excellent mechanical properties and biocompatibility.Althrough literatures reported that surface topography of Ti materials greatly affected the osteogenic differentiation of osteoblasts,its potential molecular mechanism still needs to be further investigated.On the other hand,as for clinical applications in specific population（elderly people,patients suffering from diabetes or degenerative diseases）,the possibility of implant failure obvisouly increase,which is closely related to the accumulation of excessive amounts of reactive oxygen species（ROS）in their bone tissue microenvironments.In addition,the implantation of an implant inevitably activates the innate immune response of a host.Macrophages as the main effector cells participate in the regulation of the biological functions of bone-formation related cells（mesenchymal stem cells,osteoprogenitor cells,and vascular progenitor cells）,in turn affecting osseointegration between the implant and surrounding bone tissue.Thus,under both physiological and pathological conditions,the interactions between Ti implants and cells or microenvironment play an important role in osseointegration.Considering that,this study employed several surface treatment techniques to fabricate various surface topographies on Ti materials,investigate their influenecs on cells’biological functions under physiological and pathological conditions,and clarify their potential molecular mechanisms.The study provides scientific evidence for the surface design of Ti materials and improvement of their bioformances in clinical applications.The main research contents and conclusions of the study are listed as follows:（1）Mechanism study of micro/nano hierarchical titanium surfaces mediating osteogenesisThe micro-"valley-ridge"and nano-particle composite structure was fabriacated on Ti surface by double acid etching and sol-gel technique.The material surface topography was characterized by scanning electron microscopy（SEM）.Primary osteoblasts were seeded onto titanium substrates and investigated the effects of surface topography on cell morphology,proliferation activity,alkaline phosphatase activity and extracellular matrix mineralization.ROCK-specific inhibitor and Wnt5a antibody were added to study the changes of molecular expression and to explore the potential molecular mechanism.The results proved that the micro/nano-complex structure promoted osteogenic differentiation by activating ROCK signaling pathway,stimulatingβ-catenin nuclear accumulation and high expression of Wnt5a.ROCK and Wnt5a form positive feedback loop,playing an important role in the process of osteogenic differentiation.（2）Osteogenesis potential of different titania nanotubes in oxidative stress microenvironmentTiO₂ nanotubes with various dimensions were prepared on titanium surface by anodization.The physical and chemical properties of the substrates were characterized by scanning electron microscopy（SEM）,atomic force microscopy（AFM）,X-ray diffraction（XRD）and water contact angle measurements.The in vitro oxidative stress model was constructed according cell exposure in H₂O₂（300μM）.To investigate the biological responses of osteoblasts,including protein adsorption,cell adhesion,endogenous ROS expression,cell proliferation,apoptosis and cell differentiation,on different surface morphology under normal and oxidative stress conditions.The intrinsic molecular mechanism of anti-oxidation and osteogenesis mediated by nanotube was further evaluated by qRT-PCR,western bolt and flow cytometry.The results show that in normal microenvironment,small TNTs(30 nm,TNT₃₀)were beneficial for adhesion and proliferation of osteoblasts,but large TNTs(110 nm,TNT₁₁₀)greatly increased osteogenic differentiation.However,compare to TNT₃₀ and Ti substrates,large nanotubes displayed strong capacities to improve cell adhesion,survival and differentiation of osteoblasts after H₂O₂ treatment.Meanwhile,the synergetic effect of ITGα5β1 and Wnt signals directly contributed to the superior antioxidant ability of TNT₁₁₀ substrates,and promoted osteogenic differentiation of osteoblasts under oxidative condition.（3）Biological responses of macrophages and MSCs mediated by titania nanotubes surface under oxidative stressUnder the condition of oxidative stress in vitro,macrophages（RAW264.7）were seeded onto the TNTs substrates to evaluate the size effects of TNTs on macrophage adhesion,spreading,proliferation,polarization and inflammatory reaction.Then,the interactions between macrophages（RAW264.7 cells）and MSCs on various nanotubes under oxidative condition were investigated.Finally,the molecular mechanism of inflammatory and osteogenic regulation by different TNTs under oxidative microenvironment was revealed using qRT-PCR,western bolt and immunofluorescence techniques.Cellular and molecular results proved that TNT110 greatly increased early inflammation of macrophages through activating integrin/FAK-mediated MAPK and NFκB signals and simultaneously promoted their genes expression of SDF1,IL-8 and CCL2（chemokines）when comparing with 30 nm nanotubes and titanium substrates.Co-culture results displayed that more MSCs were effectively recruited by inflammatory RAW264.7 cells on TNT110 substrates,and then secreted many anti-inflammatory factors（IL-4,IL-10,IL-13 and TGFβ1）to attenuate inflammations of RAW264.7 cells（increasing M1-to-M2 transition）.The crosstalk between MSCs and RAW264.7 cells caused by 110 nm nanotubes was determined as the key factor to promote early osteogenesis in TNT110 group.Overall,large nanotubes might be more suitable for bone injury treatment under oxidative environment.