Research On The Mechanical And Biological Properties Of Nano-hydroxyapatite In Situ Formation On The Surface Of TiO₂ Nanotubes

Owing to its excellent biocompatibility,pure titanium is generally applied in dental implants,and gradually accepted by clinic doctors and patients after a long and strict laboratory and clinical verification.Endosteal implants of pure titanium can directly link with bone tissues in structure and function,forming “osseointegration.” Implant can contact with the host bone directly,and its surface characteristics can regulate the subsequent biological effects.Therefore,the implant surface modifications become important to improve its biological properties.Recently,the surface modification has been progressed into actively guiding bone tissues reconstruction from passively adopted to the healing of surrounding tissues.Additionally,the effect of surface modification has been developed from micron scale into nanoscale.Nano-coating on implant surface can form a highly similar three dimensional rough nano surface between the surface of implant and bone tissues.This can be directly used to regulate function of cells surrounding the implant and improve the early osseointegration with good mechanical properties.In this dissertation,we prepared the nano-hydroxyapatite in situ formation on the surface of TiO₂ nanotubes based on titanium substrace?nHA/TiO₂/Ti?.We studied the effects of nHA/TiO₂/Ti on the osteoblasts behavior in vitro and early osseointegration in vivo.The main research work and results are as follows.1.A uniform three-dimensional petals curled sheet structure?examined using scanning electron microscopy,SEM?,which is HA crystal identified using EDS and XRD,was successfully generated on TiO₂ nanotube surface in situ.The surface roughness is higher than TiO₂ nanotubes and pure Ti.Its contact angle was significantly higher than that of pure TiO₂ nanotubes and Ti,indicating it had a good hydrophilicity.Nano-indentation and scratching tests showed that the mechanical properties were excellent.The bonding between the HA crystal and TiO₂ nanotubes was tight.There was a stress buffer layer between HA and TiO₂/Ti.This indicated that the surface modification was feasible.2.Immunofluorescence detection showed nHA/TiO₂/Ti promoted early adhesion of MC3T3-E1 cells.The spreading area was larger than pure Ti group for 6h,whereas less than pure Ti for 12 h,cells had more pseudopodia and abundant cytoplasm.Cells collectively grew for 24 h,and then formed a special unit of connecting ring for 48 h.This suggested that the surface of nHA/TiO₂/Ti promoted the early cell secretion and growth in their own three dimensional extracellular matrix.There were a plenty of thicker cell pseudopodia?closely combined with three-dimensional surface?with branches and cross observed by SEM.The proliferation of cells was inhibited by nHA/TiO₂/Ti and TiO₂/Ti-surface.In nHA/TiO₂/Ti group,ALP activity levels increased early and reached the peak value 3 days later;and 1 day and 3 day group were significantly higher than pure Ti and TiO₂/Ti groups.This indicated that nHA/TiO₂/Ti had a good ability to promoting cell early differentiation.Collagen and alizarin red staining showed that,the amount of collagen I and calcified nodules on nHA/TiO₂/Ti-surface significantly promoted cell secre tion.The gene expression of COL-I,ALP,OCN and RUNX2 was higher than pure Ti and TiO₂/Ti groups.COL-I,ALP and RUNX2 reached peak for 3 days later but less than or close to pure Ti and TiO₂/Ti groups for 14 days later.Although OCN was lower initially,it was higher than pure Ti and TiO₂/Ti groups for 3 days.OCN reached peak 7 days later and maintained high level.These results indicated that nHA/TiO₂/Ti could promote the differentiation of MC3T3-E1 cell and the early gene expression related mineralization.3.The results from animals in vivo showed that mineralization line on the implant surface spread quickly in nHA/TiO₂/Ti group,the speed was up to 30-40 ?m/d.Bone mineralization appposition rate?MAR?in nHA/TiO₂/Ti group were significantly higher than the pure Ti group at 2w.Bone implant contact rate?BIC%?in nHA/TiO₂/Ti group were significantly higher than the pure Ti group at 2w and 4w.Data from Micro CT showed that,compared to pure Ti group,osseointegration rate?OI%?and bone volume ratio to total volume?BV/TV%?were also obviously higher in nHA/TiO₂/Ti group at 2w,4w,and 8w.The pushout strength increased at the same timepiont.The osseointegration rate was used to correct the pushout strength,the actual contact shear strength of the implant interface in nHA/TiO 2/Ti group showed bigger at 2w than pure Ti group,while no diiference was found at 4w and 8w.This suggested nHA/TiO₂/Ti had better interface strength in early stage.The morphology and function test was in consistent.O ur results indicated that nHA/TiO₂/Ti had excellent bone conduction,and this surface modification could accelerate early osseointegration whether in bone quantity or quality.In conclusion,nHA/TiO₂/Ti had good quality in sureface topography,chemical component,physical and mechanical properties.It may promote early osteogenesis by inducing early osteoblasts adhesion and spreading,cellular differentiation to secretory osteoblasts,and the secretion of extra cellular matrix and mineralization.In addition,nHA/TiO₂/Ti showed better bone conduction,which could accelerate early osseointegration whether in bone quantity or quality.Due to its simple preparation technology,good repeatability,controllable quality,nHA/TiO₂/Ti may have a good application prospect in industrialized production as a new modification in implant surface.