1?,25-Dihydroxyvitamin D3-loaded Hierarchical Titanium Scaffold Enhanced Early Osseointegration

INTRODUCTION:Titanium?Ti?and its alloys have been extensively applied in orthopedic and dental fields as artificial bone substitutes,since them possess excellent mechanical properties,appropriate corrosion resistance and biocompatibility.Accelerated osseointegration is well considered as a critical facilitated factor for Ti implant longevity,but immensely limited by bio-inert material surfaces,high Young's modulus and restricted biological responses in vivo[1-3].Therefore,numerous researches have focused on how to improve the performance of Ti implants,promoting and accelerating osseointegration.In past decades,the researches in bone tissue engineering have applied porous structure to appropriately decrease the Young's modulus of bulk titanium implants and reduce the lesion of stress shielding effect[1,4-7].And nanomaterials with suitable surface topography could better mimic the nanostructure and affect the cell behaviors in a similar way with the natural Extracellular matrix?ECM?[9],for bone itself possesses a hierarchical structure with the initial gradient in the nanoscale[8].OBJECTIVES:1 To fabricate 3D printed porous titanium scaffold modified with TiO₂ nanotubes?p-TNTs?,for mimicking the hierarchical trabecular bone structure;2 Based on the hierarchical titanium scaffold to build nanoscale drug delivery systems?NDDSs?,evaluating its ability to control drug release and analyzing whether it could accelerate the osteogenic differentiation and improve the subsequent osseointegration.METHODS:The scaffold at macro and micro gradient was manufactured by selective laser melting process with pure titanium powder according to the computer-aided design?CAD?,which could be further anodized to construct nano-gradient framework and drug-loading districts.Sequentially,1?,25-Dihydroxyvitamin D3?VD3?was loaded into anodized TNTs,thermo-sensitive hydrogel Pluronic F-127?F-127?was coated and charged as a bio-cap to construct a nanoscale drug delivery systems?NDDSs?for control release.1 Surface characterization and in-vitro drug release:Field emission scanning electron microscopy?FE-SEM?and Focused ion beam scanning electron microscope?FIB-SEM?were used to characterize the surface morphology and cross sectional view of different porous Ti samples;the wettability of different samples was characterized with a contact angle measuring equipment;Fourier Transform infrared spectroscopy?FTIR?and X-ray photoelectron spectroscopy?XPS?were utilized to further demonstrate the presences of drug and polymer;VD3 release experiment of NDDSs was performed with a microplate reader.2 The biological experiments in vitro:the cell morphologies cultured on all groups for 24h were observed by FE-SEM;the Live/Dead Cell kit and the Cell Counting Kit-8?CCK-8?assay were applied to analyze the cell viability and proliferation of various substrates;the expression levels of osteogenic marker gene?ALP,COL-I,RUNX2,OSX?were detected by Real-time Polymerase Chain Reaction?PCR?.3Osteogenesis effect in vivo:New Zealand white rabbits were implanted with scaffolds in the distal femur,and the sequences of polychrome intravital fluorochromes were performed postoperatively at the 6th day?alizarin red,30 mg/kg?and the 10th day?calcein,10 mg/kg?,the bone repair abilities were explored with Laser Scanning Confocal Microscopy?CLSM?and Von-Gieson?VG?staining after 2 weeks.RESULTS:According to the CAD models,we obtained sheet p-Ti for in vitro and columnar p-Ti for in vivo experiments.1 Surface characterization and in-vitro drug release:the results of FE-SEM verified that a unique 3D hierarchical structure ranging from nanoscale of TNTs to microsphere particles and then to macroscale of porous structures was successfully constructed,and the cross sectional view of p-TNTs by FIB-SEM illustrated that nanotubes can emerge on both the whole surface of the microsphere and the flat substrate;the outstanding-enhanced hydrophilicity of p-TNTs was investigated by contact angle measurement,but after coating F-127,the difference between the p-Ti and p-TNTs was reduced?p<0.01?;the presences of VD3 and F-127 composite were further verified by FTIR and XPS;the VD3 released with an initial burst phase in all substrates at first 12h and subsequently slowly released between 1d and14d,which indicated that NDDSs had the ability to control the release of VD3.2 The biological experiments in vitro:the morphology of the MC3T3-E1 adhered on NDDSs exhibited more filamentous pseudopods;the results of CCK-8 and Live/Dead assay proved that,except p-Ti+VD3+F-127,other substrates owned appropriate biocompatibility and proliferation ability,but MC3T3-E1 proliferated slower after loading VD3 or/and anodizing?p<0.05?;the expression levels of osteogenic marker gene?ALP,COL-I,RUNX2,OSX?were significantly up-regulated about 4 times on NDDSs?p<0.05?.3 Osteogenesis effect in vivo:the results of VG staining illustrated that new bone?red?could form around all scaffolds,satisfactorily,the new bone formation around NDDSs bound more tightly with scaffold and bone ingrowth?13.9±2.9%?was markedly higher than other groups?p<0.05?;fluorescence sequence labelling exerted similar results with the VG staining,and it's worth mentioning that a small amount of alizarin red was observed only in the p-TNTs?3±0.8%?and p-TNTs+VD3+F-127?2.4±0.7%?,which indicated that TNTs and the controlled-released VD3 were conducive to promoting earlier osteogenesis?p<0.05?.CONCLUSIONS:We succeeded in constructing hierarchical titanium scaffold and 1?,25-Dihydroxyvitamin D3-loaded NDDSs.The results showed its good biocompatibility and significant osteogenesis enhancement.It suggests that this promising bioactive implant system may provide a far-reaching impact on accelerating and enhancing early osseointegration.