Preparation And Biological Evaluations Of Mesoporous Silica/Titania Nanotube Composite Hierarchical Nano-morphology

[Background]Dental implant technology has been widely used to repair dentition defect or missing,and has become a common restoration method.However,due to the poor bioactivity of the pure titanium material of dental implants,there are still many failed cases due to unreliable osseointegration.Therefore,how to endow titanium implants with more ideal bioactivity is a focused area in current researches.In fact,natural bone tissue presents a hierarchical structure from the microscale to the nanoscale,and contains many trace elements such as silicon,strontium,and zinc.From the perspective of biomimetics,if we could simulate bone tissue in terms of physical structure and chemical composition,the artificial materials will achieve better bioactivity.Previously,our research group had used acid etching and anodic oxidation techniques to successfully fabricate titania nanotubes（TNT）on the surface of pure titanium for simulating the hierarchical bone structure.The diameter of the TNT is adjustable from 30 nm to 100 nm,showing the ability to promote bone formation,but the TNT still has differences with nature bone in terms of physical structure,and chemical composition,which are worthy of further exploration.In recent years,mesoporous silica thin films have received extensively attention due to their high specific area,modifiable surfaces,non-toxic and degradable characteristics.We found that the pore size of mesoporous silica thin film prepared by cationic surfactant is usually less than 10 nm,which is expected to fill the gap of morphology of the material in this nanoscale.In addition,silicon is also an important chemical component in the process of bone formation.Combining the above two points,the mesoporous silica structure with a smaller pore size will be further prepared based on the TNT surface,it will form a hierarchical nano-structure,which may be more similar to bone tissue in terms of physical structure,and chemical composition.Mesoporous silica thin film can be divided into parallel mesoporous silica thin film（PMSTF）and vertical mesoporous silica thin film（VMSTF）according to the different orientations of the channels.The preparation process of the former is simple and has been extensively studied,but this film has two major limitations:（1）Due to the parallel mesochannels,most of the mesostructure is visible on the side of the film instead of the surface.（2）The surface of the titanium substrate is often processed priorly with micro-nano morphology,but this kind of film is thicker and usually covers the entire substrate surface,which may hinder the active morphology of the substrate.Unlike the PMSTF,most of mesochannels inside VMSTF run vertically,and the mesostructures can be seen clearly on the surface of the film,moreover,the thickness of this film is usually nanometers.Consider these,VMSTF may be more suitable for the construction of biomimetic hierarchical nanostructure on titanium surface.At present,the oil-in-water biphase stratification growth method has become a more commonly used method for preparing VMSTF due to its simple synthesis process and mild reaction conditions.But most of the researches are mainly carried out on the flat and non-metallic substrates（such as glass,silicon wafers,etc.）,whether a thin layer of VMSTF can be deposited on the surface of the TNT to form a composite hierarchical nano-topography（VMSTF@TNT）is the challenge and key of this study.For this reason,this study is aimed at creating the mesoporous silica/titania nanotube composite hierarchical nano-morphology,and conducting a series of biological evaluations,which may provide a theoretical basis for the further clinical application.[Objective]To explore the feasibility of preparing VMSTF@TNT hierarchical nano-morphology on pure titanium surface by oil-in-water biphase stratification growth method;Analyze the influencing factors of material synthesis and its physical and chemical properties;observe the degradation,biocompatibility and drug delivery capacity of the material;Evaluate the basic biological response of osteoblasts on the surface of the material;Analyze the adsorption behavior of human plasma proteins on the surface,together with the internal mechanism and its effect on hemostasis.[Method]Part 1:Preparation of mesoporous silica/titania nanotube composite hierarchical nano-morphology and its basic performances1.VMSTF@TNT was prepared by hydrofluoric acid etching,anodizing and oil-in-water biphase method.The biphase system was composed of a oil phase containing the TEOS as silicon source,cyclohexane as solvent and a water phase containing CTAB as template,TEA as catalyst.When the reaction was completed,all samples were calcined to remove the template agent;Field emission scanning electron microscope（SEM）,field emission transmission electron microscope（TEM）were used to observe the surface morphology of the samples,and Image J software was used to determine the pore size distribution;Infrared spectroscopy,energy spectrometer and solid-state nuclear magnetic silicon spectroscopy were applied to detect the chemical composition,distribution and the content of silanol（Si-OH）on the samples;Water contact angle measurement and XRD analysis were performed to evaluate the hydrophilicity of the sample surface and the crystal phase change of the titanium substrate.2.SEM and atomic force microscope（AFM）were used to observe the factors,which may influence the VMSTF synthesis,including reaction time,reagent（TEOS content,oil molecule type）,size of TNT（30 nm～100 nm）,other substrates（titania nano mesh,hydroxyapatite nanoparticles,hydroxylated titanium）3.To perform in vitro biodegration test,immersion experiments were conducted in phosphate buffered solution（PBS）.SEM and inductively coupled plasma emission spectrometer（ICP-AES）were used to evaluate the changes of surface morphology and the release of silicon ions at different time points,respectively;4.The osteoblast cell line MC3T3-E1 was seeded on all samples for a certain period,fluorescent staining,SEM and CCK-8 experiments were performed to observe cell adhesion,morphology and viability;Moreover,by adding silane coupling agent（BTSE）to the original biphase system,a hybrid organic-inorganic vertical mesoporous silica thin film（HMSTF）was prepared and its degradation behavior and surface cell viability were also be observed.5.The dexamethasone（DEX）,a classic small molecule bone-forming drug,was served as a model molecule for the drug loading test.DEX was qualitatively analyzed by infrared spectroscopy and SEM.The absorbance of DEX at 262 nm was measured by a microplate reader,and the loading and release rate of DEX on TNT,PMSTF and VMSTF@TNT were quantitatively compared.Part 2:Biological evaluations of mesoporous silica/titania nanotube composite hierarchical nano-morphology1.Using the TNT with 100 nm diameter as the substrate,by changing the biphase systhesis reaction time（2 h,4 h,24 h）,the samples with different content of mesoporous silica were prepared,which were recorded as 2H-VMSTF,4H-VMSTF,24H-VMSTF,,respectively.Without adding template agent,a bulk silica film with no mesostructure was prepared after the same reaction time（24 h）,which was recorded as 24H-STF.The above four groups were used to observe whether mesoporous silica thin film present physical（mesoporous morphology）and chemical（silicon content）inducements simultaneously on bone formation.2.The MC3T3-E1 cell line was seeded on the surface of different samples,and the expression level of osteogenic genes was detected by q PCR;the alkaline phosphatase staining kit was used to determine ALP activity;The mineralization level was evaluated by Alizarin Red staining.3.TNT,PMSTF and VMSTF@TNT were immersed in the healthy human plasma for 2ho and eluted with 5%SDS solution.Then Label-free proteomics analysis was performed to detect the absorbed proteins.All plasma proteins were classified according to their isoelectric point,molecular weight and biofunction;the enriched protein and biofuction were futher verified in vitro.[Results]Part 1:Preparation of mesoporous silica/titania nanotube composite hierarchical nano-morphology and its basic performances1.VMSTF was successfully deposited on the surface of TNT by the oil-in-water biphase stratification growth method.The film not only had numerous mesopores（～3 nm）which directly connected to the outside,but also deposited uniformly and continuously alongside the wall of TNT,forming a composite hierarchical nano-morphology;The chemical elements on the surface was regularly distributed as concentric circles with silicon,oxygen and titanium.The Si-OH content was about 5.56 wt%,and the water contact angle was about 7°.The titanium substrate transformed into anatase and rutile mixed crystal phase after calcination,which had been proved to present better bioactivity.2.The deposition process of VMSTF is time dependent.When the reaction time is less than 12 h,VMSTF was mainly formed in the nanotubes,and the surface roughness had not changed significantly.When the reaction time was longer than 12 h,VMSTF began to form on the outside of nanotubes,and the surface roughness was also rised obviously.Altering the amount of TEOS and the type of organic solvent only determined whether the VMSTF could be successfully synthesized,but had no significant effect on the mesostructured,the pore size is relatively constant at 2 nm～3 nm;VMSTF was also deposited on the surface of TNTs with different diameters of 30 nm～100 nm,and there was no obvious difference in mesostructures;VMSTF was successfully formed on the hydroxylated titanium sheet,titania nano mesh and hydroxyapatite nanoparticles,,but failed to deposite on the surface of polished titanium sheets.3.In the simulated physiological environment,the mesostructure of the film could maintain for 2 h,but after 24 h,it was completely degraded and the surface returned to the TNT structure.The cumulative release amount of silicon is about 10 ppm.4.After MC3T3-E1 cells were seeded on VMSTF@TNT for 2 h,the number of attached cells increased significantly compared to TNT,the cells extended well with lots of pseudopods.The cell viability after incubating for 1 d and 3 d was also significantly promoted.5.By adding BTSE to the biphase system,the hybrid organic-inorganic mesoporous silica film was successfully synthesized.The morphology was similar to that of the inorganic VMSTF.The hybrid film could maintain their structure after being immersed for21 d in PBS.With the increase of BTSE content,the water contact angle was changed from35°to 60°,but the cell activity was inhibited.6.After loading of DEX,the infrared spectrum could detect three characteristic peaks of 1620 cm^-1,1662 cm^-1,and 1702 cm^-1,which proved the DEX was successfully loaded on all samples.The loading amount on VMSTF@TNT was 1.73 mg/cm²,while TNT and PMSTF were 0.76 mg/cm² and 0.71 mg/cm²,respectively;all groups showed DEX a burst release,and the release amount reaches 80%in the hour scale.But the DEX release on the from VMSTF@TNT is relatively slow（4 h>2 h）.Part 2:Biological evaluations of mesoporous silica/titania nanotube composite hierarchical nano-morphology1.2H-VMSTF,4H-VMSTF,24H-VMSTF,24H-STF were successfully prepared,and the surface of 24H-STF had no mesostructure,which was a pure silicon dioxide layer.2.The increase of mesoporous silica content could promote osteogenesis,but after osteogenic induction for 10 days,there was no significant difference between 4H-VMSTF and 24H-VMSTF;The osteogenic effect of 24H-STF was always weaker than 24H-VMSTF,and was even similar to 2H-VMSTF with low a relatively low silicon content.3.TNT,PMSTF and VMSTF@TNT had their unique protein adsorption spectra.Most of the proteins were not high-abundance proteins in human plasma;PMSTF and VMSTF@TNT tended to adsorb acidic proteins（p I<7）,among which human serum albumin（HSA）was the most significant;Due to the molecular weights of these acidic proteins were mostly distributed in 60～80 KDa and 100～200 KDa,and were also distributed in some functional categories such as blood coagulation,complement,lipoproteins,thus,the plasma proteins on PMSTF and VMSTF@TNT showed the same distribution performances.4.Due to the incorporation of mesoporous silica,the electronegativity of PMSTF and VMSTF@TNT was enhanced.When they were contacted with the negatively charged HSA,the adsorption capacity of both was less than TNT in deionized water;But when they were immersed in HBSS,the adsorption behaviors of HSA were completely reversed.The adsorption capacity of PMSTF and VMSTF@TNT were both higher than that of TNT,and VMSTF@TNT improved more significantly.Further research found that PMSTF and VMSTF@TNT could adsorb Na⁺and Ca²⁺ions in HBSS through Si-OH groups,resulting in weakening of the surface electronegativity,which decreased the electronegativity gap with HSA,and may causing the promotion of HSA adsorption.5.PMSTF and VMSTF@TNT could promote red blood cell aggregation and platelet activation,the blood clots on the their surfaces were more stable,showing an excellcent hemostatic ability in vitro.[Conclusion]In this study,the vertical aligned mesoporous silica thin film was successfully deposited in situ on the surface of titania nanotubes by anodizing and oil-water biphase stratification strategy.Mesopores with a diameter of 3 nm were uniformly formed alongside the nanotubes with a diameter of about 100 nm,which creating a microporous-mesoporous hierarchical nano-morphology.The morphology presented high-speed degradation（<24 h）,excellent hydrophilicity（7°）,suitable silicon release（10 ppm）and high drug loading capacity（>1×）;In addition,this morphology not only enhanced the adhesion,proliferation and differentiation of osteoblasts,which reflected ideal osteogenic activity,but also promoted the enrichment of human plasma proteins,especially coagulation-related proteins,which also reflected hemostatic activity.Our work provides a new strategy for the design of biomaterial surface coatings,and shows the broad application prospects of mesoporous materials in tissue engineering.