| BackgrondThe clinical long-term existence and function of implants is closely related to their osseointegration which is the basic theory of oral implantology. Thereinto, the early osseointegration plays a pivotal role in the success rate of implant. It is reported that the total osseointegrated area covered by bone around implants which were used clinically is only 50%～75%, the most failure of implants is due to incompletely ossointegration established at the early stage which result in the damage of the bone-implant interface. Understanding of the complicated interactions between biomaterials and cells can accelerate the biomedical development. According to many reports, there are lots of factors affecting the bone implant osseointegration, and most of the scholars carried on an indepth study from the angle of the implant itself. Thereinto, the surface characteristic of implant plays an important role during the healing process at the interface of bone-implant. Many studies have approved that titanium implant could improve the protein and cell attachment to the surface after relevant surface treatment. Consequently, it can enhance the contact osteogenesis and realize the bone healing pattern of bilateral osteogenesis.Numerous studies and preliminary experiments of our research group have approved that, the surface cleanliness of implant to a lagre extent influences the biologicl activity and the early osseointegration itself. Many studies had reported that titanium surface displays greater biological activity, biocompatibility and resistance to corrosion which are all attributed to the sluggishness titanium dioxide oxide film spontaneously formed under ambient conditions. However, this oxide film would adsorb inorganic ion and organic hydrocarbons or carbon/oxygen containing species from air in 1 minute or even a few seconds, which lead to the change of the chemical composition and the decrease of surface free energy. Moreover, the phenomenon of aging exists in the titanium surface, that is the biological activity of titanium surface decreased over time and subsequently the declined osseoconduction, the suppressive chemotaxis for osteoblast to surface, the decreased proliferation and differentiation capacity. In fact, there is no product information or instruction of manufacture, and storage in these implants products used clinically, except for the expiration of sterilization, which is normally 5 years. So the implant unavoidable contact with the air which result in the surface contamination and aging. Scholars such as Kideki had approved that the contaminant adsorbed on oxide film surface mainly contain inorganic ion and organic hydrocarbon with a carbonyl moiety. Negative correlation relationship exists between the content of carbon atom on the titanium surface and the adhesive capacity of protein and cells. In consequence, the contaminant decrease the biological activity of the titanium surface, which is to the disadvantage of the early osseointegration formation and would be unsuitable for the interaction between implant and surrounding environment. Therefore, how to effectively remove the contamination on the titanium surface and maintain or enhance the biological activity for the bone formation around the implant become the main research orientation of this project.At present, the physical and chemical cleaning are main cleaning methods for titanium, but these methods could damage the titanium surface topography and chemical groups. In recent years, many studies have reported that the biological activity of titanium surface can be recovered and even enhanced by ultraviolet treatment, which could change the titanium surface chemical structure composition and effectively remove the hydrocarbon adsorbed on the titanium surface under the titanium dioxide photocatalysis without destruction of the titanium surface topography. Ultraviolet light-induced superhydrophilicity of titanium dioxide film was discovered and reported on Nature by Wang in 1997. The preliminary experiments of our research group had also approved that the enhanced cellular response on titaniun surface displayed after ultraviolet treatment, which could remove the hydrocarbon and other organic contaminant adsorbed on the surface. In addition, the scholars headed by Ogawa in the international famous Laboratory for Bone and Implant Science (LBIS) had proved the superhydrophilicity of acid etching titanium surface could generated by ultraviolet treatment. And this changed surface improved the cellular activity in vitro and bone-implant contact in animal experiment from 75% to 98.2%. However, there is no existence of TiO2 crystal form on acid etching titanium surface after ultraviolet treatment. Thus the generation of photocatalysis can be attributed to the titanium dioxide oxide film spontaneously formed under ambient condition. Some researchs had showed that the thickness of TiO2 oxide film can influence the photocatalytic activity. Another studies had approved that the photocatalysis of acid etching titanium surface could be enhanced by TiO2 particle spraying. We can conclude from these lots of researches that the photocatalysis generated by titanium dioxide oxide film spontaneously formed under ambient condition is not enough, and there is still plenty of room for improvement. Therefore, we consider how to strengthen the photocatalysis without the destruction of the titanium surface topography, consequently to remove the contaminant adsorbed on the titanium surface effectively.Actually, TiO2 as a kind of photocatalyst has been widely used in the administration of water and environment pollution. Nano-TiO2 has been widely used in the air purification, wastewater treatment and self-cleaning field for its small size effect, surface effect and so on. TiO2 nanowires is a one-dimensional nanostructure with nanoscale size in the horizontal orientation and no limitation in the longitudinal orientation. For the greater charge carrier transport efficiency and ion dispersive capacity, one-dimensional nanowires nanostructure possess stronger charge collection efficiency, which could make the photo-generated charge carriers delivery along the one-dimensional long axis direction, thus reduce the loss of photoelectron. Moreover, compared to nano-TiO2 particles and nano-TiO2 film, the research of TiO2 nanowires has been received extensively attention characterized by its smaller particle size, larger surface area, better photocatalytic performance, superior chemical stability, nontoxic and low cost.There are three main crystal form:anatase, rutile and brookite. Thereinto anatase as the strongest photocatalytic activity crystal form, because of anantase can generate photogenerated electrons and holes under a relative low photon energy for the photocatalysis. Hence, nano-anatase is usually choosed in the field of administration of water and environment pollution. However, even in anatase, the recombination of photogenerted electrons and holes is still too fast, which results in low quantum efficiencies and limits the photocatalytic potential of anatase. In fact, the quantum efficiencies of nano-anatase TiO2 is only approximately 4%. TiO2-B is a metastable monoclinic modification of TiO2 and its energy bandgap is very close to that of the anatase, while its conduction band edge is above the anatase. Therefore, the energy bandgap similarity and electronic energy level differences can boost charge transfer from one phase to the other if the anatase and the TiO2-B were combined together, as following the enhanced photocatalytic activity. Scholars such as Liu had approved that this TiO2-B/anatase core-shell nanowires posses greater photocatalysis than any single crystal form in the preliminary experiment. So as a photocatalyst, this kind of TiO2-B/anatase core-shell nanowires has obvious advantages for its surface morphology, specific surface area, the amount and crystal formIn this article, TiO2-B/anatase core-shell nanowires (TiO2-B@ANWs) was desired to prepared to clean the titanium using its better photocatalysis. We hypothesized that this method would effectively remove the contaminant adsorbed on titanium surface which is beneficial for the early cellualr response, and subsequently enhance the early osseointergration establishment, finally achieve the shortened clinical treatment periods and improved success rate of dental implant.Objective(1) To explore the effects of contaminant removel from the titanium surface after the TiO2-B@ ANWs photocalysis treatment.(2) To explore the biological activity of titanium surface after the TiO2-B@ANWs photocalysis treatment.Methods1. The preparation and characterization of TiO2-B@ANWs(1) The preparation of TiO2-B@ANWsThe stable and reproducible TiO2-B@ANWs was prepared by hydro thermal method on titanium substrate.(2) The characterization of TiO2-B@ANWsThe surface topography of nanowires was observed using field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM); The surface element component was examined using X-ray energy dispersive spectrometer (X-ray EDS); The surface crystalline phase structure was measured using X-ray diffraction (XRD); The static contact angle was detected using OCA surface contact angle analysis meter; The surface roughness was measured using surface profilometer.2. The photocatalytic degradation capacity and photocatalytic activity of TiO2-B@ ANWs was analyzed by the experiment of degradation of methylene and the indirect testing of hydroxyl radical.3. The cytological activity of titanium surface after the TiO2-B@ANWs photocalytic treatment in vitro.(1) The surface physicochemical property after the TiO2-B@ANWs photocalysis treatmentThe titanium surface topography was observed using FESEM; The surface crystalline phase structure of titanium surface was measured using XRD; The surface roughness was measured using surface profilometer; The hydrophilic status of the titanium surfaces was detected using OCA surface contact angle analysis meter; The surface chemical state of elements was analyzed using X-ray photoelectron spectroscopy (XPS).(2) The protein adsorption and cytological assayThe amount of protein adsorbed to titanium surface was measured by BCA protein kit; The cell morphology on different titanium surface was observed by scanning electron microscopy and fluorescent microscopy, the cell attachment rate was counted by fluorescent images; The cell chemotaxis migration ability to the titanium surface was measured by Transwell test; The cell proliferation activity of different titanium surfaces was examined by MTS assay; The expression of osteogenic gene ALP and OCN of different titanium surfaces was analyzed by RT-PCR technical detection, and the mineralized ability was compared by alizarin red and von Kossa staining test.4. The animal experiment of implant treated by the TiO2-B@ANWs photocalysisBeagle dogs were used as animal model and 4 Beagle dogs were random allocated into groups of 2 w and 4 w. According to random allocated priciple, three different groups of implants (SLA, UV-SLA, NWs+UV-SLA) were implanted in cycles in upper part of tibia. The specific implant sites and method as follows:the first implant was implanted in 2 cm apart from each tibial epiphyseal line, then 6 implants were successively implanted at the interval of 1 cm along the long axis of tibia in the diatal direction.Tetracycline and calcein were injected in subcutaneous position of Beagle neck for fluorescence labeling at select time. Beagle dogs were sacrified at 2 w and 4 w after operation. The condition of new bone forrmed around implants were observed using fluorescence microscopy after the hard tissue slices prepared. The relevant parameter about BIC and BA were calculated and analyzed using optical microscopy for shooting after the toluidine blue stained hard tissue slices were prepared.5. SPSS 13.0 software was used for statistical analysis, and the data were decribed as x±s. Firstly, the factorial design was adopted to analyze if the interaction effect was existed between group and time two treatment factors. Then one way ANOVA was used for statistical analysis among groups. Levene’s test was used to check homogeneity of variance. If data’variance homogeneity, LSD test was adopted to compare the mean between every two groups. If data’variance heterogeneity, the Welch test that similar to F test was used for statistical analysis among groups. Dunnett’s T3 test was adopted to compare the mean between every two groups. If there is only group as the only factor in the experiment, one way ANOVA was used for statistical analysis among groups. We hypothesized that the test was bilateral, and inspection level was a=0.05.p<0.05 was considered statistically significant.Results1. The preparation and characterization of TiO2-B@ANWs(1) The TiO2-B@ANWs nanowires was prepared by hydro thermal method on titanium substrate via a four-step synthesis. Firstly, STi@NWs nanowires wes prepared by hydrothermal method on titanium substrate. Secondly, replacement reaction was occurred between STi@NWs and dilulated hydrochloric acid to exchange Na+with H+. The STi@NWs were then transformed to HTi@NWs nanowires. Thirdly, the HTi@NWs nanowires were placed into TiCl4 solution to develop anatate nanocrystals around the HTi@NWs nanowires, that is HTi@ANWs nanowires. Finally, the HTi@ANWs nanowires was calcined to convert to TiO2-B @ANWs nanowires. After examined by crystal form structure, TiO2-B@ANWs nanowires was a core-shell recombined crystal stucture which contains the core of TiO2-B and the shell of anatase. There are four different kinds of nanowires during the process:STi@NWs、HTi@NWs、HTi@ANWs、TiO2-B@ANWs.(2) The surface morphology images of TiO2-B@ANWs were varied with the time of immersing in TiCl4 solution during the process. Villous anatase shell were formed more and more around the core of TiO2-B nanowire over time, and completely cover it at 4 h. The obvious core-shell TiO2-B@ANWs was shaped at that time. However, the density of anatase was greater at immersing in TiCl4 solution for 6 h, and multiple layer was occurred.(3) The STi@NWs, HTi@NWs, HTi@ANWs and TiO2-B@ANWs nanowires formed on titanium substrates were all blue, uniform colour and lustre, mild grind arenaceous feeling. The SEM and TEM imangs of STi@NWs and HTi@NWs were all rod-like nanowires with the average diameter from 90 nm to 110 nm. The HTi@ ANWs and TiO2-B@ANWs were all displayed similar surface morphology, with the villiform anatase formed around STi@NWs and HTi@NWs and the average diameter of approximately 130 nm.(4) In the hydrophilic assay, except the polished titanium surface was hydrophobic with the contact angle of (81.69±13.32)°, the STi@NWs, HTi@NWs, HTi@ANWs and TiO2-B@ANWs surface were all hydrophilic surfaces, with the contact angle of 0°.(5) There were three main elements on five groups titanimun surfaces:Ti, O and C. A small quantity of N, Na, Si and Cl were also detected. Some differences of element content ratio existed among different groups.(6) The surface roughness of STi@NWs, HTi@NWs, HTi@ANWs and TiO2-B @ANWs were all greater than the polished titanium surface, Ra (F=251.791, P=0.000), Rq (F=239.403, P=0.000). These four groups have no obvious difference (P>0.05).2. The photocatalytic degradation capacity and photocatalytic activity of TiO2-B@ ANWs was analyzed by the experiment of degradation of methylene and the indirect testing of hydroxyl radical.(1) Among the four different kinds of nanowires STi@NWsN HTi@NWs. HTi @ANWs and TiO2-B@ANWs formed during the process, the photocatalytic activity of TiO2-B@ANWs was the greatest.(2) The photocatalytic degradation methylene capacity of TiO2-B@ANWs enhanced over time during the heating in TiCl4 for 4 h. However, the photocatalytic degradation methylene capacity was declined when up to immersing in TiCl4 for 6 h, with a realative low value.(3) In the experiment of different concentration of 20 mg,50 mg,100 mg and 150 mg of TiO2-B@ ANWs photocatalytic degradation 500 ml methylene, the photocatalytic activity of 100 mg TiO2-B@ANWs was the greatest.(4) According to the treatment factors and negative control group in photocatalytic reaction, this experment can be divided into four groups:UV, NWs, NWs+UV and TiO2+UV. In the experiment of degradation of methylene and the indirect testing of hydroxyl radical, the photocatalytic activity of group NWs+UV was the greatest.3. The cytological activity of titanium surface after the TiO2-B@ANWs photocalytic treatment in vitro.(1) The surface physicochemical property after the TiO2-B@ANWs photocalytic treatmentThe titanium surfaces morphology of three different groups were all distributed crater-like micro-scale primary hole and alveolate nano-scale second level hole fabricated by sand blast and acid etching. There were no difference of surface crystal form and surface roughness among three groups. In the hydrophilic assay, except the control group SLA surface was hydrophobic, the UV-SLA and NWs+UV-SLA surface were all hydrophilic surfaces, with the contact angle of 0°.There were three main elements on three groups titanimun surfaces after elemental chemical state analysis using XPS:Ti, O and C. The main binding energy peak of C 1s was located in 284.8 eV which correspond to C-H and C-C. The other binding energy peak of C 1s was located in 286.4 eV which correspond to C-O. These two bingding energy value of C 1s were all declined in groups UV-SLA and NWs+UV-SLA, especially at 284.8 eV; The main binding energy peak of O 1s was located in 530.1 eV which correspond to O2- in TiO2. This bingding energy value of O 1s were all increased in groups UV-SLA and NWs+UV-SLA. The other binding energy peak of O 1s was located in 531.3 eV which correspond to Ti-OH. These two bingding energy value of O 1s were all increased sightly in groups UV-SLA and NWs+UV-SLA; The binding energy peak of Ti 2p was divided into Ti 2p3/2 located in 458.5 eV and Ti 2p1/2 located in 464.2 eV which were all correspond to Ti in TiO2. Compared to SLA, These two bingding energy peak value of Ti 2p were all increased in groups UV-SLA and NWs+UV-SLA.(2) The protein adsorption and cytological assayThe protein adsorption rate of three groups were all the greatest at 2 h during 2 h, 6 h and 24 h of incubation. Among three groups, the adsorption rate of group NWs+UV-SLA was the maximum, up to 40%. The cell morphology on three different titanium surfaces were all globular shape at beginning, then stretched out gradually during 1 h,2 h and 4 h of incubation. The cells on NWs+UV-SLA surface were the fast-growing group. After 4 h of incubation, the cells on NWs+UV-SLA surface began to extend sturdy pseudopodiums and the length of cell approximately amount to 40μm. The cell attachment rate value were all increased over time. At each selected time, the cell attachment rate of group NWs+UV-SLA was the greatest (P<0.05). The amount of cells migrated to NWs+UV-SLA titanium surface was the maximum during 24 h of incubation in Transwell test (P<0.05). The proliferation activity were all improved over time during 5 d of culture. Among three groups, the group NWs+UV-SLA was the best (P＜0.05). The expression of mineralized gene ALP of three group were all higher at 7 d than 21 d of incubation, and the group NWs+UV-SLA was the maximum at 7 d (F=17.020, P=0.003). The expression of mineralized gene OCN of three group were all higher at 21 d than 7 d of incubation, and the group NWs+UV-SLA was the best at 21 d (F=32.417, P=0.001). In the alizarin red and von Kossa stainning mineralized capacity test, the titanium surface of group NWs+UV-SLA was stained deepest, followed by UV-SLA, finally the SLA group.4. The animal experiment of implant treated by the TiO2-B@ANWs photocalysisDifferent amount of fluorescent stainning new bone trabecula was formed in bone defective area of trapezoid groove between threads of implants at 2 w and 4 w in three groups (SLA, UV-SLA and NWs+UV-SLA). The amount of bone in three groups was greater at 4 w than 2 w, especially the group NWs+UV-SLA. The bone defective area of trapezoid groove in group NWs+UV-SLA were almost filled with new bone at 4 w, followed by UV-SLA, finally the SLA group. Blue-stained new bone trabecula was formed in bone defective area of trapezoid groove between threads of implants at 2 w and 4 w in three groups. Most of the new bone trabecula grew along side walls of trapezoid groove of implant. Agree with the fluorescent image, the new bone area in bone defective area of trapezoid groove between threads of three groups implants was greater at 4 w than 2 w. The bone trabecula became bulky and mutual fusion. There were significant differences among the BIC values of three groups at 2 w and 4 w. Group NWs+UV-SLA was amount to 48% and 68% at 2 w and 4 w respectively, which were all greater than group UV-SLA and SLA (P＜0.05). The BIC values of group UV-SLA were higher than group SLA (P＜0.05). There were significant differences among the BA values of three groups at 2 w and 4 w. Group NWs+UV-SLA were amount to 29% and 47% at 2 w and 4 w respectively, which were all greater than group UV-SLA and SLA (P<0.05). The BA values of group UV-SLA were higher than group SLA (P<0.05).Conclusion1. The TiO2-B@ANWs with greater photocatalytic activity was successfully prepared on titanium surface using hydrothermal method.2. In the vitro test, it has been approved that the biological activity of titanium surface was improved after TiO2-B@ ANWs photocatalytic treatment.3. In the animal experiment, it has been approved that the titanium implant surface improved the new bone formation around the implant after TiO2-B@ANWs photocatalytic treatment, which is beneficial for the early oeesointegration estabishment. |
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