CN113198048A

CN113198048A - Titanium dioxide nanotube and preparation method and application thereof

Info

Publication number: CN113198048A
Application number: CN202110478709.2A
Authority: CN
Inventors: 程文丹; 李慧武; 王琛; 荆珏华; 刘言畅; 蔡德超; 谢武昆; 许新忠; 张积森; 叶曙明; 于浩然
Original assignee: Second Affiliated Hospital of Anhui Medical University
Current assignee: Second Affiliated Hospital of Anhui Medical University
Priority date: 2021-04-30
Filing date: 2021-04-30
Publication date: 2021-08-03

Abstract

The invention discloses a titanium dioxide nanotube and a preparation method and application thereof, relating to the technical field of biomedical nano materials, and the technical scheme is as follows: the prepared titanium dioxide nanotube has the height of 2 mu m, the inner diameter of 128nm and the roughness of 84nm, and has the directive function of promoting the differentiation of stem cells to osteoblasts when the elastic deformation amount is 0.9 percent under the push-out test of a metal rod with the loading rate of 0.5mm/min and the diameter of 3 mm. The prepared titanium dioxide nanotube has the height of 2 mu m, the inner diameter of 92nm and the roughness of 64nm, has the directive function of promoting the differentiation of stem cells to fat cells when the elastic deformation amount is 0.3 percent under the push-out test of a metal rod with the loading rate of 0.5mm/min and the diameter of 3mm, and provides a foundation for effectively controlling the elastic deformation of titanium metal and the application of generated mechanical signals.

Description

Titanium dioxide nanotube and preparation method and application thereof

Technical Field

The invention relates to the technical field of biomedical nano materials, in particular to a titanium dioxide nanotube and a preparation method and application thereof.

Background

With the rapid development of society and the accelerated aging process of population, various high-energy bone wounds and chronic bone diseases are increasing, which become one of the main reasons threatening the health of people and causing injuries and disabilities. Under the premise of rapid development of medical biotechnology, the orthopedic implant is produced at the same time and plays a vital role in the treatment of orthopedic diseases, and both manpower and material resources for research and development of the orthopedic implant and the use amount of the orthopedic implant are in a trend of obvious increase. Because of the complex problems of bone repair, limb function reconstruction, prosthesis repair, postoperative infection control and the like, research on the internal plant performance is always a great challenge in orthopedics clinical and even in the whole medical industry.

The titanium metal material is one of the most commonly used implant materials in orthopedics clinic because of its characteristics of high strength and good biocompatibility. The traditional research on the porous titanium metal implant mainly focuses on the aspects of alloy component improvement, scaffold pore size, pore communication rate control, metal surface modification and the like, and the osseointegration of the implant-host bone interface is increased. Few studies on the physical deformation of titanium and the regeneration of bone tissue have been reported. Research proves that titanium metal generates nonlinear elastic deformation under appropriate stress stimulation, the deformation is influenced by physical factors such as elastic modulus, form, diameter, three-dimensional framework and the like, in the process of load walking or bouncing and the like, the titanium metal bracket generates extremely fine elastic deformation due to cyclic reciprocating stress, and bone cells and stem cells are cells sensitive or responsive to mechanical signals. Mechanical factors regulate stem cell self-renewal and lineage differentiation even in the absence of biochemical stimuli.

However, it has not been reported that whether the mechanical signal caused by the elastic deformation of titanium metal can cause the change of cytoskeleton adhered on the surface of the titanium metal, thereby affecting the cell differentiation and how to control the signal to make the cell differentiate to the direction beneficial to bone regeneration.

Disclosure of Invention

In order to solve the defects in the prior art, the invention aims to provide a titanium dioxide nanotube and a preparation method and application thereof.

The technical purpose of the invention is realized by the following technical scheme:

in a first aspect, there is provided a titanium dioxide nanotube having a height of 1.8 to 2.2 μm, an inner diameter of 74 to 148nm, a roughness of 50 to 110nm, and an elastic deformation amount of 0.3 to 0.9% under a metal bar push-out test at a loading rate of 0.5mm/min and a diameter of 3 mm.

Preferably, the titanium dioxide nanotube has a height of 2 μm, an inner diameter of 128nm, a roughness of 84nm, and an elastic deformation amount of 0.9% under a metal bar push-out test at a loading rate of 0.5mm/min and a diameter of 3 mm.

Preferably, the titanium dioxide nanotube has a height of 2 μm, an inner diameter of 92nm, a roughness of 64nm, and an elastic deformation amount of 0.3% under a metal bar push-out test at a loading rate of 0.5mm/min and a diameter of 3 mm.

In a second aspect, there is provided a method for preparing a titanium dioxide nanotube as described in any one of the first aspect, comprising the steps of:

fixing the pretreated sample as an anode while using a platinum sheet as a cathode in an electrolyte aqueous solution of 0.15MNH4F and 90% ethylene glycol, maintaining the distance between the cathode and the working electrode at 40 mm;

anodizing the anode titanium sheet in the electrolyte for 1 hour under the voltage of a direct current power supply of 30-70V, wherein the temperature of the water bath is 20 ℃;

washing the anode with deionized water for 30 minutes after the anode oxidation, and washing the anode with anhydrous alcohol in an ultrasonic cleaning machine for 15 minutes;

and (3) sterilizing the cleaned sample for 1 hour by using 120-degree high pressure sterilization to obtain the titanium dioxide nanotube.

In a third aspect, there is provided a use of the titanium dioxide nanotubes of the first aspect in osteogenic growth.

In a fourth aspect, there is provided a use of the titanium dioxide nanotube according to the first aspect for promoting differentiation of stem cells into osteoblasts.

Preferably, the expression of osteopontin, osteoblast secretory protein and RUNX2 gene by the titanium dioxide nanotube is simultaneously improved.

Preferably, the titanium dioxide nanotubes have increased expression of osteogenic genes RUNX2, ALP, cyanate, and OSX.

Preferably, the f-actin in the titanium dioxide nanotube group is up-regulated.

In a fifth aspect, there is provided a use of the titanium dioxide nanotube according to the first aspect for promoting differentiation of stem cells into adipocytes.

Compared with the prior art, the invention has the following beneficial effects:

1. the titanium dioxide nanotube prepared by the invention has the height of 2 mu m, the inner diameter of 128nm and the roughness of 84nm, and has the directive function of promoting the differentiation of stem cells to osteoblasts when the elastic deformation amount is 0.9 percent under the push-out test of a metal rod with the loading rate of 0.5mm/min and the diameter of 3 mm;

2. the prepared titanium dioxide nanotube has the height of 2 mu m, the inner diameter of 92nm and the roughness of 64nm, and has the directional effect of promoting the differentiation of stem cells to fat cells when the elastic deformation amount is 0.3 percent under the push-out test of a metal rod with the loading rate of 0.5mm/min and the diameter of 3 mm;

3. the invention provides a foundation for effectively controlling the elastic deformation of the titanium metal and the application of the generated mechanical signals, and provides a condition for independently generating osteogenesis without depending on chemical stimulation such as growth factors;

4. the titanium dioxide nanotube provided by the invention can effectively support the mechanics of the new tissue, and simultaneously can convert the elastic deformation of the stent caused by external circulating stress into cytoskeleton change which is beneficial to cell differentiation and proliferation, thereby helping to realize the biomechanical bionic manufacture of the titanium metal stent.

Drawings

The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:

FIG. 1 is a scanning electron microscope image of nanotubes prepared at different constant pressures in an embodiment of the present invention;

FIG. 2 is a schematic view of the diameter of a nanotube prepared under different constant pressures in an embodiment of the present invention;

FIG. 3 shows TiO in an example of the present invention₂A result graph of the nanotube promoting the osteogenic gene expression of the bone marrow mesenchymal stem cells;

FIG. 4 shows TiO of different tube diameters in the embodiment of the present invention₂And (3) a result graph of the effect of the nanotubes on the osteogenic markers.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.

Example 1: preparing TiO with different pipe diameters₂Nanotube and method of manufacturing the same

The anodic oxidation method is to prepare the nanotube by using the titanium anodic oxidation principle and the balance of the generation speed and the dissolution of the titanium surface oxide layer. Compared with a template method and a hydrothermal method, the anodic oxidation method can directly form a fixed nanotube on the surface of the titanium electrode, can control the diameter of the nanotube through parameter adjustment, and has low cost and strong operability.

The method comprises the following steps of totally dividing the titanium sheets into six groups, wherein the first group is subjected to anodic oxidation under the voltage of 30V after the titanium sheets are pretreated, the second group is subjected to anodic oxidation under the voltage of 40V after the titanium sheets are pretreated, the third group is subjected to anodic oxidation under the voltage of 50V after the titanium sheets are pretreated, the fourth group is subjected to anodic oxidation under the voltage of 60V after the titanium sheets are pretreated, the fifth group is subjected to anodic oxidation under the voltage of 70V after the titanium sheets are pretreated, the contrast group is the sixth group, the test pieces are only subjected to conventional titanium sheet pretreatment, and anodic oxidation is not performed.

Pure titanium sheets (purity 99.9%, 3.5 x 3.5cm, thickness 2mm) were used as substrates and polished to 400 grit and 1500 grit using No. silicon carbide sandpaper. Then the sample was washed sequentially with acetone, anhydrous alcohol, deionized water and finally dried at room temperature for 3 h.

Preparing and mixing electrolyte as a preparation system; the pre-treated sample was fixed as an anode while using a platinum sheet as the counter anode in an electrolyte aqueous solution of 0.15MNH4F and 90% ethylene glycol. And controlling the voltage of a direct current power supply, and respectively carrying out anodic oxidation on the anode titanium sheet in the electrolyte for 1 hour under the voltages of 30V, 40V, 50V, 60V and 70V. After the anodic oxidation, the anode was rinsed with deionized water for 30 minutes and washed with anhydrous alcohol in an ultrasonic cleaner for 15 minutes. Finally, all samples were sterilized with 120 ° autoclaving for 1h, and then the media was humidified prior to use. And controlling the temperature of the water bath at 20 ℃ in the process of anodic oxidation of the titanium sheet, and keeping the distance between the cathode and the working electrode at 40 mm. The reaction temperature of the system is controlled by changing the temperature of the water bath of the low-temperature water bath.

Characterization of the samples: samples prepared with different voltages were rinsed with ethanol and deionized water for 15 minutes and then dried at room temperature. Scanning electron microscopy was used to characterize the surface structure and measure the inside diameter and height of the nanotubes after plating with thin gold samples. Meanwhile, the elemental composition of the nanotubes was analyzed by x-ray energy dispersion analysis. The surface morphology and surface roughness of the samples were studied using atomic force microscopy. Three different regions were selected from each sample and repeated three times.

Titanium oxide sheet apparatus 1h at different constant voltages (30, 40, 50, 60, 70V). A uniformly distributed self-assembled nanotube array was observed with a scanning electron microscope. As shown in FIG. 1, the height of the nanotubes in all samples of the present invention was about 2 μm, while the inner diameter of the nanotubes was about 74nm (30V), 92nm (40V), 112nm (50V), 128nm (60V) and 148nm (70V). Then, X-ray energy dispersion analysis was performed to analyze the elemental composition of the nanotubes. It is shown that the nanotubes have only two elements, O and Ti. The nanotube structure was examined by Atomic Force Microscopy (AFM), and the arithmetic mean deviation of the nanotube profile was measured as surface roughness (Ra). The data show that nanotube surface roughness increases with increasing diameter (i.e., anodization voltage), as shown in fig. 2.

Example 2: influence of TiO2 nanotubes with different tube diameters on mesenchymal stem cell function

Primary cell extraction: four week old male Sprague-Dawley (SD) rats were sourced from the Experimental animals center in the ninth national Hospital, Shanghai. Rat bone marrow mesenchymal stem cells BMSCs were aseptically isolated from femur and tibia. Bone marrow mesenchymal stem cells were further purified and expanded in α -minimal essential culture containing 10% (v/v) fetal bovine serum FBS, 100mg/m streptomycin Gibco and 100U/mL penicillin Gibco, and cultured in a medium consisting of 95% air and 5% CO₂Incubation at 37 ℃ in a humidified atmosphere of composition.

Cell passage: the medium was refreshed every 2 days, cells trypsinized, 80% confluent passage. All cells used in the present invention were between passage 3 and passage 5. Osteogenic induction medium consisted of growth medium supplemented with 100nM dexamethasone, 10mM beta glycerophosphate, and 50mM ascorbic acid. Washing lacquer cells with 3ml PBS, adding 2ml pancreatin digestive juice, observing the cells under a microscope to be cloudy, adding 1ml calf serum to terminate the digestive reaction, repeatedly blowing the cells, mixing uniformly, transferring into a graduated centrifuge tube, centrifuging at 1500rpm for 5min, and discarding the supernatant. Resuspending the cells with complete medium, pumping and mixing evenly, adding a new culture flask, and adjusting the complete medium amount to 5 ml/flask.

Freezing and storing cells: cells in the logarithmic growth phase were taken, washed with PBS and trypsinized. The digestion reaction was interrupted by adding calf serum and centrifuged at 1500rpm for 5 min. The supernatant was discarded and 2ml of DMEM complete medium was added to resuspend the cells. Dropwise adding 2ml of the frozen stock solution, subpackaging, sealing and marking. Placing into a freezing storage box, storing at-20 deg.C for 1 hr, and storing at-150 deg.C.

And (3) treating the titanium sheet: referring to the above, the test piece is divided into six groups, the first group is subjected to anodic oxidation at a voltage of 30V after the titanium sheet is pretreated, the second group is subjected to anodic oxidation at a voltage of 40V after the titanium sheet is pretreated, the third group is subjected to anodic oxidation at a voltage of 50V after the titanium sheet is pretreated, the fourth group is subjected to anodic oxidation at a voltage of 60V after the titanium sheet is pretreated, the fifth group is subjected to anodic oxidation at a voltage of 70V after the titanium sheet is pretreated, the comparison group is the sixth group, the test piece is only subjected to conventional titanium sheet pretreatment, and anodic oxidation is not performed.

Cell inoculation: respectively inoculating bone marrow mesenchymal stem cells into culture dishes with titanium sheets of a test group and a control group, culturing with a conventional fetal bovine serum culture medium, and placing the culture dishes in CO₂In the incubator, the culture solution is replaced every 2 to 3 days.

And (3) detection by a scanning electron microscope: and (3) carrying out composite culture on the inoculated bone marrow mesenchymal stem cells and 6 groups of test pieces, and carrying out scanning electron microscope detection after 1d of culture. And observing the growth condition and the cell morphology of the cells on the surface of the material.

ALP detection: alkaline phosphatase is a class of enzymes that are widely found in animals. The normal osteoblasts are continuously secreted in the maturation process, the content of the normal osteoblasts can be used as a marker of the osteoblast osteogenesis function, the osteoblast mineralization deposition process can be represented to a certain degree, and the osteoblast mineralization deposition marker is an important parameter commonly used in osteoblast culture and detection, is used for detecting the differentiation capacity of cells, and can be used for directly representing the biocompatibility of the material on the osteoblasts.

Westernblot detection: inoculating bone marrow mesenchymal stem cells to the five groups of test pieces, performing composite culture for 3d, 7d and 14d, removing culture solution, flushing the bone marrow mesenchymal stem cells with 4 ℃ precooled PBS, digesting the cells with pancreatin, centrifuging, washing for 2-3 times, and collecting the cells in an EP tube. Cells were washed three times with PBS, lysed with RIPA buffer, and lysed on ice for 30 minutes with protease and phosphatase inhibitor cocktail. Lysates were collected by centrifugation at 12000rpm and centrifuged for 15min at 4 ℃. The concentration of total protein in the supernatant was determined using a dicaprylic acid (BCA) protein assay kit (Beyotime) as indicated by the instructions. The loading buffer was added to the protein sample, followed by cooking at 95 ℃ for 15 minutes. In the Westernblotting assay, 10. mu.L of the protein preparation was loaded onto a 12.5% SDSPAGE gel, electrophoresed at 120V for 1h, and then electrotransferred at 250mA to a polyvinylidene fluoride (PVDF) membrane for 2 h. The antibody was then blocked with 5-10% skim dry milk in TBST for 1 hour at room temperature on a shaker and diluted once in dilution buffer (Beyotime) overnight at 4 ℃. Next, after washing three times with TBST for 5 minutes, the fluorescent conjugated secondary antibody diluted with dilution buffer was added to the membrane and incubated at room temperature for 1 h. The protein band is detected by a bicolor infrared fluorescence imaging system. If the bands of the internal reference protein are unified, the membrane is peeled off and re-detected with another primary antibody, and the same process is followed.

(1) Protein extraction: adding RIPA lysate and liquid into cells, mixing well, and standing on ice for 10 min. 12000rpm, centrifugation for 10 min. Transfer supernatant to another EP tube. Protein concentration was measured and stored at-20 ℃ for later testing.

(2) Protein sample preparation and spotting: adding 5 xSDS loading buffer solution into each protein sample according to a proportion, mixing uniformly, heating at 95 ℃ for 5min, centrifuging at 12000rpm for 2min, sucking supernatant, adding into gel sample holes, adjusting the protein concentration to 20 ul/hole, and adding 10ul of pre-stained protein molecular weight marker into one hole.

(3) Electrophoresis: connecting the electrophoresis device with a power supply, performing electrophoresis at 40V for about 30min until the voltage of the dye strip is changed to 60V after entering the separation gel, continuing electrophoresis at constant voltage for 90min until the bromoku blue reaches the bottom of the gel, and turning off the power supply.

(4) Film transfer: the electrophoresed protein gel was equilibrated in sufficient transfer buffer for 15 min. The PVDF membrane of the desired size is cut off, immersed in methanol for 30 seconds, rinsed with ultra-pure water for 5 minutes with shaking, and equilibrated in a sufficient amount of transfer buffer for 15 minutes. Film transfer: the sandwich was made in the following order: cathode-filter paper-gel-PVDF membrane-filter paper-anode. Placing the prepared sandwich into a transfer tank, adding sufficient transfer buffer solution to completely soak the sandwich in the liquid, connecting with a source, transferring at 300mA and 4 ℃ for 50-60 min. The PVDF membrane was removed and the gel was stained with Coomassie brilliant blue to observe the effect of the transfer.

(5) And (3) sealing: and (3) placing the PVDF membrane in a hybridization bag, adding enough sealing liquid, shaking and incubating for one hour at room temperature, removing the sealing liquid, and shaking and washing paint for 3 times by using enough washing liquid.

(6) Primary antibody incubation: diluting the primary antibody with a primary antibody diluent according to a ratio of 1:1000, adding the diluted primary antibody into a hybridization bag, and oscillating and incubating the hybridization bag in a refrigerator at 4 ℃ overnight. The primary antibody incubation was removed and washed 5 times with sufficient PBS wash for 10min each time with shaking.

(7) And (3) secondary antibody incubation: HRP-labeled secondary antibodies were diluted 1:5000 with blocking solution (total volume 5ml), added to the hybridization bag, and incubated at room temperature for 1 hour with shaking. The secondary antibody incubation was removed and washed with sufficient wash solution for 3X 10min with shaking and 10min in TBS. And (5) color development treatment.

7 days after osteogenic induction, ALP staining was first performed to evaluate osteogenic differentiation of MSCs. The staining results showed that MSCs cultured on TiO2 nanotubes had higher ALP activity than cells cultured on smooth titanium substrates (control) as shown in fig. 3A. Statistical analysis of the stained area showed that the capacity of the nanotubes to induce osteogenic differentiation was significantly enhanced compared to the control group. Meanwhile, we observed that the larger the diameter of the TiO2 nanotubes in the diameter range of the present experiment, the stronger the ability to induce osteogenic differentiation as shown in fig. 3B. Therefore, the 70V group was used in subsequent experiments to better show the results. Next, the expression of osteogenic genes on day 3 and day 7 was analyzed. Bone marrow mesenchymal stem cells cultured on the titanium dioxide nanotubes for 3 days and 7 days showed significant promotion of the expression of osteogenic genes (RUNX2, ALP, cyanate, and OSX) as compared to the control group, as shown in fig. 3D. Westernblot results confirmed that protein expression of RUNX2 and OSX was also increased. As shown in fig. 3C. It was also found that f-actin was upregulated in the titanium dioxide nanotube group. Thus, the titanium dioxide nanotubes are directed towards osteoblast differentiation, which is related to the diameter of the nanotubes. The results also indicate that F-actin is involved in this process. The titanium dioxide nanotube promotes osteogenic differentiation of the mesenchymal stem cells. In fig. 3: a, ALP staining was performed on a smooth titanium substrate and five different nanotube substrates. Osteogenic medium induced cells 7 d. B, statistical analysis of the stained area using ImageJ. On day 7, MSCs were analyzed for C-osteogenic associated proteins (RUNX2 and OSX) and F-actin using western blotting. Mice were tested for RUNX2(D), alp (e), ocn (f), osx (g) mRNA expression on days 3, 7 using qRT-PCR. And (4) a nano-tube group. Data represent the mean SD of three samples. P <0.05, P <0.01, and P < 0.001.

Example 3: osteogenesis of nanotube titanium implants of different calibers

An animal model is constructed at the proximal end of the femur of an adult rat, and the titanium rod implant with the surface provided with the nanotubes with different scales is successfully implanted; the bone healing state and characteristics of the bone interface of the implant implanted into the animal body are researched by Micro-CT scanning, mechanical testing, morphology and immunohistochemistry methods, and the osteogenesis promoting effect and interface combination of the nanocrystallized implant are discussed in the tissue layer.

Preparing an implant: the implants for animal experiments are divided into five groups. The first group is subjected to anodic oxidation under the voltage of 30V after the titanium rod is pretreated, the second group is subjected to anodic oxidation under the voltage of 50V after the titanium rod is pretreated, the third group is subjected to anodic oxidation under the voltage of 70V after the titanium rod is pretreated, the fourth group is subjected to anodic oxidation under the voltage of 90V after the titanium rod is pretreated, the comparison group is the fifth group, and the test piece is only subjected to conventional titanium rod pretreatment and is not subjected to anodic oxidation.

Establishing an object model: animals selected for the experiment were healthy adult SD rats, 30 animals each, 6 animals each. Respectively implanting a titanium rod oxidized at a voltage of 30V, a titanium rod oxidized at a voltage of 50V, a titanium rod oxidized at a voltage of 70V, a titanium rod oxidized at a voltage of 90V and a titanium rod not oxidized.

The implant implantation step: adult healthy SD rats were anesthetized by intraperitoneal injection and fixed on an operating table in a prone position; removing hair near the incision of bilateral hind limb operation, disinfecting with conventional iodophor alcohol, and cutting skin and fascia; separating muscles blunt layer by layer, turning up fascia and periosteum, and exposing the upper section of the femur; removing cortex on one side by using a low-speed ball drill, positioning, preparing holes step by step, and communicating with a medullary cavity; when preparing the hole, the hole is cooled by normal saline, so that the bone tissue burns caused by overhigh temperature can be prevented; each femur was prepared with 1 hole and one titanium rod was implanted. The wound is closed by layered suture, and penicillin is injected into the muscle within three days after the operation, twice a day. Animals are sacrificed at different time points after operation, the implant standard wood is taken integrally together with the thighbone, and the specimens are fixed by 4% formaldehyde according to different experimental contents and purposes.

Implant and osseointegration strength test results: the mechanical test results of the five groups of test pieces are greatly improved, the bonding strength of the titanium implant and the bone of the experimental group is still obviously higher than that of the contrast group, and the nano surface is still advantageous in the aspects of new bone formation, matrix mineralization, mechanical embedding and the like.

And (3) performing morphological examination on tissues around the implant: the sacrifice and the material drawing are carried out at three time points of 2 weeks, 4 weeks and 6 weeks after the implantation of the titanium bar implant into the femur of the rat, and the conditions of cell growth, collagen secretion, osteoid deposition and new bone formation of the bone interface of the implant are observed through staining. After the implants are implanted in the body for 2 weeks, 4 weeks and 6 weeks, the formation of new bones is observed on the surfaces of five groups of implants; wherein new bones on the surfaces of the 70V group and the 90V group are completely formed, a bone trabecula structure is formed locally, and bone cells exist in the middle; and only a thin layer of new bone-like structure appears on the surface of the control group, the thickness is small, and the continuity is insufficient.

Scanning electron microscope results: a new reticular bone structure is fully distributed between the test group implant and the autogenous bone, a gap area formed after the new reticular bone structure is woven and filled with the old bone is absorbed, and the new bone tends to extend towards the marrow cavity; there was no significant space between the implants of the 70V and 90V groups and the old bone, the implants formed a tight contact with the bone; in the control group, it was observed that there was still a slight gap between the implant and the autogenous bone, and only a small amount of new bone was deposited in the gap near the marrow cavity.

And (3) Micro-CT detection result: after animal surgery, 2w, 4w and 6w of rats are sacrificed and are taken for Micro-CT detection, and three-dimensional reconstruction images established by airborne software can observe that the yellow new bone mass of each group is continuously increased along with the time, but at each time point, the coverage area of the new bone of the experimental group on the titanium rod is obviously more than that of the control group. According to the measured data of the onboard software, statistical analysis is carried out, as the implantation time of the titanium rod is prolonged, BV/TV (bone volume/total volume), Tb.Th (trabecular bone thickness) and TbN (trabecular bone number) of the two groups are continuously increased, particularly, the increase from 4w to 6w is very obvious, and the three parameters of the experimental group are higher than those of the control group (P <0.05) at each time point. In contrast, although both BS/BV (bone surface area in bone volume) and tb.sp (trabecular bone spacing) of each group decreased with increasing implantation time, the magnitude of the decrease in the experimental group was significantly lower at each time point than the control group (P < 0.05). The titanium rod which is formed into the nanotube through anodic oxidation can better promote the formation and regeneration of the surrounding bone tissues.

Example 4: influence of elastic deformation of titanium sheet on cell biological behavior

Elastic deformation test, titanium dioxide nanotubes prepared under 60V: a height of 2 μm, an inner diameter of 128nm, a roughness of 84nm, and an elastic deformation amount of 0.9% under a push-out test of a metal rod having a loading rate of 0.5mm/min and a diameter of 3 mm. Titanium dioxide nanotubes prepared at 40V: a height of 2 μm, an inner diameter of 92nm, a roughness of 64nm, and an elastic deformation amount of 0.3% under a push-out test of a metal rod having a loading rate of 0.5mm/min and a diameter of 3 mm. The two titanium dioxide nanotubes are respectively inoculated with cells (hMSCs) and subjected to related detection.

The results show the effect on osteogenic differentiation of cells: the expression of osteogenesis related markers OPN (osteopontin), OCN (osteoblast secretory protein) and RUNX2 were examined. As shown in fig. 4, the expression of 0.3% and 0.9% groups was increased to different degrees compared with the control groups OPN, OCN, RUNX2, and the increase of expression of 0.9% group OPN, OCN, RUNX2 was more significant, indicating that the titanium dioxide nanotube with elastic deformation amount of 0.9% has stronger directivity: promoting the differentiation of stem cells into osteoblasts. And the titanium dioxide nanotube with the elastic deformation amount of 0.3 percent also has stronger directivity: promote the differentiation of stem cells into adipocytes.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. A titanium dioxide nanotube is characterized in that the height of the titanium dioxide nanotube is 1.8-2.2 μm, the inner diameter is 74-148 nm, the roughness is 50-110nm, and the elastic deformation amount is 0.3-0.9% under the push-out test of a metal rod with the loading rate of 0.5mm/min and the diameter of 3 mm.

2. The titanium dioxide nanotube according to claim 1, wherein the titanium dioxide nanotube has a height of 2 μm, an inner diameter of 128nm, a roughness of 84nm, and an elastic deformation amount of 0.9% in a push-out test of a metal rod having a loading rate of 0.5mm/min and a diameter of 3 mm.

3. The titanium dioxide nanotube according to claim 1, wherein the titanium dioxide nanotube has a height of 2 μm, an inner diameter of 92nm, a roughness of 64nm, and an elastic deformation amount of 0.3% in a push-out test of a metal rod having a loading rate of 0.5mm/min and a diameter of 3 mm.

4. A method for preparing titanium dioxide nanotubes as claimed in any one of claims 1 to 3, comprising the steps of:

5. The use of a titanium dioxide nanotube as claimed in claim 1 in osteogenic growth.

6. Use of a titanium dioxide nanotube according to claim 2 for promoting differentiation of stem cells into osteoblasts.

7. The use according to claim 6, wherein the expression of osteopontin, osteoblast secretory protein, RUNX2 gene is simultaneously increased by the titanium dioxide nanotubes.

8. The use according to claim 6, wherein said titanium dioxide nanotubes have an increased expression of osteogenic genes RUNX2, ALP, cyanate and OSX.

9. The use according to claim 6, wherein f-actin is upregulated in said titanium dioxide nanotube group.

10. The use of a titanium dioxide nanotube according to claim 3 for promoting differentiation of stem cells into adipocytes.