CN115444980A - Metal ion doped titanium dioxide nano coating, preparation method and application thereof in nerve and bone tissue repair - Google Patents
Metal ion doped titanium dioxide nano coating, preparation method and application thereof in nerve and bone tissue repair Download PDFInfo
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- CN115444980A CN115444980A CN202110638401.XA CN202110638401A CN115444980A CN 115444980 A CN115444980 A CN 115444980A CN 202110638401 A CN202110638401 A CN 202110638401A CN 115444980 A CN115444980 A CN 115444980A
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Abstract
The invention discloses a metal ion doped titanium dioxide nano-coating, a preparation method and application thereof in nerve and bone tissue repair. The nano coating takes the titanium dioxide nanowires which are staggered with each other as a carrier, and metal ions exist on the surface of the titanium dioxide nanowires in the form of ions or oxides or are doped into titanium dioxide crystal lattices in the form of ions; the metal ion is alkaline earth metal ion and/or transition metal ion, preferably Ca 2+ 、Mg 2+ 、Mn 2+ At least one of; the metal ion contentThe mass fraction of the rice coating is 0.1-10%, preferably 0.1-6%. The metal ion doped titanium dioxide nano coating has good biological activity, and can activate nerve cell integrin by releasing metal ions, promote nerve cell differentiation and nerve peptide release, and potentially regulate and control bone tissue repair.
Description
Technical Field
The invention relates to a metal ion doped titanium dioxide nano coating, a preparation method and application thereof in nerve and bone tissue repair, belonging to the field of biomedical materials.
Background
In the human skeletal system, nervous tissue is distributed mainly in areas of active bone metabolism, and its normal function is essential for maintaining the skeletal microenvironment stable and promoting fracture healing. For example, calcitonin Gene Related Peptide (CGRP) secreted from nerve cells can promote bone tissue regeneration after human fracture, and is favorable for bone integration of bone implant Materials after surgery (Materials Today,2018,21 (4): 362-376). However, when the nervous system is diseased or functionally damaged, it results in insufficient innervation around the fracture, which causes the fracture site to often show low callus strength, delayed fracture healing and nonunion, severely restricting the bone repair process. Therefore, the development of a novel bone repair material having a good nerve regeneration function is of great significance in promoting bone repair of patients with nervous system neuropathy or injury.
The metallic titanium material and the alloy thereof have good biocompatibility, chemical stability and corrosion resistance, and are clinically common bone implant materials. However, the titanium substrate surface is biologically inert, resulting in insufficient capacity to promote osteogenesis and nerve regeneration. The results of the present studies show that cell adhesion to the surface of the implant and intracellular and extracellular signaling are regulated by the activity of cell membrane integrins. In the inactive state, the integrin molecules are in a closed, folded conformation with low affinity for extracellular matrix proteins. After the activated integrins are combined with extracellular matrix proteins, intracellular structural proteins can be further recruited to form adhesion plaques, so that intracellular signal channels are activated, and the behaviors of migration, proliferation, differentiation and the like of cells are regulated and controlled. Therefore, by activating cell integrins, the activity of osteoblasts and nerve cells is expected to be regulated, and the aim of promoting the rapid repair of bone and nerve tissues is fulfilled.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a metal ion doped titanium dioxide nano coating, a preparation method and application thereof in nerve and bone tissue repair. The metal ion doped titanium dioxide nano coating has good bioactivity, and can activate nerve cell integrin by releasing metal ions, promote nerve cell differentiation and neuropeptide release, and potentially regulate and control bone tissue repair. Meanwhile, the metal ions released by the nano coating can activate the integrin of osteoblasts, thereby promoting osteogenic differentiation of the osteoblasts.
In a first aspect, the present invention provides a metal ion doped titanium dioxide nanocoating. The nano coating takes the titanium dioxide nanowires which are staggered with each other as a carrier, and metal ions exist on the surface of the titanium dioxide nanowires in the form of ions or oxides or are doped into the titanium dioxide crystal lattices in the form of ions. The titanium dioxide nanowire structure has the characteristics of large specific surface area and high hydrophilicity, and can promote the surface of the titanium dioxide nanowire structure to adsorb extracellular matrix proteins. These adsorbed extracellular matrix proteins facilitate the activation of cell membrane integrins and produce synergistic effects with metal ions. Particularly, the introduction of metal ions in the nano coating of the invention reserves the structure of the titanium dioxide nano wire, and can further promote the adhesion and functional expression of cells on the surface of the cell.
The metal ion is alkaline earth metal ion and/or transition metal ion, preferably Ca 2+ 、Mg 2+ 、Mn 2+ At least one of (a). Compared with K + 、Na + The monovalent metal ions and the divalent metal ions have high charge density and strong electron accepting capability and are easier to be bonded with integrinThe resultant, i.e. divalent, ions have a strong ability to activate integrins. And, ca 2+ 、Mg 2+ 、Mn 2+ The content of metal ions inside and outside cells is high, and the biocompatibility is good.
The mass fraction of the metal ions in the nano coating is 0.1-10%, preferably 0.1-6%. The metal ion doped titanium dioxide coating with the content range can dissolve out metal ions with proper concentration, can activate cell integrins, does not obviously inhibit cell functions such as cell differentiation and mineralization and the like, and promotes tissue repair.
Preferably, the thickness of the metal ion doped titanium dioxide nano coating is 2-5 μm. The coating in this thickness range remains in the nanowire form. If the thickness of the coating is too low, the coating is in a nano-leaf or nano-pore shape, and if the thickness of the coating is too high, the coating is easy to peel off from the base material.
Preferably, the diameter of the titanium dioxide nanowire is 20-60nm.
Preferably, the metal ion doped titanium dioxide nanocoating releases metal ions to activate integrins, in particular neuronal and/or osteoblastic integrins. The coating of the present invention uses Ca 2+ 、Mg 2+ 、Mn 2+ The charge density and the electron accepting ability of the metal ions present an activation capability suitable for the application of the coating in nerve and bone tissue repair.
In a second aspect, the invention provides a method for preparing the metal ion doped titanium dioxide nano-coating. The preparation method comprises the following steps:
(1) Immersing a titanium base material in a sodium hydroxide aqueous solution, and growing a sodium titanate nano layer on the surface of the titanium base material in situ by adopting a hydrothermal reaction method;
(2) Sequentially soaking the base material with the surface in-situ grown sodium titanate nano-layer prepared in the step (1) in hydrochloric acid and an aqueous solution containing metal ions to dope the metal ions in the sodium titanate nano-layer through ion exchange;
(3) And (3) annealing the titanium substrate obtained in the step (2) to obtain the metal ion doped titanium dioxide nano coating.
Preferably, in the step (1), the concentration of the sodium hydroxide aqueous solution is 0.1-10mol/L; the hydrothermal reaction temperature is 150-300 ℃, and the reaction time is 8-30h.
Preferably, in the step (2), the concentration of the hydrochloric acid is 0.01-1mol/L, and the soaking time is 0.5-4h; the concentration of the aqueous solution containing the metal ions is 0.001-1mol/L, and the soaking time is 0.1-24h.
Preferably, in the step (3), the annealing temperature is 400-700 ℃ and the annealing time is 0.5-4h.
Preferably, the titanium substrate is a medical titanium substrate and comprises pure titanium or a titanium alloy.
In a third aspect, the invention provides a metal ion doped TiO capable of promoting nerve and bone tissue repair 2 And (4) nano coating.
Drawings
A in FIG. 1 is TiO 2 And Mn 2+ Doped TiO 2 2 XRD pattern of nano coating, B is Mn 2+ Doped TiO 2 2 Ion elution amount of nano coating, C is TiO 2 And Mn 2+ Doped TiO 2 2 SEM photograph of the nanocoating;
a in FIG. 2 is Mn 2+ Doped TiO 2 2 Nano TEM picture and element surface distribution diagram, B is Mn 2+ Doped TiO 2 2 Mn 2p of nano coating 3/2 High resolution XPS spectra with C being Mn 2+ Doped TiO 2 2 XRD pattern of anatase (101) crystal face of nano coating;
a in FIG. 3 is TiO 2 And Mn 2+ Doped TiO 2 2 The integrin alpha 5 (Itga 5) gene expression quantity of nerve cells on the surface of the nano coating, B is integrin beta 3 (Itgb 3) gene expression quantity, C is Tubulin secretion quantity, and D is neuropeptide CGRP secretion quantity;
a in FIG. 4 is TiO 2 And Mn 2+ Doped TiO 2 2 The integrin expression amount of osteoblasts on the surface of the nano coating, B is ALP activity, C is extracellular matrix mineralization expression amount, and D is osteocalcin OCN expression amount;
a in FIG. 5 is TiO 2 And Mg 2+ Doped TiO 2 2 XRD pattern of nano coating, B is Mg 2+ Doped TiO 2 2 Ion elution amount of nano coating, C is Mg 2+ Doped TiO 2 2 SEM photograph of the nanocoating;
a in FIG. 6 is TiO 2 And Mg 2+ Doped TiO 2 2 The integrin alpha 5 (Itga 5) gene expression quantity of nerve cells on the surface of the nano coating, B is integrin beta 3 (Itgb 3) gene expression quantity, C is Tubulin secretion quantity, and D is neuropeptide CGRP secretion quantity;
a in FIG. 7 is TiO 2 And Mg 2+ Doped TiO 2 2 The integrin expression amount of osteoblasts on the surface of the nano-coating, B is ALP activity, C is extracellular matrix mineralization, and D is Osteocalcin (OCN) expression amount.
Detailed Description
The present invention is further illustrated by the following examples, which are to be understood as merely illustrative of, and not restrictive on, the present invention. Unless otherwise specified, each percentage means a mass percentage.
The present disclosure provides a metal ion doped titanium dioxide nanocoating that can promote nerve and bone tissue repair. The bio-coating has a nanowire structure (as shown by C in fig. 1 and C in fig. 5). The coating structure of the nano-wire can promote cell adhesion and is beneficial to functional expression of nerve regeneration, osteogenic differentiation and the like.
The nano coating takes the titanium dioxide nanowires which are staggered with each other as a carrier, and metal ions exist on the surface of the titanium dioxide nanowires in the form of ions or oxides and/or are doped into the titanium dioxide crystal lattices in the form of ions. Compared with the coating form of nano leaves and nano holes, the titanium dioxide in the nano line form is easier to adsorb extracellular matrix proteins. These adsorbed extracellular matrix proteins facilitate the activation of cell membrane integrins. The titanium dioxide is mainly anatase type and may contain a small amount of rutile type. The titanium dioxide of the anatase phase has higher biocompatibility.
The integrin structure contains multiple Metal ion binding sites, such as Metal Ion Dependent Adhesion Site (MIDAS), adjacent MIDASA site (ADA to MIDAS) and cooperative Metal ion binding site (Syn)An experimental metal binding site, syMBS). The metal ion of the coating of the invention may be Ca 2+ 、Mg 2+ 、Mn 2+ And at least one of alkaline earth metal or transition metal ions. When a certain concentration of Ca 2+ 、Mg 2+ And Mn 2+ After binding to them, metal ions activate integrins and promote cell adhesion and differentiation.
The mass fraction of the metal ions in the nano coating is 0.1-10%, preferably 0.1-6%. The doping amount of the metal ions is too high, so that excessive metal ions are easily dissolved out, and integrin is excessively activated, thereby reducing the activity of cells. Compared with TiO not doped with metal ions 2 Nano coating, metal ion doped TiO obtained by the invention 2 The nano coating has better biological activity, can stimulate nerve cells and osteoblasts to differentiate, is a potential biomedical material, and can be used for research and development of bone repair materials of patients with nervous system pathological changes or injuries.
The following is an exemplary illustration of the metal ion doped TiO of the present invention for promoting nerve and bone tissue repair 2 A preparation method of a nano coating. Adopting a hydrothermal reaction, ion exchange and high-temperature annealing three-step method, absorbing metal ions on the surface of the sodium titanate nanowire through ion exchange in the process, and preparing metal ion doped titanium dioxide (TiO) with a nanowire structure on the surface of the titanium substrate in situ after subsequent annealing 2 ) The coating provides possibility for researching and developing novel coating materials for repairing bone-nerve tissues.
And (3) constructing a sodium titanate nano layer on the surface of the titanium substrate in situ by utilizing a hydrothermal reaction. Immersing the medical titanium substrate in a sodium hydroxide aqueous solution, and growing a sodium titanate nano layer on the surface of the medical titanium substrate in situ by adopting a hydrothermal reaction method. The nanolayers are comprised of sodium titanate nanowires that are interlaced with each other. The shape of the nano layer can be adjusted and controlled by controlling the time and the concentration of the hydrothermal reaction so as to form the nano wire. The coating can be formed on the surface of pure titanium or titanium alloy. The reaction temperature of the hydrothermal reaction method is 150-300 ℃, and preferably 180-250 ℃. The hydrothermal reaction is carried out for 8-30h, preferably 12-18h. The concentration of sodium hydroxide is 0.1-10mol/L, preferably 0.5-2mol/L. The concentration of the sodium hydroxide aqueous solution is too low, the hydrothermal reaction temperature is too low or the heat preservation time is too short, and a coating with a proper nano structure is not easy to form on the surface of the base material. The concentration of sodium hydroxide is too high, the hydrothermal reaction temperature is too high or the heat preservation time is too long, the thickness of the obtained nano coating is larger, and the binding force between the coating and the base material can be reduced.
And doping metal ions in the sodium titanate nano coating by using an ion exchange method. Soaking the prepared sodium titanate nano layer in hydrochloric acid solution for a period of time, namely using H + Exchange of Na in sodium titanate + This facilitates the conversion of the sodium titanate to titanium dioxide upon subsequent annealing. The concentration of the hydrochloric acid solution is 0.01-1mol/L, preferably 0.05-0.5mol/L, and the soaking time is 0.5-4h, preferably 1-3h. Too low hydrochloric acid concentration or too short soaking time are not favorable for forming TiO after annealing 2 . Too high a hydrochloric acid concentration or soaking time may reduce the bonding force of the annealed coating and the substrate. Then soaking in water solution containing metal ions for a period of time to obtain nano coating containing metal ions, and mixing the metal ions with H + The exchange forms a nanowire coating doped with metal ions. The concentration of the metal ion solution is 0.001-1mol/L, preferably 0.005-0.5mol/L; the soaking time is 0.1-24h, preferably 0.5-15h. If the concentration of the ionic solution is too low or the soaking time is too short, metal ions are not easy to be doped into TiO 2 The coating, too high concentration of ionic solution or too long soaking time easily causes the coating to be doped with excessive metal ions, which is not favorable for the function of surface cells.
And annealing the nano coating doped with the metal ions. The annealing temperature is 400-700 ℃, preferably 450-600 ℃, and the annealing time is 0.5-4h, preferably 1-2h. Annealing can convert the sodium titanate into highly biocompatible titanium dioxide. After annealing, the sodium titanate is converted into titanium dioxide, and metal ions exist on the surface of the titanium dioxide nanowire in the form of ions/oxides or are doped into the inside of titanium dioxide crystal lattices in the form of ions.
The nano coating obtained by the invention has good biological activity, can activate nerve cell integrin by releasing metal ions (preferably divalent metal ions), promotes nerve cell differentiation and neuropeptide release, and potentially regulates and controls bone tissue repair. Meanwhile, the metal ions released by the nano coating can activate the integrin of osteoblasts, thereby promoting osteogenic differentiation of the osteoblasts.
The present invention will be described in further detail with reference to examples. It is also to be understood that the following examples are illustrative of the present invention and are not to be construed as limiting the scope of the invention, and that insubstantial modifications and adaptations of the invention by those skilled in the art based on the foregoing description are intended to be included within the scope of the invention. The specific process parameters and the like of the following examples are also only one example of suitable ranges, i.e., those skilled in the art can select the appropriate ranges through the description herein, and are not limited to the specific values exemplified below.
Example 1
A：Mn 2+ Doped TiO 2 2 Preparation of the Nanocaoatings
And (3) placing the titanium sheet polished to be smooth by the abrasive paper in a sodium hydroxide aqueous solution, and growing a sodium titanate nano coating on the surface of the titanium sheet in situ by adopting a hydrothermal reaction method. The volume of the aqueous solution of sodium hydroxide used in the hydrothermal reaction method was 60mL, and the concentration of sodium hydroxide was 1mol/L. The hydrothermal reaction time is 15h, and the hydrothermal reaction temperature is 220 ℃. Respectively coating the coating prepared by hydrothermal reaction in hydrochloric acid solution and MnCl by adopting an ion exchange method 2 Soaking in water solution for a period of time, and preparing Mn by high-temperature annealing method 2+ Doped TiO 2 2 And (4) nano coating. The concentration of the hydrochloric acid solution is 0.1mol/L, and the soaking time is 2h. MnCl 2 The water concentration is 0.01mol/L (soaking time is 1 h) or 0.1mol/L (soaking time is 5 h). The annealing temperature is 600 ℃, and the annealing time is 1h.
In the same manner, but omitting MnCl 2 Solution soaking process to prepare undoped Mn 2+ Of TiO (2) 2 And (4) nano coating.
MnCl used 2 The concentration of the aqueous solution is 0.01mol/L, the soaking time is 1h, and the sample formed by annealing is marked as TiO 2 -Mn1. MnCl used 2 The concentration of the aqueous solution is 0.1mol/L, the soaking time is 5h, and the sample formed by annealing is marked as TiO 2 -Mn2. Not passing through MnCl 2 The sample formed by soaking in aqueous solution and direct annealing is marked as TiO 2 。
And after the preparation of the coating is finished, analyzing the composition of the nano coating. The major phase of the three coatings was anatase TiO as shown by the XRD pattern shown as A in FIG. 1 2 Containing a small amount of rutile type TiO 2 。
The coatings were analyzed for ion release using 0.9wt.% NaCl, and soaking times of 1, 4, and 7 days. From the ion release amount shown as B in FIG. 1, tiO was found 2 Mn1 and TiO 2 Mn2 as the soaking time increases, its Mn 2+ Elution amount is increased and TiO 2 Mn2 vs. TiO 2 The ion elution amount of-Mn 1 is larger.
As can be seen from the SEM photograph shown as C in FIG. 1, the surface topography of all three coatings is nanowires, and the diameter of each nanowire is 20-60nm. EDS test results show that TiO 2 Mn1 and TiO 2 The content of Mn in Mn2 was 1.9wt.% and 3.9wt.%, respectively.
From the TEM and the elemental surface distribution photograph shown in a in fig. 2, mn element was uniformly distributed on the surface of the nanowire, and from the XPS result shown in B in fig. 2, it was found that expensive Mn was present in the nanocoating layer 3+ And Mn 4+ It is known that doped Mn may exist on the surface of the nanowire in the form of an oxide containing Mn in a higher valence state. From the XRD results shown in FIG. 2C, it is understood that the diffraction peak corresponding to the (101) crystal plane of anatase is shifted by Mn doping, and that Mn is incorporated into TiO 2 Inside the crystal lattice and change the interplanar spacing.
B：Mn 2+ Doped TiO 2 2 Nano coating for promoting nerve cell integrin expression, cell differentiation and neuropeptide secretion detection
Rat adrenal pheochromocytoma PC12 cells were used for nerve cell-related experiments.
(1) Integrin expression
The density is 2.5 multiplied by 10 5 PC12 cells per well were seeded on the surface of the sterilized samples. After 3d of culture, RNA was extracted using Trizol kit (Invitrogen, USA) and passed through R with PrimeScript RT kit (Takara, japan)Reverse transcription of NA produces cDNA. Real-time quantitative polymerase chain reaction was performed using a model 9700 real-time PCR system (ABI, USA) and the results were recorded. According to 2 -ΔΔCt The method analyzes and calculates the detection result. The genes tested included Ita5 and Itb3.
(2) Cell differentiation assay
PC12 cells were cultured at 2X 10 4 The density of the/pores is seeded on the coating surface. After incubation for 4d, the samples were fixed in 4% formaldehyde solution for 30min and washed 2 times with PBS. Subsequently, the sample was placed in a well plate, subjected to a membrane disruption treatment for 10min at room temperature with PBS containing 0.5% Triton X-100, treated with BSA blocking solution at 37 ℃ for 1h, and washed 3 times with PBS. beta-Tubulin primary antibody (TUJ 1, abcam, USA) was added, incubated overnight at 4 ℃ and the samples were washed 3 times with PBS. Secondary antibodies (Alexa Fluor 488, abcam, USA) were added and treated for 1h at room temperature, washing the samples 3 times with PBS. Nuclei were stained with DAPI (Abcam, USA) for 5min in the dark, followed by washing 3 times with PBS. Cells were visualized using a fluorescence microscope (Leica, germany) for photographs and the fluorescence intensity of Tubulin was statistically analyzed using Image J software.
(3) Amount of CGRP neuropeptide secreted
The density is 5 multiplied by 10 4 PC12 cells per well were seeded on the surface of the sterilized sample. After incubation for 2 days, the content of CGRP in the culture solution was determined by ELISA.
A and B in FIG. 3 are integrin gene expression results of PC12 cells cultured on different sample surfaces for 3d, and it is found that gene expression levels of Itga5 and Itgb3 in cells are dependent on the nano-coating Mn 2+ The increase in elution amount indicates that the metal ion is doped with TiO 2 Mn dissolved out from nano coating 2+ Is favorable for activating integrin of PC12 cells.
Tubulin is a marker of nerve cell differentiation, and tubulin formation plays an important role in maintaining neurite outgrowth. FIG. 3C shows the statistics of the fluorescence intensity of Tubulin Tubulin after PC12 cells were cultured on the surface of different samples for 4 days. Mn along with coating 2+ The release amount is increased, and the fluorescence intensity of the microtubulin of the PC12 cell is increased. The above experimental results confirm Mn 2+ Can promote nerve cell differentiation and tubulin formation.
Neuropeptides produced by the nervous system play an important regulatory role in the growth and reconstruction process of bones, and CGRP is a neuropeptide with higher activity and is considered as the most important neuropeptide for regulating bone reconstruction. D in FIG. 3 is the amount of CGRP secretion from PC12 cells on the surface of different samples. Mn along with coating 2+ The dissolution amount is increased, the CGRP expression amount of the cells on the surface of the coating is in an increasing trend, and the expression amount is consistent with the differentiation trend of the cells.
As a result, mn is known 2+ Doped TiO 2 2 The coating can be coated by dissolving out Mn 2+ Activating integrin of the PC12 cell, further promoting differentiation, and the PC12 cell with better differentiation degree has the potential of regulating osteoblast activity.
C：Mn 2+ Doped TiO 2 2 Nano-coating for promoting osteoblast integrin expression and cell differentiation
Osteoblast-related experiments were performed using mouse preosteoblasts MC3T 3-E1.
(1) Integrin expression level
Will have a density of 5X 10 3 MC3T3-E1 cells per well were seeded on the surface of the sterilized sample (1 cm. Times.1 cm). After incubation for 2d, the supernatant was discarded and the sample was washed 2 times with PBS. And adding a paraformaldehyde solution with the mass volume ratio of 4% for fixation for 15min, and washing the sample 3 times by using PBS. BSA blocking solution was used for 1h at 37 ℃. 100. Mu.L of a dilution of 1:100 antibodies to Ita5 and Itb1 (CST, USA) were stained. The samples were removed and washed 3 times with PBS, and 100 μ L dilution 1:200 fluorescent secondary antibodies (CST, USA) were incubated at 37 ℃ for 30min. Cells were then observed and photographed using CLSM. The fluorescence intensities of Ita5 and Itb1 were statistically analyzed using Image J software.
(2) Alkaline phosphatase (ALP) Activity
MC3T3-E1 cells were seeded onto the surface of the sterilized samples. After incubation for 7d or 14d, 200. Mu.L of 0.2% Triton X-100 in PBS was added to each well and lysed on ice for 15min. The lysed suspension was then centrifuged for 5min and the supernatant collected and transferred to a 96-well plate. mu.L of the supernatant was mixed with 150. Mu.L of p-nitrophenylphosphate (pNPP) and incubated for 10min in the absence of light. And measuring the OD value of the supernatant by using a microplate reader. In addition, the BCA method was used to detect the total protein content in the cells, and the ALP activity was expressed in. Mu.M/min. G.
(3) Extracellular matrix mineralization
MC3T3-E1 cells were seeded onto the surface of the sterilized samples. After incubation for 14d and 21d, the samples were washed 2 times with PBS and fixed for 15min by adding paraformaldehyde solution. Alizarin red (ARS, sigma-Aldrich) was then added, incubated at 37 ℃ for 30min, washed 3 times with pbs, and 500 μ L of Cetylpyridinium chloride (CPC, sigma-Aldrich) solution was added per well for evaluation of the amount of calcium deposited on the cells, and incubated at room temperature for 15min. OD of the supernatant at 590nm was measured using a microplate reader.
(4) Osteocalcin (OCN) expression level
Will have a density of 5X 10 3 MC3T3-E1 cells per well were seeded on the surface of the sterilized sample (1 cm. Times.1 cm). After incubation for 14d, the supernatant was discarded and the sample was washed 2 times with PBS. A4% paraformaldehyde solution was added for fixation for 15min, and the sample was washed 3 times with PBS. BSA blocking solution was used for 1h at 37 deg.C. 100. Mu.L of a dilution of 1:100 antibodies to OCN (CST, USA) were stained. The samples were removed, washed 3 times with PBS, and 100 μ L of a dilution of 1:200 fluorescent secondary antibody (CST, USA) were incubated at 37 ℃ for 30min. Cells were then observed and photographed using CLSM. The fluorescence intensity of the OCN was statistically analyzed using Image J software.
FIG. 4A shows the statistics of the fluorescence of integrins α 5 and β 1 of preosteoblasts cultured on the material surface for 2 days, and the fluorescence intensity of Itga5 and Itgb1 in cells is found to follow the nano-coating Mn 2+ The increase in elution amount indicates that the metal ion is doped with TiO 2 Mn dissolved out from nano coating 2+ Facilitates the activation of the integrins of MC3T3-E1 cells.
FIG. 4B shows ALP activity of preosteoblasts cultured on the material surface for 7d and 14d, showing that ALP activity of cells is dependent on Mn of the nano-coating 2+ The amount of elution gradually increased.
C in FIG. 4 is the result of mineralizing extracellular matrix of preosteoblasts after being cultured on the surface of the material for 14d and 21d, and the mineralizing amount of MC3T3-E1 cells on the surfaces of different nano coatings is found to show the following trend: tiO 2 2 -Mn2＞TiO 2 -Mn1＞TiO 2 Indicates that the metal ion is doped with TiO 2 Mn dissolved out from nano coating 2+ Promotes the mineralization of the extracellular matrix of MC3T 3-E1.
D in FIG. 4 is the result of statistics of fluorescence intensity of OCN (late marker of osteogenic differentiation) after culturing the preosteoblasts on the surface of the material for 14D, and the fluorescence intensity of OCN in the cells is found along with the nano-coating Mn 2+ The increase of the elution amount shows an upward trend, and the metal ion doped TiO is shown 2 Mn dissolved out from nano coating 2+ Promotes the formation of OCN by MC3T3-E1 cells.
Thus, mn 2+ Doped TiO 2 2 The coating can dissolve Mn 2+ Activating the integrins of MC3T3-E1 cells, and further promoting osteogenic differentiation.
Example 2
A：Mg 2+ Doped TiO 2 2 Preparation of the Nanocaoatings
And (3) placing the titanium sheet polished to be smooth by the abrasive paper in a sodium hydroxide aqueous solution, and growing a sodium titanate nano coating on the surface of the titanium sheet in situ by adopting a hydrothermal reaction method. The volume of the sodium hydroxide aqueous solution used in the hydrothermal reaction method is 60mL, and the concentration of the sodium hydroxide aqueous solution is 1mol/L. The hydrothermal reaction time is 15h, and the hydrothermal reaction temperature is 220 ℃. Respectively preparing the coating prepared by hydrothermal reaction in hydrochloric acid solution and MgCl by ion exchange method 2 Soaking in water solution for a period of time, and preparing Mg by high-temperature annealing method 2+ Doped TiO 2 2 And (4) nano coating. The concentration of hydrochloric acid is 0.1mol/L, and the soaking time is 2h. MgCl 2 The water concentration is 0.1mol/L, and the soaking time is 5h. The annealing temperature is 600 ℃, and the annealing time is 1h.
In the same way, but omitting MgCl 2 Solution soaking process to prepare undoped Mg 2+ Of TiO 2 2 And (4) nano coating.
MgCl used 2 Concentration of 0.1mol/L and soaking time of 5h and sample formed by annealingArticle label TiO 2 -Mg. Not passing through MgCl 2 The sample formed by concentration soaking and direct annealing is marked as TiO 2 。
XRD pattern, tiO as shown in A of FIG. 5 2 The main phase of Mg is anatase TiO 2 Containing a small amount of rutile type TiO 2 。
From the ion release amount shown as B in FIG. 5, tiO was found 2 Mg as the soaking time increases, mg 2+ The elution amount of (2) is increased.
From the SEM photograph shown in FIG. 5C, tiO was found 2 The surface morphology of Mg is nanowires, the diameter of which is 20-60nm. XPS test results show that TiO 2 The Mg content in Mg was 1.5wt.%.
B：Mg 2+ Doped TiO 2 2 Nano coating for promoting nerve cell integrin expression, cell differentiation and neuropeptide secretion detection
The neural cells used and the relevant experimental procedures were the same as in example 1.
In FIG. 6, A and B are the integrin gene expression results of PC12 cells cultured on different sample surfaces for 3 d. Compared with TiO 2 ，TiO 2 The gene expression of Itga5 and Itgb3 in Mg surface cells increased significantly with the nanocoating, indicating that TiO 2 -Mg dissolved from Mg nanocoating 2+ Is favorable for activating integrin of PC12 cells.
C in FIG. 6 is the result of statistics of the fluorescence intensity of tubulin after PC12 cells were cultured on the surface of different samples for 4 days. Compared with TiO 2 ，TiO 2 Increase in the fluorescence intensity of PC12 cell tubulin on the Mg surface, indicating Mg 2+ Can promote nerve cell differentiation and tubulin formation.
D in FIG. 6 is the amount of CGRP secretion from PC12 cells on the surface of different samples. Compared with TiO 2 ，TiO 2 The CGRP expression of the-Mg surface PC12 cell is obviously improved and is consistent with the differentiation trend of the cell.
In summary, mg 2+ Doped TiO 2 2 The coating can be formed by dissolving out Mg 2+ Activating integrins of PC12 cells to promote their differentiation and to a good degreeThe PC12 cell has the potential of regulating the activity of osteoblast.
C：Mg 2+ Doped TiO 2 2 Nano-coating for promoting osteoblast integrin expression and cell differentiation
The osteoblasts used and the relevant experimental procedures were the same as in example 1.
Fig. 7 a shows statistics of the fluorescence of integrins α 5 and β 1 after 2d incubation of preosteoblasts on the material surface. Compared with TiO 2 ，TiO 2 Increase in fluorescence intensity of Itga5 and Itgb1 in Mg surface cells, tiO 2 -Mg eluted from Mg nanocoating 2+ Facilitates the activation of the integrins of MC3T3-E1 cells.
B in FIG. 7 is ALP activity of preosteoblasts after 7d and 14d incubation on the material surface, and TiO was found 2 ALP activity of Mg surface cells is obviously higher than that of TiO 2 。
C in FIG. 7 is the results of mineralization of the extracellular matrix after culturing of preosteoblasts on the surface of the material for 14d and 21d, compared to TiO 2 ，TiO 2 The mineralization quantity of Mg surface cells is obviously improved, indicating that TiO 2 -Mg dissolved from Mg nanocoating 2+ Promotes the mineralization of the extracellular matrix of the MC3T 3-E1.
D in FIG. 7 is the statistic result of OCN fluorescence intensity after culturing the preosteoblasts on the surface of the material for 14 days, and TiO is found 2 The fluorescence intensity of the-Mg surface cells is obviously higher than that of TiO 2 Indicating TiO 2 The Mg nano-coating can promote the formation of OCN by MC3T3-E1 cells.
In summary, mg 2+ Doped TiO 2 2 The coating can be formed by dissolving out Mg 2+ Activating the integrins of MC3T3-E1 cells, and further promoting osteogenic differentiation.
Claims (10)
1. The metal ion doped titanium dioxide nano coating is characterized in that the nano coating takes titanium dioxide nanowires which are staggered with each other as a carrier, and metal ions exist on the surface of the titanium dioxide nanowires in the form of ions or oxides or are doped into titanium dioxide crystal lattices in the form of ions; the metal ion is alkaline earth metal ion and/or transition metal ion, preferably Ca 2+ 、Mg 2+ 、Mn 2+ At least one of; the mass fraction of the metal ions in the nano coating is 0.1-10%, preferably 0.1-6%.
2. The metal ion-doped titanium dioxide nanocoating according to claim 1, wherein the thickness of the metal ion-doped titanium dioxide nanocoating is 2-5 μ ι η.
3. The metal ion doped titanium dioxide nanocoating according to claim 1 or 2, wherein the diameter of the titanium dioxide nanowires is 20-60nm.
4. The metal ion doped titanium dioxide nanocoating according to any one of claims 1 or 3, wherein the metal ion doped titanium dioxide nanocoating releases metal ions to activate integrins, in particular neuronal and/or osteoblast integrins.
5. The method for preparing a metal ion doped titanium dioxide nanocoating according to any one of claims 1 to 4, characterized in that it comprises the steps of:
(1) Immersing a titanium substrate in a sodium hydroxide aqueous solution, and growing a sodium titanate nano layer on the surface of the titanium substrate in situ by adopting a hydrothermal reaction method;
(2) Sequentially soaking the base material with the surface in-situ grown sodium titanate nano-layer prepared in the step (1) in hydrochloric acid and an aqueous solution containing metal ions to dope the metal ions in the sodium titanate nano-layer through ion exchange;
(3) And (3) annealing the titanium substrate obtained in the step (2) to obtain the metal ion doped titanium dioxide nano coating.
6. The production method according to claim 5, wherein in the step (1), the concentration of the aqueous sodium hydroxide solution is 0.1 to 10mol/L; the hydrothermal reaction temperature is 150-300 ℃, and the reaction time is 8-30h.
7. The preparation method according to claim 5 or 6, wherein in the step (2), the concentration of the hydrochloric acid is 0.01-1mol/L, and the soaking time is 0.5-4h; the concentration of the aqueous solution containing the metal ions is 0.001-1mol/L, and the soaking time is 0.1-24h.
8. The production method according to any one of claims 5 to 7, wherein in the step (3), the annealing temperature is 400 to 700 ℃ and the annealing time is 0.5 to 4 hours.
9. The production method according to any one of claims 5 to 8, wherein the titanium substrate is a medical titanium substrate, and comprises pure titanium or a titanium alloy.
10. Use of the metal ion doped titanium dioxide nanocoating according to any one of claims 1 to 4 in nerve and bone tissue repair.
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