Preparation method of gradient porous titanium dioxide bone repair material

Abstract

A preparation method of a gradient porous titanium dioxide bone repair material comprises the following steps: respectively dissolving tetrabutyl titanate and protamine serving as a template agent in an acidic aqueous solution to prepare TiO2 sol and a protamine solution, and mixing the TiO2 sol and the protamine solution to form precursor sol; the molar ratio of the tetrabutyl titanate to the protamine is 1: (0.1-0.25); treating the precursor sol by adopting a gradient freeze-drying technology, and controlling formation of a pore gradient structure; and carrying out gradient heating calcination on the freeze-dried product to obtain the titanium dioxide particles with aperture gradient. According to the method, the pore structure of the prepared material is in three-stage gradient distribution which continuously changes from inside to outside, and is matched with an irregular spherical submicron structure of which the surface is disorderly distributed to form a surface appearance with good bone induction capability, so that the material is suitable for repairing oral bone defects and has excellent biocompatibility and bone integration capability.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a preparation method of a gradient porous titanium dioxide bone repair material. Background Art

[0002] During the process of bone tissue regeneration, the surface topological structure of the material plays a key regulatory role in cell behavior. Research shows that macropores with a size of 5 - 10 μm on the titanium dioxide surface promote the migration and vascularization of osteoblasts, while micropores with a size of 0.3 - 1 μm can enhance cell adhesion and differentiation. However, traditional homogeneous porous structures (such as pores with a size of 200 - 500 μm prepared by the pore-forming agent method) are difficult to simultaneously meet the requirements of mechanical support and cell infiltration. Although three-dimensional printing technology can construct gradient pores, the weak interlayer bonding force results in a compressive strength of less than 15 MPa, and the minimum resolution is only 50 μm, which cannot simulate the fine structure of the natural bone Haversian system (inner layer 5 - 7 μm → outer layer 0.2 - 0.5 μm).

[0003] There are significant bottlenecks in existing gradient pore preparation technologies: chemical etching is difficult to control the continuous change of pores; multi-layer composite sintering leads to interfacial stress concentration; the equipment for electric field-assisted deposition is complex and costly. Especially for freeze-drying technology, although pores can be formed through ice crystal templates, a single freezing temperature (usually -50 °C) results in a discrete pore size distribution, with a coefficient of variation exceeding 40%, and sub-micron pore regulation cannot be achieved.

[0004] As a natural cationic polypeptide, protamine provides a new way for pore regulation with its unique properties: its isoelectric point pH = 10 - 12 makes it carry a strong positive charge under acidic conditions (pH = 1 - 3), and has a strong electrostatic interaction with negatively charged TiO 2 sol; the good matching of its thermal decomposition temperature (200 - 300 °C) and the TiO 2 crystallization temperature (above 400 °C) can retain the integrity of the pore structure. However, existing research only uses protamine to prepare homogeneous porous materials, and there is no application report on its use in constructing gradient pores. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method of a gradient porous titanium dioxide bone repair material. The method can make the pore structure of the prepared material show a three-level gradient distribution that continuously changes from the inside to the outside. Combined with the irregular spherical sub-micron structure with disordered surface distribution, a surface topography with good bone induction ability can be formed.

[0006] To achieve the above invention purpose, the technical solution adopted by the present invention is: a preparation method of a gradient porous titanium dioxide bone repair material, comprising the following steps:

[0007] (1) Dissolve tetrabutyl titanate and protamine as a template agent in acidic aqueous solutions respectively to prepare a TiO 2 sol and a protamine solution, and mix the TiO 2 sol with the protamine solution to form a precursor sol; the molar ratio between tetrabutyl titanate and protamine is 1:(0.1 - 0.25);

[0008] (2) Treat the precursor sol obtained in step (1) by gradient freeze-drying technology to control the formation of a pore gradient structure;

[0009] (3) Gradually heat the product after freeze-drying in step (2) for calcination to obtain titanium dioxide particles with a pore size gradient.

[0010] Preferably, in step (2), the gradient freeze-drying is divided into three stages:

[0011] Deep freezing stage: Rapidly freeze at -80°C to -84°C for 3 - 5 hours;

[0012] Transition regulation stage: Heat up to -40°C to -44°C and maintain for 2 - 4 hours;

[0013] Surface solidification stage: Keep at -20°C to -24°C for 1 - 2 hours.

[0014] Preferably, in step (3), the specific process of gradient heating calcination is: Gradually heat to 600 - 800°C at a rate of 1 - 3°C / min in an air atmosphere and hold for 2 - 4 hours.

[0015] Preferably, in step (3), the specific process of gradient heating calcination is: Gradually heat to 600°C at a rate of 1°C / min in an air atmosphere.

[0016] Preferably, in step (1), the molecular weight of the protamine is 3 - 8 kDa, the isoelectric point pH value is 10 - 12; the concentration of the protamine solution is 10 - 12 mg / mL, pH is 6.5 - 7; the pH of the tetrabutyl titanate hydrolysis solution is 6.5 - 7.

[0017] Preferably, in step (1), the acidic aqueous solution is obtained by adjusting the pH of deionized water to 1 - 3 with dilute hydrochloric acid or acetic acid; or the acidic aqueous solution is a nitric acid aqueous solution with a concentration of 0.1 - 0.5 M.

[0018] Preferably, in step (1), irregular spherical structures with a diameter of 100 - 200 nm are disorderly distributed on the surface of the precursor sol.

[0019] Preferably, the pore structure of the titanium dioxide particles obtained in step (3) presents a three-level gradient distribution that changes continuously from the inside to the outside, with the pore size of the inner layer being 400 to 10,000 nm, the middle layer being 80 to 200 nm, and the outer layer being 1 to 10 nm.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention proposes a method for preparing a gradient porous titanium dioxide bone repair material based on a protamine template method. The protamine carries a strong positive charge in an acidic environment and reacts with TiO2 to form a protonated porous titanium dioxide bone repair material. 2 Sol phase adsorption forms a disordered, irregular spherical submicron structure; combined with the three-level gradient freeze-drying technology, the pore structure of the final product presents a three-level gradient distribution that changes continuously from the inside to the outside, forming a surface morphology with good bone induction ability. Subsequently, a gradient temperature calcination process is used to break the amide bond of protamine at 200-300°C, and decompose it into CO without residue. 2 , H 2 O and NH 3 , avoiding the toxic residue of traditional templates (such as residual benzene derivatives from PMMA cracking), while inducing titanium dioxide to form anatase / rutile particles, achieving a synergistic improvement in the biosafety and functionality of bone repair materials. The material prepared by the present invention is suitable for oral bone defect repair and has excellent biocompatibility and bone integration ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the pore size distribution data of the material prepared in Example 1;

[0023] Figure 2 is a scanning electron microscope image of the material prepared in Example 1;

[0024] Figure 3 The osteogenic differentiation of the material prepared in Example 1 in vitro;

[0025] Figure 4 This is the bone tissue formation of the material prepared in Example 1 in the in vivo experiment. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below in conjunction with specific embodiments and drawings.

[0027] Example 1

[0028] A method for preparing a gradient porous titanium dioxide bone repair material, characterized in that it comprises the following steps:

[0029] (1) Hydrolyze tetrabutyl titanate solution and protamine solution as a template agent in a certain proportion in an acidic aqueous solution to form a precursor sol;

[0030] (1-1) Use dilute hydrochloric acid or acetic acid to adjust the pH of deionized water to 2.0, and dissolve protamine (Sigma C7854) with a molecular weight of 7 kDa and an isoelectric point pH = 11 to 10 mg / mL;

[0031] (1-2) Mix tetrabutyl titanate (Alfa 046893) and absolute ethanol in a volume ratio of 1:4, and slowly add it dropwise to a 0.2 M nitric acid aqueous solution, and stir magnetically for 2 h to form TiO 2 sol (Zeta potential -22 mV);

[0032] (1-3) Mix the protamine solution and TiO 2 sol in a molar ratio of titanium / protein of 1:0.1, ultrasonically treat for 30 min and then stand for aging for 12 h to obtain a milky white composite sol;

[0033] (2) Use gradient freeze-drying technology to process the precursor sol obtained in step (1) to control the formation of a pore gradient structure;

[0034] (2-1) Inject the precursor sol obtained in step (1) into a polytetrafluoroethylene mold and place it in a Christ Alpha 2-4LDplus freeze-dryer;

[0035] (2-2) Execute a three-stage freezing program:

[0036] Deep freezing: Rapidly freeze at -80 °C for 3 h (ice crystal growth rate 5000 nm / min) to form a macroporous template with axial alignment;

[0037] Transition regulation: Raise the temperature to -40 °C and maintain for 2 h (heating rate 0.5 °C / min) to reduce the pores to 60-200 nm;

[0038] Surface solidification: Maintain at -20 °C for 1 h (lateral growth of ice crystals is restricted) to form 1-8 nm surface micropores;

[0039] (3) Gradiently heat and calcine the product after freeze-drying in step (2) to obtain titanium dioxide particles with a pore size gradient;

[0040] (3-1) Place the freeze-dried body in a muffle furnace (Nabertherm L3 / 11), raise the temperature to 600 °C at a rate of 1 °C / min, and keep it for 3 h to complete the following transformation;

[0041] ① 200-300 °C: Protamine thermally decomposes to generate gas escape channels;

[0042] ②400 - 500 °C: Amorphous TiO 2 Phase transition to anatase;

[0043] ③Above 600 °C: Trigger the formation of rutile phase, anatase / rutile mass ratio 80:16;

[0044] (3 - 2) Cool with the furnace to obtain white porous particles.

[0045] The white porous particles obtained in this example were detected for pore size and distribution by mercury intrusion porosimetry to obtain pore size distribution data, see Figure 1 ; It can be seen from the figure that the micro - pore sizes of the materials on the figure are mainly distributed in the regions of 1 - 10 nm, 80 - 200 nm, and 400 - 10000 nm, showing a continuous "nano - sub - micron - micron" three - level gradient change from the outside to the inside.

[0046] The surface of the white porous particles obtained in this example was observed by scanning electron microscopy, see Figure 2 ; It can be seen from the figure that the surface of the white porous particles has irregular spherical structures with diameters of 100 - 200 nm and a pore structure with good uniformity and connectivity in distribution.

[0047] The white porous particles obtained in this example were added to the conditioned medium for culturing mesenchymal stem cells. After culturing for 7 and 14 days, the alkaline phosphatase expression of mesenchymal stem cells under co - culture conditions was detected by alkaline phosphatase staining to reflect the osteogenic differentiation tendency of the cells. The results showed that the mesenchymal stem cells had good osteogenic differentiation, see Figure 3 .

[0048] The white porous particles obtained in this example were implanted into the paravertebral muscles of beagle dogs. After 12 weeks, the samples were taken out, fixed with paraformaldehyde, dehydrated with gradient ethanol, embedded in hard tissue, sectioned, and stained with methylene blue - basic fuchsin. The obtained sections were observed under an electron microscope. The results showed that 13% of bone tissue was formed around the bone tissue, see Figure 4 .

[0049] Example 2

[0050] A preparation method of a gradient porous titanium dioxide bone repair material, characterized by comprising the following steps:

[0051] (1) Hydrolyzed solution of tetrabutyl titanate and protamine solution as a template agent were dissolved in an acidic aqueous solution in a certain proportion to form a precursor sol;

[0052] (1 - 1) Use dilute hydrochloric acid or acetic acid to adjust the pH of deionized water to 2.5, and dissolve protamine (Sigma C7854) with a molecular weight of 7 kDa and an isoelectric point pH = 11 to 11 mg / mL;

[0053] (1 - 2) Mix tetrabutyl titanate (Alfa 046893) and absolute ethanol in a volume ratio of 1:4, and slowly drop the mixture into 0.3M nitric acid aqueous solution. Stir magnetically for 2h to form TiO 2 sol (Zeta potential - 22mV);

[0054] (1 - 3) Mix the protamine solution and TiO 2 sol in a molar ratio of titanium / protein of 1:0.2. After ultrasonic treatment for 30min, let it stand for aging for 12h to obtain a milky white composite sol;

[0055] (2) Use gradient freeze - drying technology to treat the precursor sol obtained in step (1) to control the formation of pore gradient structure;

[0056] (2 - 1) Inject the precursor sol obtained in step (1) into a polytetrafluoroethylene mold and place it in a Christ Alpha 2 - 4LDplus freeze - dryer;

[0057] (2 - 2) Execute a three - stage freezing program:

[0058] Deep freezing: Rapidly freeze at - 80°C for 4h (ice crystal growth rate 6000nm / min) to form a macroporous template with axial arrangement;

[0059] Transition regulation: Raise the temperature to - 42°C and maintain for 3h (heating rate 0.6°C / min) to reduce the pores to 70 - 250nm;

[0060] Surface solidification: Maintain at - 22°C for 1.5h (lateral growth of ice crystals is restricted) to form 1 - 9nm surface micropores;

[0061] (3) Gradiently heat - calcine the product after freeze - drying in step (2) to obtain titanium dioxide particles with pore size gradient;

[0062] (3 - 1) Put the freeze - dried body into a muffle furnace (Nabertherm L3 / 11), and raise the temperature to 700°C at a rate of 2°C / min, and keep it for 3h to complete the following transformations;

[0063] ① 200 - 300°C: The protamine thermally decomposes to produce gas escape channels;

[0064] ② 400 - 500°C: Amorphous TiO 2 transforms to anatase phase;

[0065] ③ Above 600°C: Trigger the formation of rutile phase, and the anatase / rutile mass ratio is 87:17;

[0066] (3 - 2) Cool with the furnace to obtain white porous particles.

[0067] The pore size and distribution of the white porous particles obtained in this example were detected by mercury intrusion porosimetry. The results were the same as those in Example 1, also showing a continuous "nano - sub - micron - micron" three - level gradient change from the outside to the inside.

[0068] Scanning electron microscopy observation of the surface of the white porous particles obtained in this example shows that the surface of the white porous particles has irregular spherical structures with diameters of 100 - 200 nm and a pore structure with good uniformity and connectivity in distribution.

[0069] The white porous particles obtained in this example were added to the conditioned medium for culturing mesenchymal stem cells. After culturing for 7 and 14 days, the alkaline phosphatase expression of mesenchymal stem cells under co - culture conditions was detected by alkaline phosphatase staining to reflect the osteogenic differentiation tendency of the cells. The results showed that the mesenchymal stem cells had good osteogenic differentiation.

[0070] The white porous particles obtained in this example were implanted into the paravertebral muscles of beagle dogs. After 12 weeks, the samples were taken out, fixed with paraformaldehyde, dehydrated with gradient ethanol, embedded in hard tissue, sectioned, and stained with methylene blue - basic fuchsin. The obtained sections were observed under an electron microscope. The results showed that 12% of bone tissue was formed around the bone tissue.

[0071] Example 3

[0072] A preparation method of a gradient porous titanium dioxide bone repair material, characterized by comprising the following steps:

[0073] (1) Hydrolyzed tetrabutyl titanate solution and protamine solution as a template agent were dissolved in an acidic aqueous solution in a certain proportion to form a precursor sol;

[0074] (1 - 1) Use dilute hydrochloric acid or acetic acid to adjust the pH of deionized water to 3, and dissolve protamine (Sigma C7854) with a molecular weight of 7 kDa and an isoelectric point pH = 11 to 12 mg / mL;

[0075] (1 - 2) Mix tetrabutyl titanate (Alfa 046893) and absolute ethanol in a volume ratio of 1:4, and slowly drop it into a 0.4 M nitric acid aqueous solution, and magnetically stir for 2 h to form TiO 2 sol (Zeta potential - 22 mV);

[0076] (1 - 3) Mix the protamine solution and TiO 2 sol in a titanium / protein molar ratio of 1:0.25, ultrasonically treat for 30 min, and then stand for aging for 12 h to obtain a milky white composite sol;

[0077] (2) Use gradient freeze - drying technology to process the precursor sol obtained in step (1) to control the formation of the pore gradient structure;

[0078] (2-1) Inject the precursor sol obtained in step (1) into a polytetrafluoroethylene mold and place it in a Christ Alpha 2-4LDplus freeze dryer.

[0079] (2-2) Execute a three-stage freezing procedure:

[0080] Deep freezing: Rapidly freeze at -84 °C for 5 h (ice crystal growth rate 7000 nm / min) to form a macroporous template with axial alignment.

[0081] Transition regulation: Raise the temperature to -44 °C and hold for 4 h (heating rate 0.7 °C / min) to reduce the pore size to 80 - 300 nm.

[0082] Surface curing: Maintain at -24 °C for 2 h (restricted lateral growth of ice crystals) to form surface micropores of 1 - 10 nm.

[0083] (3) Gradiently heat and calcine the product after freeze-drying in step (2) to obtain titanium dioxide particles with a pore size gradient.

[0084] (3-1) Place the freeze-dried body in a muffle furnace (Nabertherm L3 / 11), raise the temperature to 800 °C at a rate of 3 °C / min, and hold for 3 h to complete the following transformations:

[0085] ① 200 - 300 °C: Protamine thermally decomposes to generate gas escape channels.

[0086] ② 400 - 500 °C: Amorphous TiO 2 Transforms to the anatase phase.

[0087] ③ Above 600 °C: Trigger the formation of the rutile phase, with an anatase / rutile mass ratio of 92:18.

[0088] (3-2) Cool with the furnace to obtain white porous particles.

[0089] The pore size and distribution of the pores of the white porous particles obtained in this example are detected by the mercury intrusion method. The results are the same as in Example 1, also showing a continuous "nano - submicron - micron" three-stage gradient change from the outside to the inside.

[0090] By observing the surface of the white porous particles obtained in this example with an electron scanning microscope, it can be seen that the surface of the white porous particles has irregular spherical structures with a diameter of 100 - 200 nm and a pore structure with good uniformity and connectivity in distribution.

[0091] The white porous particles obtained in this example were added to the conditioned medium for culturing mesenchymal stem cells. After culturing for 7 and 14 days, the alkaline phosphatase expression of mesenchymal stem cells under co-culture conditions was detected by alkaline phosphatase staining to reflect the osteogenic differentiation tendency of the cells. The results showed that the mesenchymal stem cells had good osteogenic differentiation.

[0092] The white porous particles obtained in this example were implanted into the paravertebral muscles of beagle dogs. After 12 weeks, the samples were taken out, fixed with paraformaldehyde, dehydrated with gradient ethanol, embedded in hard tissue, sectioned, and stained with methylene blue-basic fuchsin. The obtained sections were observed under an electron microscope. The results showed that 13% of bone tissue was formed around the bone tissue.

[0093] In summary, the present invention utilizes the strong positive charge of protamine in an acidic environment and the electrostatic adsorption of negatively charged TiO 2 sol formed by the hydrolysis of tetrabutyl titanate to construct an irregular spherical submicron structure with bone induction ability, and forms a three-level gradient pore distribution that continuously changes from the inside to the outside through a three-level gradient freeze-drying technique. The gradient heating calcination process (200 - 300 °C) is used to decompose protamine without residue into non-toxic small molecules (CO 2 , H 2 O, NH 3 ), and simultaneously induces titanium dioxide to form an anatase / rutile composite crystal form, solving the problem of toxic residue of traditional templating agents and enhancing the biological functionality of the material. This material has both a gradient porous structure and a nano surface morphology, and exhibits excellent biocompatibility, bone integration ability, and the synergistic optimization characteristics of biological safety - functionality in the repair of oral bone defects.

Claims

1. A method for preparing a gradient porous titanium dioxide bone repair material, characterized in that: The following steps are involved: (1) dissolving tetrabutyl titanate and protamine as a template in an acidic aqueous solution to form a TiO2 sol and a protamine solution, respectively, and mixing the TiO2 sol and the protamine solution to form a precursor sol; the molar ratio of tetrabutyl titanate to protamine is 1:(0.1-0.25); (2) using a gradient freeze-drying technique to process the precursor sol obtained in step (1) to control the formation of a pore gradient structure; (3) The freeze-dried product of step (2) is subjected to gradient temperature calcination to obtain titanium dioxide particles with a pore size gradient.

2. The method for preparing a gradient porous titanium dioxide bone repair material according to claim 1, characterized in that: In step (2), the gradient freeze-drying is divided into three stages: Deep freezing stage: quick freezing at -80℃ to -84℃ for 3-5 hours; Transition and regulation stage: raise the temperature to -40℃ to -44℃ and maintain for 2-4 hours; Surface curing stage: keep at -20℃ to -24℃ for 1-2 hours.

3. The method for preparing a gradient porous titanium dioxide bone repair material according to claim 1 or 2, characterized in that: In step (3), the specific process of gradient temperature calcination is: in an air atmosphere, the temperature is increased to 600-800° C. at a gradient rate of 1-3° C. / min, and kept at this temperature for 2-4 hours.

4. The method for preparing a gradient porous titanium dioxide bone repair material according to claim 3, characterized in that: In step (3), the specific process of gradient temperature calcination is: increasing the temperature to 600° C. at a gradient of 1° C. / min in an air atmosphere.

5. The method for preparing a gradient porous titanium dioxide bone repair material according to claim 1 or 2, characterized in that: In step (1), the molecular weight of the protamine is 3-8 kDa, and the isoelectric point pH is 10-12; the concentration of the protamine solution is 10-12 mg / mL, and the pH is 6.5-7; the pH of the tetrabutyl titanate hydrolyzate is 6.5-7.

6. The method for preparing a gradient porous titanium dioxide bone repair material according to claim 1 or 2, characterized in that: In step (1), the acidic aqueous solution is obtained by adjusting the pH of deionized water to 1-3 with dilute hydrochloric acid or acetic acid; or the acidic aqueous solution is a nitric acid aqueous solution with a concentration of 0.1-0.5M.

7. The method for preparing a gradient porous titanium dioxide bone repair material according to claim 1 or 2, characterized in that: In step (1), irregular spherical structures with a diameter of 100-200 nm are randomly distributed on the surface of the precursor sol.

8. The method for preparing a gradient porous titanium dioxide bone repair material according to claim 1 or 2, characterized in that: The pore structure of the titanium dioxide particles obtained in step (3) presents a three-level gradient distribution that changes continuously from the inside to the outside, with the pore size of the inner layer being 400 to 10,000 nm, the middle layer being 80 to 200 nm, and the outer layer being 1 to 10 nm.

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