CN114748687B

CN114748687B - Preparation and application of in-situ induced biomimetic mineralized ZIF-8 nano material

Info

Publication number: CN114748687B
Application number: CN202210339866.XA
Authority: CN
Inventors: 王冰倩; 汪振星; 孙家明; 曾宇阳; 刘绍恺; 周牧冉
Original assignee: Tongji Medical College of Huazhong University of Science and Technology
Current assignee: Tongji Medical College of Huazhong University of Science and Technology
Priority date: 2022-04-01
Filing date: 2022-04-01
Publication date: 2023-03-21
Anticipated expiration: 2042-04-01
Also published as: CN114748687A

Abstract

The invention provides a preparation method and application of an in-situ induced biomimetic mineralized ZIF-8 nano material. The method specifically comprises the steps of mixing and stirring zinc nitrate hexahydrate, 2-methylimidazole and methanol according to the molar ratio of 1. The in-situ induced biomimetic mineralized ZIF-8 nano material prepared by the invention has the original ZIF-8 performance, has good cell affinity and osteogenesis inducing performance, and can be applied to a bone tissue biological scaffold as a modification material to promote bone regeneration.

Description

Preparation and application of in-situ induced biomimetic mineralized ZIF-8 nano material

Technical Field

The invention relates to the field of medical materials, in particular to a preparation method and application of an in-situ induced biomimetic mineralized ZIF-8 nano material.

Background

Zeolite imidazolate framework material ZIF-8 is a metal organic framework Material (MOF) which is composed of zinc metal ions and organic ligand 2-methylimidazole (2-HmIM) of imidazole derivatives. Because ZIF-8 has the characteristics of low cytotoxicity, stable structure, large specific surface area and the like, and is synthesized in a water phase, the environment cannot be polluted, so that the ZIF-8 can be used for adsorbing pollutants and is widely applied to the fields such as: gas adsorption separation, catalysis, drug loading, removal of pollutants in water and the like; among them, ZIF-8 itself has very excellent adsorption capacity and can adsorb various substances through a porous structure on the surface by a physical diffusion method, so ZIF-8 is widely concerned in the biomedical fields of biocatalysis, sensing, drug delivery and the like. However, the application of ZIF-8 in the field of bone tissue engineering is rather limited, one reason is that the negative Zeta potential on the surface of ZIF-8 influences the affinity of ZIF-8 to cells and forms a microenvironment which is not beneficial to the growth and differentiation of the cells. Secondly, even though ZIF-8 can degrade and release free zinc element capable of promoting osteogenesis in the biological fluid environment, a large amount of ZIF-8 can generate cytotoxicity and influence organisms.

Disclosure of Invention

The invention provides a preparation method and application of an in-situ induced biomimetic mineralized ZIF-8 nano material aiming at the problem that the application of the existing ZIF-8 material (nano-scale zeolite imidazolate framework-8) in the field of bone tissue engineering is rather limited, the method comprises the steps of treating the prepared ZIF-8 nano material in simulated biological body fluid and competitively combining with ions existing in a solution to form bone-like calcium hydroxy phosphate, using the ZIF-8 nano material as a nucleation site to form favorable biomimetic mineralized crystal deposition, releasing free zinc ions at the same time, and cooperatively promoting osteogenesis.

In order to achieve the aim, the invention provides a preparation method of an in-situ induced biomimetic mineralized ZIF-8 nano material, which is characterized by comprising the following specific steps:

(1) Respectively dissolving zinc nitrate hexahydrate and 2-methylimidazole in methanol to prepare a zinc nitrate hexahydrate solution and a 2-methylimidazole solution, dropwise adding the dissolved zinc nitrate hexahydrate solution into the 2-methylimidazole solution at a stable and slow speed for reaction, and magnetically stirring the whole process; the molar ratio of zinc nitrate hexahydrate, 2-methylimidazole and methanol in the reaction system is 1;

(2) Stirring the reaction solution obtained in the step (1) at a constant speed for 4-6 hours, pouring the obtained milky white suspension into a centrifuge tube, centrifuging the milky white suspension in a centrifuge at 3500-5000 rpm for 5-10 minutes, taking out the milky white suspension, and removing the supernatant to obtain ZIF-8 white precipitate;

(3) Adding equal amount of methanol solution into the centrifugal tube containing the ZIF-8 white precipitate in the step (2) for washing for 2-4 times to remove unreacted ligand, wherein each washing is to disperse the ZIF-8 white precipitate uniformly, then centrifugally washing for 10-12 minutes at the rotating speed of 8000-10000 rpm, and discarding the supernatant;

(4) Putting the washed ZIF-8 white precipitate in the step (3) into a sealed container, drilling holes with the diameter of 2-5 mm on a container cover, putting the container into a drying box at 60-70 ℃, drying overnight, pouring out the dried ZIF-8, grinding the dried ZIF-8 white precipitate into fine powder, namely ZIF-8 nano material, and storing the ZIF-8 nano material in a sealed manner at normal temperature;

(5) Putting the ZIF-8 nano material prepared in the step (4) into a dialysis bag, adding 1-3ml of SBF simulated body fluid with the pH value of 7.40-7.45 into each 100mg of the ZIF-8 nano material for mixing and dispersing, then soaking the dialysis bag filled with the ZIF-8 nano material into the SBF simulated body fluid, placing the SBF simulated body fluid on a shaking table of a constant-temperature incubator at 37 ℃, and mineralizing for at least 1 day by controlling the speed of the shaking table to be 200-250 r/min; wherein the volume of the SBF simulated body fluid soak solution is more than 10 times of the total volume of the ZIF-8 nano material, and the ZIF-8 nano material is completely soaked;

(6) Mineralizing for at least 1 day on a shaking table, washing the ZIF-8 nano material in the dialysis bag by deionized water, standing, removing supernatant, freezing in a refrigerator at-20 ℃ for 8-16 h, and freeze-drying in a freeze dryer for 10-12 h to obtain the in-situ induced biomimetic mineralization ZIF-8 nano material; when the mineralization time is longer than 1 day, replacing the SBF simulated body fluid for soaking the dialysis bag filled with the ZIF-8 nano material the next day.

The invention has the following excellent technical scheme: the formulation of SBF-mimicking body fluids per liter included the following agents:

serial number	Name (R)	Quality of
			1	NaCl	8.035g
2	NaHCO ₃	0.355g
			3	KCl	0.225g
4	K ₂ HPO ₄ ·3H ₂ 0	0.231g
			5	MgCl ₂ ·6H ₂ 0	0.311g
6	1.0M HCl	39ml
			7	CaCl ₂	0.292g
8	NaSO4	0.072g
			9	Tris	6.118g
10	1.0M HCl	0-5ml

The invention has the following excellent technical scheme: the methanol concentration in the step (1) and the step (3) is more than 99.7 percent.

The invention has the following excellent technical scheme: and (3) repeating the washing step in the step (3) for 3 times, wherein in each washing step, the reactant from which the supernatant is discarded is placed in a covered centrifugal tube, an equal amount of methanol solution is added, the centrifugal tube is covered, and the centrifugal tube is shaken up and down to uniformly disperse the ZIF-8 white precipitate.

The invention has the advantages that: the specific preparation process of the 1LSBF simulated body fluid is as follows:

a. adding 700ml of deionized water into a 1L glass volumetric flask, putting the glass volumetric flask into a heat collection type magnetic stirrer, and raising the temperature and keeping the temperature at 36.5 +/-1.5 ℃; adding the No. 1-8 reagents a little by a plurality of times according to the sequence in the table, and continuously stirring by using a glass rod in the whole process;

b. continuously adding deionized water into the volumetric flask, and fixing the volume to 900ml;

c. inserting a pH electrode, the pH of the solution being 2.0 +/-1.0 at this time, and adding Tris slowly until the pH is 7.45;

d. alternately adding 1.0M HCl and Tris to maintain the pH value between 7.40 and 7.45 and the temperature at 36.5 +/-0.2 ℃ until the Tris is completely added;

e. taking out the electrode, adding deionized water to a constant volume of 1000ml to obtain 1L of SBF simulated body fluid, and storing the prepared SBF simulated body fluid in a refrigerator at 4-10 ℃ for use within 30 days.

The invention has the following excellent technical scheme: in the preparation process of the SBF simulated body fluid, a starting pH meter is used for monitoring the whole process, and the solution is discarded and the preparation is restarted when the solution is turbid in the preparation process and the storage process.

The invention also provides an application of the in-situ induced biomimetic mineralized ZIF-8 nano material prepared by the method, which is characterized by comprising the following steps: the in-situ induced biomimetic mineralized ZIF-8 nano material is applied to a modification material of a bone tissue engineering biological scaffold.

The further technical scheme of the invention is as follows: and mixing the in-situ induced biomimetic mineralized ZIF-8 nano material with a film forming material, and directly coating the mixture on the outer surface of the bone tissue engineering biological scaffold to form a coating, or pressing the mixture into a film to be pasted on the outer surface of the bone tissue engineering biological scaffold.

The further technical scheme of the invention is as follows: and mixing the in-situ induced biomimetic mineralized ZIF-8 nano material with a biological scaffold printing material, and forming the bone tissue engineering biological scaffold through 3D printing.

Due to the abundant metal cations and inorganic anions in the biological fluid; among them, phosphate group has been proved that metal elements in the ZIF-8 skeleton have high affinity, can change ion coordination balance to form insoluble zinc phosphate precipitate, and the body fluid contains calcium element more active than zinc element. Due to the fact that basic conditions capable of competing and combining with body fluid components exist in the ZIF-8 structure, the ZIF-8 structure combines the advantages of a porous structure, and has potential sites for mineral nucleation growth, ion adsorption and crystallization deposition in organisms.

According to the invention, a ZIF-8 nano material is prepared by a special proportion, then the ZIF-8 nano material is immersed into simulated body fluid SBF for treatment, due to the attack of active ions in the SBF, the ions compete for coordination, a deposit containing hydroxyl-like calcium phosphate is generated, and the deposit is used as a nucleation site to form favorable biomimetic mineralized crystal deposition, namely, the ZIF-8 is subjected to self-mineralization. Compared with unmineralized ZIF-8, the mineralized product Zeta potential is positive, and the sediment of the calcium hydroxyphosphate-like promotes the adhesion, growth and differentiation of osteoblasts, so that the calcium hydroxyphosphate-like growth has good cell affinity and osteogenesis inducing performance. Compared with other osteogenesis additives, the ZIF-8 has the potential of synergy of self-mineralization and drug release in a body fluid environment, and the ZIF-8 can still keep the original performance of serving as a drug slow-release carrier after mineralization treatment; the prepared in-situ induced biomimetic mineralization ZIF-8 nano material is applied to a bone tissue biological scaffold as a modification material, and after the modified bone tissue biological scaffold is implanted into a body, the ZIF-8 nano material can be continuously mineralized and release free zinc ions at the same time, and the two materials synergistically promote osteogenesis; and the in-situ induced biomimetic mineralization ZIF-8 nano material can mineralize the surfaces of other biological scaffold materials without biomimetic mineralization performance, such as PCL biological scaffold materials, so that the bone induction capability is endowed, and the bone regeneration is better promoted.

Drawings

FIG. 1 is a morphology under an electron microscope of a ZIF-8 nanomaterial prepared in the example;

FIG. 2 is a graph of X-ray diffraction characterization of the invention before and after ZIF-8 mineralization;

FIG. 3 is a scanning spectrum of ZIF-8 nanomaterial prepared in example;

FIG. 4 is a Zeta potential analysis before and after the mineralization of ZIF-8 in the examples;

FIG. 5 is a graph of nanoparticle size analysis before and after ZIF-8 mineralization in the examples;

FIG. 6 is a graph of Fourier-IR spectra before and after the ZIF-8 mineralization in the examples;

FIG. 7 is a graph showing the experiment of inducing bone marrow mesenchymal cells to form bone after ZIF-8 mineralization in the examples;

FIG. 8 is a graph comparing in vivo osteogenesis capacity of pure PCL scaffolds implanted into rat skull defect model animals in example 2 with ZIF-8/PCL scaffolds and a blank group.

Detailed Description

The invention is further illustrated with reference to the following figures and examples.

In the following examples, the method of formulating a Simulated Body Fluid (SBF) is as follows: (taking configuration 1L as an example)

a. Reagents required (strictly in order)

b. A1L glass volumetric flask was charged with 700ml of deionized water (dH) ₂ 0) Then putting the mixture into a heat collection type magnetic stirrer, raising the temperature and keeping the temperature at 36.5 +/-1.5 ℃; adding the No. 1-8 reagents in a small amount for multiple times according to the sequence, and continuously stirring by using a glass rod in the whole process; if the solution is turbid in the process, restarting;

c. adding deionized water into a volumetric flask, and fixing the volume to 900ml;

d. inserting a pH electrode, wherein the pH of the solution is 2.0 +/-1.0;

e. slowly adding Tris, and continuing to add the Tris after the temperature and the pH value are stable until the pH value is just lower than 7.45 after a small amount of Tris is added;

f. alternately adding 1.0M HCl and Tris to maintain the pH value between 7.40 and 7.45 and the temperature at 36.5 +/-0.2 ℃ until all Tris is added;

g. taking out the electrode, adding deionized water to a constant volume of 1000ml;

h. storing the prepared SBF solution in a refrigerator at 4-10 ℃ and using up within 30 days; the use was stopped when turbidity appeared.

Embodiment 1 provides a preparation method of an in-situ induced biomimetic mineralized ZIF-8 nano material, which is characterized by comprising the following specific steps:

(1) Respectively dissolving zinc nitrate hexahydrate and 2-methylimidazole in methanol to prepare a zinc nitrate hexahydrate solution and a 2-methylimidazole solution, dropwise adding the dissolved zinc nitrate hexahydrate solution into the 2-methylimidazole solution at a stable and slow speed for reaction, and magnetically stirring the whole process; the molar ratio of zinc nitrate hexahydrate, 2-methylimidazole and methanol in the reaction system is 1; the methanol concentration is more than 99.7%;

(2) Stirring the reaction solution in the step (1) at a constant speed for 4 hours, pouring the obtained milky white suspension into two 50mL centrifuge tubes, balancing, putting the centrifuge tubes into a centrifuge, centrifuging at 3500rpm for 5 minutes, taking out, and carefully discarding the supernatant to obtain ZIF-8 white precipitate, wherein the weight difference between the two centrifuge tubes cannot be more than 500 mg;

(3) And (3) adding methanol solution which is equal to the ZIF-8 white precipitate into the two centrifuge tubes in the step (2) for washing, wherein the washing process comprises the following steps: covering the centrifugal tube, shaking the centrifugal tube up and down to uniformly disperse the ZIF-8 white precipitate, then centrifugally washing the centrifugal tube at the rotating speed of 8000rpm for 10 minutes, and removing the supernatant; repeating the washing process for three times, wherein the methanol concentration is more than 99.7%;

(4) Covering the opening of the centrifugal tube filled with the ZIF-8 white precipitate after washing in the step (3), drilling holes with the diameter of 2-5 mm on the opening, putting the pipe into a drying oven, drying the pipe at 60 ℃ for one night, pouring out the dried ZIF-8, grinding the dried ZIF-8 into fine powder by using an agate mortar, namely a ZIF-8 nano material, and sealing and storing the material at normal temperature;

(5) Putting the ZIF-8 nano material prepared in the step (4) into a dialysis bag, adding 1-3ml of SBF simulated body fluid prepared by the method into each 100mg of the ZIF-8 nano material for mixing and dispersing, then soaking the dialysis bag filled with the ZIF-8 nano material into the SBF simulated body fluid prepared by the method, placing the SBF simulated body fluid on a shaking table of a constant-temperature incubator at 37 ℃, and mineralizing for at least 1 day by controlling the speed of the shaking table to be 200-250 r/min; wherein the volume of the SBF simulated body fluid soak solution is more than 10 times of the total volume of the ZIF-8 nanometer material, and the ZIF-8 nanometer material is completely soaked; when the mineralization time is longer than 1 day, replacing the SBF simulated body fluid used for soaking the dialysis bag filled with the ZIF-8 nano material the next day;

(6) Respectively sampling at the time of mineralization for 1 hour, 3 hours, 6 hours, 12 hours, 1 day, 7 days, 14 days and 21 days, respectively washing the ZIF-8 nano material in the dialysis bag by deionized water, standing, removing supernatant, freezing in a refrigerator at the temperature of-20 ℃ for 10 hours, and freeze-drying in a vacuum drying freezer at the temperature of-60 ℃ and under the pressure of 5Pa overnight to obtain the in-situ induced biomimetic mineralization ZIF-8 nano material with different mineralization times.

The inventors of the present application carried out the following comparative experiments on the in-situ induced biomimetic mineralized ZIF-8 nanomaterial prepared in example 1 and the unmineralized ZIF-8 nanomaterial:

test 1: the morphology of the ZIF-8 nano material prepared in the step (4) in the embodiment is observed by a transmission electron microscope, as shown in FIG. 1, the ZIF-8 nano material prepared in the embodiment under the transmission electron microscope is in a typical under-mirror hexagonal shape, and the three-dimensional shape is a rhombic dodecahedron. Zn and N are characteristic elements contained in the ZIF-8 crystal.

Test 2: aiming at the in-situ induced biomimetic mineralization ZIF-8 nanomaterial prepared in 1 hour, 3 hours, 6 hours, 12 hours and 1 day of mineralization in example 1, the inventor of the application respectively observes the characterization of X-ray diffraction in the mineralization process of the ZIF-8 nanomaterial by an X-ray diffractometer, and as shown in fig. 2 in detail, it can be seen from fig. 2 that the peak intensity generated at the (011) plane of the ZIF-8 crystal is reduced along with the extension of soaking time, which indicates that the ZIF-8 crystal structure has defects, which indicates that the ZIF-8 structure is degraded in SBF simulated body fluid and the coordination combination of atoms is changed.

Test 3: the inventor of the application aims at that the in-situ induced biomimetic mineralized ZIF-8 nano material prepared by mineralizing for 12 hours, 1 day, 4 days, 7 days, 14 days and 21 days in the example 1 and the unmineralized ZIF-8 nano material are subjected to energy spectrum scanning under a transmission electron microscope, the energy spectrum scanning diagram is shown in fig. 3, and the energy spectrum scanning of the unmineralized ZIF-8 nano material (the first diagram in fig. 3) can be seen through fig. 3 to show that the synthesized nanocrystal uniformly contains metallic Zn elements and N elements in a ligand framework. After being soaked and mineralized for 12 hours by SBF (the second picture in figure 3), the adsorption and deposition of Ca ions appear on the ZIF-8 structure, but the combination of the P element is not obvious; after 1 day of mineralization (third picture in fig. 3), the mineralization sites formed by ZIF-8 itself nucleate and grow, and Ca, P ions and Zn ions start to grow together uniformly; after 7 days of mineralization (third panel in fig. 3), the mineralized ZIF-8 was continuously agglomerated with a volume significantly greater than the samples from the first few time points, and the mineralization deposition process was found to be ongoing during the subsequent 14, 21 day samples.

Test 4: the inventor of the application carries out Zeta potential observation on the in-situ induced biomimetic mineralized ZIF-8 nano material and the unmineralized ZIF-8 nano material prepared by mineralizing for 21 days in the embodiment 1 through a dynamic light scattering instrument, the observation result is shown in figure 4, the Zeta potential on the surface of the mineralized ZIF-8 material is changed from negative to positive, the mineralized ZIF-8 nano material not only has better cell compatibility, but also has a promoting effect on the deposition of mineralized substances.

Test 5: the inventor of the application carries out nano-particle size detection on nano-particles of an in-situ induced biomimetic mineralized ZIF-8 nano material and an unmineralized ZIF-8 nano material prepared by mineralizing for 21 days in example 1 through a dynamic light scattering instrument, as shown in FIG. 5, pure ZIF-8 is nano-particles with small particle size, and although the particles are influenced by an agglomeration phenomenon, the particle size is slightly larger than actual data, but is still far smaller than the particle size of the mineralized ZIF-8. The average particle size of the mineralized ZIF-8 reaches 500-600nm, which shows that the ZIF-8 has continuously increased sediment in the mineralization process and is more obvious in interconnection and agglomeration.

Test 6: the inventors of the present application performed fourier infrared transform spectroscopy detection on the in-situ induced biomimetic mineralized ZIF-8 nanomaterial prepared by mineralization for 21 days in example 1 and the unmineralized ZIF-8 nanomaterial, respectively, and the infrared spectrogram of the detection was as shown in fig. 6, and the generated crystal mixture was composed of biomimetic apatite deposition and a small amount of zinc phosphate in the ZIF-8 degradation by-products. The above results indicate that ZIF-8 particles spontaneously mineralize during degradation.

Test 7: the inventor of the application carries out a cell osteogenesis induction experiment aiming at an in-situ induction biomimetic mineralized ZIF-8 nano material and an unmineralized ZIF-8 nano material which are prepared by mineralizing for 7 days in 1, and the specific experiment is as follows:

(1) Human bone marrow mesenchymal stem cells (rBMSCs) are cultured at a concentration of not less than 1.5X 10 ⁵ Culturing the strain on a 6-well plate for 24 hours or growing until 60-70% of the strain is fused to form a culture medium without osteogenic induction liquid;

(2) The ZIF-8 nanomaterial prepared in the step (4) of example 1 and the in-situ induced biomimetic mineralized ZIF-8 nanomaterial prepared after mineralization 7 in the step (6) of example 1 were added to the culture medium without osteogenic induction solution in the step (1) at a concentration of 1mg/ml, respectively, and cultured for 14 days, and then fixed in 4% paraformaldehyde for 30min. After the fixative solution is removed, alizarin Red (Alizarin Red) is added to show the mineralization degree of the extracellular matrix, deionized water is used for removing excessive Alizarin dye, the experimental comparison results are shown in figure 7, the first picture in figure 7 is a control group without adding ZIF-8, the second picture in figure 7 is an osteogenesis experimental picture with adding ZIF-8 nano material, the third picture in figure 7 is an osteogenesis experimental picture with adding in-situ induced biomimetic mineralized ZIF-8 nano material, through experimental comparison, the rBMSCs with the mineralized ZIF-8 are cultured for 14 days to show more mineral nodules, while the ZIF-8 group only shows a small amount of mineral nodules, and the control group has no mineral deposition. The results show that the mineralized ZIF-8 has more excellent capacity of inducing osteogenic differentiation in a culture medium without osteogenic inducing components.

Example 2: the in-situ induced biomimetic mineralization ZIF-8 nano material prepared in the embodiment 1 is applied to modification of a bone tissue engineering biological scaffold, and specifically, the in-situ induced biomimetic mineralization ZIF-8 nano material and a PCL material are mixed and printed by 3D to form the bone tissue engineering biological scaffold, and the method specifically comprises the following steps:

(1) Mixing the in-situ induced biomimetic mineralization ZIF-8 nano material mineralized for seven days in the embodiment 1 with polycaprolactone Particles (PCL) according to a mass ratio of 1;

(2) 3D printing the melted mixed material by adopting a near-field melt electrostatic direct writing technology to obtain a PCL biological scaffold containing ZIF-8; the MEW printing parameters are: interval of 1-2mm, square wave frequency of 15-20 times, speed of 8-10mm/s, and acceleration of 48-50mm/s ² And 2 superposed layers.

(3) Sequentially passing the PCL biological scaffold containing ZIF-8 through CaCl ₂ Soaking the solution in deionized water, drying, and sequentially passing through K ₂ HPO ₄ The bionic mineralized bone repair scaffold is obtained by soaking the solution and deionized water, drying and pretreating.

Experimental studies were carried out on the biomimetic mineralized bone repair scaffold implanted in animals in example 2:

(1) Male SD rats of 6 weeks of age were selected and randomly divided into two groups, a blank control group and an experimental group. The bionic mineralized bone repair scaffold prepared in example 1 was placed in the left hole of the skull of the rat in the experimental group, and the right hole was in the common PCL bioscaffold. Rats were weighed and anesthetized with a intraperitoneal injection of 10% chloral hydrate at 0.4ml/100 g;

(2) After the surgical site was sterilized, a longitudinal full-thickness incision of about 4cm was made along the median sagittal line of the SD rat cranium. Two round (5 mm in diameter and 2mm in thickness) skull defects are symmetrically manufactured by using a trephine with the diameter of 5mm, and are sequentially washed by normal saline;

(3) A sterile bioscaffold (5 mm diameter and 2mm thickness) was placed directly over the skull defect. After the implantation, the operation incision is sutured, and the fascia layer must be sutured tightly to prevent the material from moving, falling off and shifting;

(4) Animals were sacrificed 4 weeks post-surgery. Rat calvaria were harvested and fixed in 10% strength paraformaldehyde solution. The 10% paraformaldehyde fixed cranium (n =4 per group) was taken and the bone tissue defect area (5 mm diameter and 2mm thickness) was assessed by Micro-CT scanning. As shown in fig. 8, the bone defect of the biomimetic mineralized bone repair scaffold is basically closed, which is much higher than the new bone generation of PCL group, which indicates that the in-situ induced biomimetic mineralized ZIF-8 nano material in the application can increase the bone regeneration capability of the biological scaffold when used in bone tissue engineering.

The above description is only one embodiment of the present invention, and the description is specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. The preparation method of the in-situ induced biomimetic mineralized ZIF-8 nano material is characterized by comprising the following specific steps:

(1) Respectively dissolving zinc nitrate hexahydrate and 2-methylimidazole in methanol to prepare a zinc nitrate hexahydrate solution and a 2-methylimidazole solution, dripping the dissolved zinc nitrate hexahydrate solution into the 2-methylimidazole solution at a stable and slow speed for reaction, and magnetically stirring the whole process; the molar ratio of zinc nitrate hexahydrate, 2-methylimidazole and methanol in the reaction system is 1;

(2) Stirring the reaction solution in the step (1) at a constant speed for 4-6 hours, pouring the obtained milky white suspension into a centrifuge tube, centrifuging the milky white suspension in a centrifuge at 3500-5000 rpm for 5-10 minutes, taking out the milky white suspension, and removing supernatant to obtain ZIF-8 white precipitate;

(4) Putting the cleaned ZIF-8 white precipitate in the step (3) into a sealed container, drilling a hole with the diameter of 2-5 mm on a container cover, putting the container into a drying box at the temperature of 60-70 ℃, drying overnight, pouring out the dried ZIF-8, grinding the dried ZIF-8 white precipitate into fine powder, namely ZIF-8 nano material, and sealing and storing at normal temperature;

(5) Putting the ZIF-8 nano material prepared in the step (4) into a dialysis bag, adding 1-3ml of SBF simulated body fluid with the pH value of 7.40-7.45 into each 100mg of the ZIF-8 nano material for mixing and dispersing, then soaking the dialysis bag filled with the ZIF-8 nano material into the SBF simulated body fluid, placing the SBF simulated body fluid on a shaking table of a constant-temperature incubator at 37 ℃, and mineralizing for at least 1 day by controlling the speed of the shaking table to be 200-250 r/min; wherein the volume of the SBF simulated body fluid soak solution is more than 10 times of the total volume of the ZIF-8 nanometer material, and the ZIF-8 nanometer material is completely soaked; wherein the formulation of SBF-mimicking body fluid per liter comprises the following agents: (1) 8.035 g NaCl, (2) 0.355 g NaHCO ₃ 、③0.225 g KCl、④0.231g K ₂ HPO ₄ ·3H ₂ 0、⑤0.311 g MgCl ₂ ·6H ₂ 0、⑥39 ml 1.0M HCl、⑦0.292 g CaCl ₂ 、⑧0.072 g NaSO ₄ (9) 6.118 g Tris, 0-5 ml 1.0M HCl in R;

the specific preparation process of each liter of SBF simulated body fluid is as follows:

a. adding 700ml of deionized water into a 1L glass volumetric flask, putting the glass volumetric flask into a heat collection type magnetic stirrer, and raising the temperature and keeping the temperature at 36.5 +/-1.5 ℃; adding the reagents (1) - (8) in sequence for a few times, and continuously stirring by using a glass rod in the whole process;

c. inserting a pH electrode, the solution pH being 2.0 + -1.0 at this time, and adding Tris slowly until pH 7.45;

e. taking out the electrode, adding deionized water to a constant volume of 1000ml to obtain 1L of SBF simulated body fluid, storing the prepared SBF simulated body fluid in a refrigerator at 4-10 ℃, and using up the SBF simulated body fluid within 30 days;

2. The preparation method of the in-situ induced biomimetic mineralized ZIF-8 nanomaterial according to claim 1, wherein the preparation method comprises the following steps: the methanol concentration in the step (1) and the step (3) is more than 99.7 percent.

3. The preparation method of the in-situ induced biomimetic mineralized ZIF-8 nanomaterial according to claim 1, wherein the preparation method comprises the following steps: and (3) repeating the washing step in the step (3) for 3 times, wherein each washing step is to place the reactant from which the supernatant is discarded into a covered centrifugal tube, add an equal amount of methanol solution into the capped centrifugal tube, cover the centrifugal tube, and shake the centrifugal tube up and down to uniformly disperse the ZIF-8 white precipitate.

4. The preparation method of the in-situ induced biomimetic mineralized ZIF-8 nanomaterial according to claim 1, wherein the preparation method comprises the following steps: in the preparation process of the SBF simulated body fluid, a pH meter is used for monitoring the whole process, and the solution is discarded and starts to be prepared again when the solution is turbid in the preparation process and the preservation process.

5. Use of an in situ induced biomimetic mineralized ZIF-8 nanomaterial prepared according to any of claims 1 to 4, characterized in that: the in-situ induced biomimetic mineralized ZIF-8 nano material is applied to a modification material of a bone tissue engineering biological scaffold; mixing the in-situ induced biomimetic mineralized ZIF-8 nano material with a film forming material, and directly coating the mixture on the outer surface of the bone tissue engineering biological scaffold to form a coating, or pressing the mixture into a film to be pasted on the outer surface of the bone tissue engineering biological scaffold; or mixing the in-situ induced biomimetic mineralized ZIF-8 nano material with a biological scaffold printing material, and forming the bone tissue engineering biological scaffold through 3D printing.