CN110694103A

CN110694103A - Preparation method of composite bioactive ceramic bracket for bone regeneration repair and product thereof

Info

Publication number: CN110694103A
Application number: CN201911136646.1A
Authority: CN
Inventors: 蔡晴; 郭立英; 易蜜; 全静茹; 杜志云; 杨小平
Original assignee: BEIJING OYA BORUI TECHNOLOGY Co Ltd
Current assignee: BEIJING OYA BORUI TECHNOLOGY Co Ltd
Priority date: 2019-11-19
Filing date: 2019-11-19
Publication date: 2020-01-17
Anticipated expiration: 2039-11-19
Also published as: CN110694103B

Abstract

The invention discloses a preparation method of a composite bioactive ceramic bracket for bone regeneration and repair and a product thereof. The preparation method comprises the following steps: roughening the surface of the organic foam template; obtaining at least one impregnation liquid of a calcium silica sol-gel precursor solution, a calcium phosphorus sol-gel precursor solution and suspension slurry; repeatedly dipping the organic foam template subjected to surface roughening treatment by using suspension slurry, or alternately dipping by using at least two dipping liquids, and sintering the dipped compound to obtain a ceramic support; the composite bioactive ceramic bracket taking calcium silicon/calcium phosphorus as a main body is prepared by a preparation method combining an organic foam impregnation method and a sol-gel method for preparing the biological ceramic, and the prepared ceramic bracket has the advantages of good performance, adjustable degradation rate and performance, controllable size and appearance, high porosity and capability of meeting the requirements and requirements of wider bone defect regeneration and repair.

Description

Preparation method of composite bioactive ceramic bracket for bone regeneration repair and product thereof

Technical Field

The invention relates to the technical field of biological materials, in particular to a preparation method of a composite bioactive ceramic bracket for bone regeneration and repair and a product thereof.

Background

Bone injury repair, colloquially, is the repair of defective tissue by the use of biological materials when bone injury occurs. In the traditional treatment, the biological medical material is used for repairing, the biological activity of the material is poor, the material can only be used as a filler in a body, and the normal physiological function cannot be realized. The new tissue engineering technology utilizes stem cells to differentiate to form various tissues or organs completely composed of autologous cells of patients and biodegradable materials, and realizes the recovery in the true sense. Firstly, extracting a small amount of stem cells from the body of a patient to perform in-vitro amplification, preparing a model which is completely the same as the damaged part of the patient, and shaping a degradable biological material according to the model to form a 'bracket' closely matched with the defect part of the patient; the expanded stem cells are then placed on a "scaffold" to induce differentiation into osteoblasts, "growing" osteogenic tissue, and then implanted entirely into the defect site. With the lapse of time, the scaffold material will be gradually degraded and absorbed by the human body, and the cells will continue to carry out bone reconstruction at the defect site, finally realizing complete repair.

At present, the scaffold materials applied to clinical application comprise natural bone-derived scaffold materials, metal scaffolds, artificial bionic scaffold materials and the like, but natural bone tissue materials are difficult to obtain, and the application range of the metal scaffolds is limited due to poor biocompatibility, so that the scaffold materials prepared from the bioceramic for guiding regeneration and repair of bone defects are always the research focus and hot spot in the field of bone tissue engineering.

In the prior art, a method of mixing and sintering biological ceramic powders with different proportions is usually adopted to prepare a composite biological ceramic support, and common methods for preparing the ceramic support mainly include a pore-forming agent method, a 3D printing method or an organic foam impregnation method, but regardless of the pore-forming agent method, the 3D printing method or the organic foam impregnation method, the prepared ceramic support is a calcium-phosphorus or calcium-silicon single-component ceramic support, the degradation rate of the calcium-phosphorus ceramic support is low, the calcium-silicon ceramic support is locally alkaline due to too high degradation rate, inflammation reaction is easily caused, and the performances of the single-component calcium-phosphorus or calcium-silicon ceramic support are poor in brittleness and the like.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a preparation method of a composite bioactive ceramic bracket for bone regeneration repair, which combines an organic foam impregnation method and a sol-gel method to prepare a bioceramic so as to prepare a calcium-phosphorus/calcium-silicon composite bioactive ceramic bracket with better performance, and the degradation rate of the ceramic bracket is adjustable.

The second purpose of the invention is to provide a composite bioactive ceramic bracket for bone regeneration repair, which has the advantages of controllable degradation rate and performance, controllable size and shape, high porosity and capability of meeting the requirements and requirements of wider bone defect regeneration repair.

In order to achieve the first object, the invention provides the following technical scheme:

a preparation method of a composite bioactive ceramic bracket for bone regeneration repair comprises the following steps:

roughening the surface of the template: dipping the organic foam template by using alkali liquor, cleaning and drying for later use;

obtaining a steeping fluid: the impregnation liquid is at least one of a calcium-silica sol-gel precursor solution, a calcium-phosphorus sol-gel precursor solution and a suspension slurry, and the suspension slurry is formed by adding the biological ceramic nano powder into the calcium-silica sol-gel precursor solution or the calcium-phosphorus sol-gel precursor solution;

dipping with a dipping solution: repeatedly dipping the organic foam template subjected to surface roughening treatment by using suspension slurry, or alternately dipping by using at least two dipping liquids, and then drying to obtain a dipped compound;

and (3) sintering: and (3) placing the impregnated compound in an air atmosphere, and sintering at 800-1300 ℃ for 1-6 h to obtain the ceramic support.

By adopting the technical scheme, the composite bioactive ceramic bracket is prepared by combining an organic foam impregnation method and a sol-gel method, adopting organic foam as a template and then adopting impregnation liquid impregnation and sintering methods, wherein the degradation rate and the ion dissolution rate of the ceramic bracket are between those of calcium-phosphorus ceramic and calcium-silicon ceramic with single components and can be regulated and controlled, the brittleness and other properties of the ceramic bracket are obviously improved compared with those of the ceramic bracket with single components of calcium-phosphorus or calcium-silicon, and the requirements and requirements of wider bone defect regeneration and repair can be met.

In the preparation process, the organic foam template can be repeatedly impregnated by the impregnation liquid with the same components to adjust the degradation rate of the ceramic support and the release rate and release amount of bioactive ions, and the impregnation liquid with different components can be alternately impregnated to adjust the degradation rate of the ceramic support and the release rate and release amount of bioactive ions, so that the performances of the support, such as brittleness, structure, strength and the like, can be adjusted. The ceramic scaffold prepared by the invention can provide a suitable microenvironment for cell adhesion, proliferation and differentiation, is beneficial to bone defect regeneration and repair, and has no risk in the aspects of immunological rejection reaction caused by similar animal-derived bone repair materials and the like.

Preferred modes of the template surface roughening treatment: soaking the organic foam template in a sodium hydroxide solution with the concentration of 12-17% for 2-6 h, then respectively cleaning with absolute ethyl alcohol and clear water for 2-5 times, and finally drying at 50-70 ℃ for later use. In the invention, a wide variety of organic foam templates are available, and organic porous foam with high porosity and continuous through pore structure is preferably used as the template, such as polyurethane, polyvinyl chloride, polystyrene and the like. The surface roughening treatment of the template can be used for cleaning the foam template on one hand, and on the other hand, the template is soaked by using a sodium hydroxide solution, so that the hole wall of the organic foam template can be corroded, the hole wall is rough, and the slurry can be more easily adsorbed. In the invention, the optimized surface roughening treatment mode is favorable for obtaining the ceramic scaffold with the macroporous structure and the rough ceramic pore wall to a certain extent, thereby being favorable for providing a proper microenvironment for cell adhesion, proliferation and differentiation.

Further, when the dipping solution is used for alternate dipping, a calcium silica sol-gel precursor solution and a calcium phosphorus sol-gel precursor solution are adopted; further, the number of repeated or alternate dipping is preferably 2 to 5.

By adopting the technical scheme, the ceramic support prepared by repeatedly dipping the organic foam template by using the suspension slurry and the biological ceramic support prepared by alternately dipping the organic foam template by using the calcium-silicon sol-gel precursor solution and the calcium-phosphorus sol-gel precursor solution have obvious difference in degradation rate, the release rate and the release amount of calcium, silicon and phosphorus ions are different, and the degradation rate and the release amount of bioactive ions of the support can be regulated and controlled by the calcium-phosphorus/calcium-silicon composite ratio, the composite mode and the like, so that the performances of the support such as brittleness, structure, strength and the like can be regulated and controlled better. The ceramic scaffold obtained by adopting the two impregnation methods has good performances such as brittleness, structure, strength and the like, and provides a suitable microenvironment for cell adhesion, proliferation and differentiation, thereby greatly enriching the selectivity of the biological ceramic scaffold.

The organic foam template is alternately immersed in the calcium-silicon sol-gel precursor solution and the calcium-phosphorus sol-gel precursor solution to prepare the calcium-phosphorus/calcium-silicon composite biological ceramic scaffold, such as a calcium-phosphorus/calcium-silicon/calcium-phosphorus composite biological ceramic scaffold or a calcium-phosphorus/calcium-silicon/calcium-phosphorus composite biological ceramic scaffold. The single calcium-phosphorus ceramic scaffold has slow degradation rate and belongs to space occupying repair; the single calcium-silicon ceramic stent is often degraded too fast to cause local alkalinity, and inflammatory reaction and other symptoms are easy to cause; compared with a calcium-phosphorus or calcium-silicon ceramic bracket with a single component, the degradation rate and the ion dissolution rate of the calcium-phosphorus/calcium-silicon composite bioactive ceramic bracket are between those of the calcium-phosphorus ceramic bracket and the calcium-silicon ceramic bracket, and are relatively suitable and controllable; compared with the calcium-phosphorus or calcium-silicon ceramic bracket with single component, the brittleness and performance of the calcium-phosphorus/calcium-silicon composite bioactive ceramic bracket are also obviously improved, and the requirements of wider bone defect regeneration and repair can be met.

Further, the calcium-silica sol-gel precursor solution and the calcium-phosphorus sol-gel precursor solution both comprise precursor substances containing bioactive ions required for preparing the target ceramic scaffold, and the precursor substances comprise at least two of a calcium source substance, a silicon source substance and a phosphorus source substance; still further, the calcium source material is selected from at least one of soluble nitrate, acetate or chloride salt containing calcium; the silicon source material is tetraethyl silicate; the phosphorus source substance is any one of triethyl phosphate, trimethyl phosphate and phosphorus pentoxide.

By adopting the technical scheme, the main component of the bone mineral is Hydroxyapatite (HA), but the bone mineral also contains abundant doping elements such as silicon, magnesium, zinc, manganese and the like besides calcium and phosphorus elements, and the precursor substance containing bioactive ions can directly influence the size and the stacking density of hydroxyapatite nanocrystals, can indirectly influence the metabolism of minerals by activating alkaline phosphatase, and plays an important role in the formation process of new bones.

Further, the calcium-silica sol-gel precursor solution, the calcium-phosphorus sol-gel precursor solution and the suspension slurry each further include a dopant ion selected from at least one of magnesium, zinc, manganese and strontium ions.

By adopting the technical scheme, the sol-gel precursor solution has strong adjustability in preparation, and in addition to bioactive substances such as a calcium source substance, a silicon source substance, a phosphorus source substance and the like, the composite ceramic scaffold is prepared by adding doping ions (the doping ions are selected from at least one of magnesium, zinc, manganese and strontium ions) and sintering to obtain a single or multiple ion-doped biological ceramic porous scaffold, so that the composition of the composite ceramic scaffold is closer to that of natural bone mineral, and the composite ceramic scaffold has more excellent bone conduction and bone induction properties.

Further, when the repeated impregnation or the alternate impregnation is carried out, each impregnation comprises the following steps:

a. completely soaking the organic foam template in the soaking solution, vacuumizing and maintaining for at least 5min, and then taking out;

b. centrifuging the organic foam template taken out in the step a for 30-120 s at the rotating speed of 200-1000 rpm, and removing redundant impregnation liquid on the organic foam template;

c. and drying the centrifuged organic foam template at the temperature of 60-120 ℃ for 1-6 h.

By adopting the technical scheme, the organic foam template is vacuumized in the dipping process, namely, the air in the gaps of the template is pumped out, so that the dipping solution enters the gaps of the organic foam template, and the dipping solution is favorably adhered in the gaps of the organic foam template. Redundant (hanging or dripping) impregnation liquid on the impregnated organic foam template is removed by adopting a centrifugal separation method, so that the condition that the support is not uniform due to the fact that the redundant impregnation liquid is accumulated at the bottom of the organic foam template is avoided; the test shows that the residual impregnating liquid on the foam template can be effectively retained while the residual impregnating liquid on the foam template is removed at the rotating speed of 200-1000 rpm. In the present invention, the excess impregnation liquid may be removed by pressing the impregnated template.

Each impregnation comprises the steps of impregnation, centrifugation, drying and the like, impregnation liquid is firstly led into gaps of the organic foam template, then in order to maintain the uniformity of the impregnation liquid in the template, redundant impregnation liquid is removed in a centrifugation mode, and finally the impregnation liquid in the gaps is solidified on the organic foam template through drying, so that effective substances in the impregnation liquid can be fully and uniformly adhered to the organic foam template. The preparation method is simple and easy to implement, relatively simple in process flow, low in cost and easy to realize batch preparation.

Further, the preparation of the calcium silica sol-gel precursor solution comprises the following steps: adding a calcium source substance into a volatile organic solvent, preparing a salt alcohol solution, adding a silicon source substance after the salt alcohol solution is completely dissolved, and stirring to obtain a calcium-silica sol-gel precursor solution; the concentration of the saline alcohol solution is 20-60 wt%; the silicon source substance accounts for 20-60 wt% of the calcium-silica sol-gel precursor solution;

still further, the preparation of the calcium silica sol-gel precursor solution comprises the following steps: adding a calcium source substance into ethanol, preparing a saline solution, adjusting the pH value of the saline solution to 4-11 after the calcium source substance is completely dissolved, adding a silicon source substance, and stirring in a water bath at 35-40 ℃ to obtain a calcium-silica sol-gel precursor solution; the concentration of the saline alcohol solution is 30-40 wt%; the silicon source substance accounts for 30-40 wt% of the calcium-silica sol-gel precursor solution. In the present invention, the volatile organic solvent may be an alcohol solution, such as: methanol, ethanol or propanol, preferably ethanol, and optionally calcium source material can be dissolved in deionized water. When the pH value of the saline alcohol solution is adjusted to 4-11, ammonia water or hydrochloric acid can be used for adjusting.

By adopting the technical scheme, when the calcium source substance is dissolved by the ethanol, the ethanol is absolute ethanol (analytically pure), so that the calcium source substance (salt) is fully dissolved in the ethanol, the absolute ethanol is used as an organic solvent and is used as an intermediate in the reaction process, and the absolute ethanol is easy to remove in the later period and has no great influence on the whole system. According to the invention, the calcium-silica sol-gel precursor solution is prepared according to a specific proportion, and the prepared calcium-silica sol-gel precursor solution has good performance and is beneficial to being compounded with the calcium-phosphorus sol-gel precursor solution.

Further, the molar ratio of calcium to phosphorus in the calcium-phosphorus sol-gel precursor solution is 1.2-1.8.

By adopting the technical scheme, the calcium salt and the phosphorus salt are respectively dissolved in deionized water or absolute ethyl alcohol and are fully dissolved to prepare a phosphorus source solution and a calcium source solution; and then dropwise adding the calcium source solution into the phosphorus source solution to prepare a calcium-phosphorus sol-gel precursor solution with the molar ratio of calcium to phosphorus of 1.2-1.8, wherein in order to ensure that the calcium and the phosphorus are fully mixed, the dropwise adding process can be carried out in an ice-water mixed bath at the temperature of 2-6 ℃, and meanwhile, the rapid stirring is assisted.

Further, the preparation of the suspension slurry comprises the following steps:

adding the biological ceramic nano powder into the sol-gel precursor solution, and fully mixing to obtain dispersed suspension slurry; the biological ceramic nano powder accounts for 20-80 wt% of the suspension slurry, preferably 60-70 wt%; the biological ceramic nano powder is selected from at least one of hydroxyapatite, beta-tricalcium phosphate, whitlockite or clay.

By adopting the technical scheme, at least one of Hydroxyapatite (HA), beta-tricalcium phosphate (beta-TCP), whitlockite or clay (the main component is aluminosilicate) is added into the sol-gel precursor solution to prepare suspension slurry, then the suspension slurry is used for repeatedly dipping the organic foam template, and the special chemical composition and crystallinity of the prepared ceramic bracket after sintering are favorable for bone defect repair. In the invention, different kinds of biological ceramic nano powder are dispersed and compounded in the sol-gel, different doping ions for promoting bone biological activity are introduced into the sol-gel precursor solution, the components are matched with each other, and the ceramic scaffold prepared by combining a specific preparation process has basic requirements of good biocompatibility, cell affinity, proper degradation rate, processability and the like, and more importantly, the ceramic scaffold can provide proper promoting factors for tissue regeneration, accelerate functional reconstruction, and realize the regulation and control of the composition, degradation rate, performance and bone biological activity of the composite ceramic scaffold.

Further, the aperture range of the organic foam template is 50-500 mu m; still further, the pore size range of the organic foam template is preferably 200-400 μm.

By adopting the technical scheme, the aperture of the organic foam template is controlled to be 50-500 microns, preferably 200-400 microns, so that the effective components in the sol-gel precursor solution or the suspension slurry can conveniently enter the pores of the organic foam template or be adhered to the organic foam template during subsequent impregnation; according to the invention, the organic foam template with a specific pore size range is selected, so that the ceramic scaffold with the pore size of 30-400 microns can be prepared, the ceramic scaffold with a continuous through pore structure and a rough ceramic pore wall can be prepared by combining a specific template surface roughening treatment process, the pore size of 30-400 microns is favorable for cell migration and tissue ingrowth, the rough pore wall is favorable for cell adhesion, and micropores on the pore wall are favorable for transportation of nutrient substances and discharge of cell metabolic wastes, so that the ceramic scaffold can be used as an excellent material for regeneration and repair of bone defects. The ceramic prepared by the invention can be adjusted, and an appropriate pore structure and morphology can be constructed according to specific requirements, so that an appropriate ceramic support is constructed, and the ceramic support is convenient to adapt to different use conditions.

In order to achieve the second object, the invention provides the following technical scheme:

the ceramic support prepared by the preparation method of the composite bioactive ceramic support for bone regeneration and repair uses calcium phosphorus/calcium silicon composite as a main body, has two or more osteogenic bioactive substances of calcium silicate, bioactive glass, hydroxyapatite, tricalcium phosphate and complex phase calcium phosphate, has high porosity, adjustable degradation rate and performance and controllable size and shape, and can meet the requirements and requirements of wider bone defect regeneration and repair.

The calcium-phosphorus/calcium-silicon composite bioactive ceramic bracket prepared by the invention has flexible and various application forms, and can be formed into a required three-dimensional bracket by utilizing an organic porous template with a set shape and size according to the filling requirement of target bone defects; or cutting and forming the three-dimensional bracket after forming; the bone filler can also be prepared into porous small particles with different sizes by crushing and sieving, and is similar to commercial Bio-Os bone filling materials, and the porous small particles are filled into bone defects of non-bearing parts for bone guide regeneration repair.

The ceramic scaffold has high porosity and highly communicated pore structure, and can be compounded with organic polymer biological materials such as natural polymers including collagen and synthetic polymers including polylactic acid according to requirements, so that the scaffold performance is further improved, and degradation and ion release behaviors are regulated and controlled, so that the ceramic scaffold is suitable for requirements and requirements of wider bone defect regeneration and repair.

In conclusion, the invention has the following beneficial effects:

firstly, the biological ceramic scaffold prepared by combining an organic foam impregnation method and a sol-gel method for preparing biological ceramic is prepared by using a calcium-phosphorus/calcium-silicon composite biological ceramic scaffold, the degradation rate and the ion dissolution rate of the biological ceramic scaffold are between those of the calcium-phosphorus ceramic scaffold and the calcium-silicon ceramic scaffold and can be regulated, and the performances of brittleness and the like of the calcium-phosphorus/calcium-silicon composite biological active ceramic scaffold are obviously improved compared with those of a calcium-phosphorus or calcium-silicon ceramic scaffold with a single component;

secondly, the impregnation method of the impregnation liquid for the organic foam template is variable, the organic foam template can be repeatedly impregnated by adopting the suspension slurry with the same component, and the organic foam template can also be alternately impregnated by adopting different calcium-silica sol precursor solution-gel and calcium-phosphorus sol-gel precursor solution, so that the degradation rate of the bracket and the release rate and release amount of bioactive ions can be adjusted, the structure, strength, brittleness and other properties of the bracket can be adjusted, and the requirements and requirements of wider bone defect regeneration and repair can be met;

thirdly, besides the precursor substances with bioactivity, such as a calcium source substance, a silicon source substance, a phosphorus source substance and the like, different doping ions which promote the bioactivity of the bone, such as magnesium, zinc, strontium, manganese and the like, are introduced, and the biological ceramic porous scaffold doped with single or multiple ions is obtained after sintering, so that the composition of the composite ceramic scaffold is closer to that of natural bone mineral and has more excellent bone conduction and bone induction properties;

the ceramic scaffold prepared by the method has the basic requirements of good biocompatibility, cell affinity, proper degradation rate, processability and the like, and more importantly, the ceramic scaffold can provide proper promoting factors for tissue regeneration, provide proper microenvironment for cell adhesion, proliferation and differentiation, is beneficial to bone defect regeneration repair and accelerates function reconstruction;

and fifthly, organic porous foam is used as a template, and then the ceramic support is prepared by repeated impregnation or alternate impregnation of impregnation liquid and combination of a specific sintering method.

Drawings

FIG. 1 is a macroscopic scale view of a composite ceramic scaffold provided by the present invention;

FIG. 2 is an electron microscope observation image of the composite ceramic scaffold of example 1 of the present invention, in which FIGS. A and B are aperture morphology diagrams at different magnifications, and FIG. C is a graph of the degree of crystallization observed under a high magnification;

FIG. 3 is a graph showing the results of the osteogenic activity test in some examples of the present invention and in comparative examples.

Detailed Description

The present invention will be described in further detail with reference to examples.

Preparation example

Preparation example-preparation of a calcium silica sol-gel precursor solution:

dissolving 0.032mol of calcium nitrate tetrahydrate in 7.5ml of absolute ethyl alcohol (analytically pure), mixing, stirring for 2h until all calcium nitrate is dissolved, and preparing a salt alcohol solution; then regulating the pH value of the hydrochloric acid solution to 8 by using a hydrochloric acid solution (the concentration is 1mol/l), finally adding 0.032mol of tetraethyl silicate, and stirring the mixture in a water bath at 37 ℃ for 24 hours to obtain the calcium-silica sol-gel precursor solution.

Preparing a calcium phosphorus sol-gel precursor solution:

mixing 18.2g of triethyl phosphate, 54.6g of deionized water and 54.6g of absolute ethyl alcohol, and carrying out oil bath hydrolysis at the temperature of 80 ℃ for 24 hours to obtain a phosphorus source solution; dissolving 27.4g of calcium nitrate tetrahydrate in absolute ethyl alcohol at the temperature of 30 ℃ in a water bath to prepare 50ml of calcium source solution; dropwise adding the calcium source solution into the phosphorus source solution under the condition of ice-water mixed bath at 4 ℃ and fully stirring, and continuously stirring for 24 hours after dropwise adding is finished to obtain the calcium-phosphorus sol-gel precursor solution with the calcium-phosphorus ratio of 1.5.

Preparation example two

The difference between the second preparation example and the first preparation example is that: (1) in the calcium silica sol-gel precursor solution, the concentration of the salt alcohol solution is 20 wt%; the silicon source substance accounts for 60wt% of the calcium-silica sol-gel precursor solution;

(2) in the calcium-phosphorus sol-gel precursor solution, the calcium-phosphorus ratio is 1.7;

the remainder was in accordance with the preparation example.

Preparation example three

The difference between the third preparation example and the first preparation example is that: (1) in the calcium silica sol-gel precursor solution, the concentration of the salt alcohol solution is 60 wt%; the silicon source substance accounts for 20 wt% of the calcium-silica sol-gel precursor solution;

(2) in the calcium-phosphorus sol-gel precursor solution, the calcium-phosphorus ratio is 1.2;

the remaining homogeneous preparations remained consistent.

Preparation example four

The difference between the fourth preparation example and the first preparation example is that: in preparation example four: adding calcium nitrate tetrahydrate, magnesium nitrate hexahydrate and manganese chloride into ethanol according to the molar ratio of 8:1:1 to obtain a calcium-silica sol-gel precursor solution doped with magnesium ions and manganese ions, wherein the rest uniform preparation examples are consistent.

Preparation example five preparation of suspension slurry:

taking the calcium silica sol-gel precursor solution in the first preparation example for later use;

dissolving 0.1mol of calcium nitrate tetrahydrate in 150ml of deionized water to obtain a solution I, dissolving 0.064mol of diammonium hydrogen phosphate (NH4)2HPO4 in 150ml of deionized water to obtain a solution II, and uniformly mixing the solution I and the solution II to obtain a mixed solution; then heating the temperature of the mixed solution to 90 ℃, adjusting the pH value of the solution to 10, reacting for 3 hours at 90 ℃, centrifugally separating at 4000rpm, washing with deionized water for 3 times, then washing with ethanol for 3 times to obtain wet Hydroxyapatite (HA) containing absolute ethyl alcohol, and drying the wet hydroxyapatite in an oven to obtain hydroxyapatite nano powder; adding 12g of Hydroxyapatite (HA) powder into 60ml of the standby calcium silica sol-gel precursor solution, performing ultrasonic dispersion to fully and uniformly disperse the Hydroxyapatite (HA) powder in the calcium silica sol-gel precursor solution, and continuously stirring for 24 hours to obtain the suspension slurry.

Preparation example six

The difference between the sixth preparation example and the fifth preparation example is as follows: in preparation example six: hydroxyapatite (HA) powder accounts for 20 wt% of the suspension slurry, and the five uniformly prepared examples are consistent.

Preparation example seven

The difference between the seventh preparation example and the fifth preparation example is as follows: preparation example seven: hydroxyapatite (HA) powder accounts for 80wt% of the suspension slurry, and the five uniformly prepared examples are consistent.

Preparation example eight

Adding calcium nitrate tetrahydrate and manganese chloride in a molar ratio of 9:1, mixing, completely dissolving with ethanol (absolute ethanol), then adding 7.57ml of tetraethyl silicate (TEOS), adjusting the pH value to 11 with hydrochloric acid solution (1mol/L), stirring in water bath at 37 ℃ for 24h to obtain calcium silica sol-gel precursor solution doped with manganese ions;

0.94g of clay was mixed with 3.5ml of ethanol, and after shear dispersion at high speed (12000rpm), 5g of the mixture was added to the calcium silicate sol-gel precursor doped with manganese ions prepared above, and stirring was continued for 2 hours to obtain a suspension slurry.

Examples

Example 1

roughening the surface of the template: soaking the organic foam template in 15% sodium hydroxide solution for 3h, then respectively cleaning with absolute ethyl alcohol and clear water for 4 times, and finally drying at 60 ℃ for later use;

obtaining a steeping fluid: taking the calcium silica sol-gel precursor solution and the calcium phosphorus sol-gel precursor solution prepared in the first preparation example; dipping with a dipping solution: alternately dipping the template subjected to surface roughening treatment for 3 times between the calcium-silica sol-gel precursor solution and the calcium-phosphorus sol-gel precursor solution prepared in the first preparation example, and specifically performing the following operations:

for the first time: a. completely soaking the template subjected to surface roughening treatment in a calcium-silica sol-gel precursor solution for 5min, vacuumizing the organic foam template in the soaking process, pumping out air in gaps of the template, and taking out the template; b. centrifuging the organic foam template taken out in the step a at the rotating speed of 600rpm for 60 seconds, and removing redundant impregnation liquid on the organic foam template; c. drying the centrifuged organic foam template at 60 ℃ for 3 h;

and (3) for the second time: a. completely soaking the organic foam template dried in the first step c in the calcium-phosphorus sol-gel precursor solution, keeping for 5min, vacuumizing the organic foam template in the soaking process, pumping out air in gaps of the template, and then taking out the organic foam template; b. centrifuging the organic foam template taken out in the step a at the rotating speed of 600rpm for 60s, and removing redundant impregnation liquid on the organic foam template; c. drying the centrifuged organic foam template at 60 ℃ for 3 h;

and thirdly: a. completely soaking the organic foam template dried in the second step c in the calcium-silica sol-gel precursor solution, keeping for 5min, vacuumizing the organic foam template in the soaking process, pumping out air in the gaps of the template, and then taking out the organic foam template; b. centrifuging the organic foam template taken out in the step a at the rotating speed of 600rpm for 60 seconds, and removing redundant impregnation liquid on the organic foam template; c. drying the centrifuged organic foam template at 60 ℃ for 3h to obtain a compound;

and (3) sintering: and (3) placing the compound dried for the third time in an air atmosphere, and sintering for 2h at 1000 ℃ to obtain the calcium-silicon/calcium-phosphorus/calcium-silicon composite bioactive ceramic scaffold.

Example 2

roughening the surface of the template: soaking the organic foam template in 17% sodium hydroxide solution for 4h, then respectively cleaning with absolute ethyl alcohol and clear water for 5 times, and finally drying at 50 ℃ for later use;

for the first time: a. completely soaking the template subjected to surface roughening treatment in a calcium-silica sol-gel precursor solution for 10min, vacuumizing the organic foam template in the soaking process, pumping out air in gaps of the template, and taking out the template; b. centrifuging the organic foam template taken out in the step a at the rotating speed of 200rpm for 120 seconds, and removing redundant impregnation liquid on the organic foam template; c. drying the centrifuged organic foam template at 120 ℃ for 1 h;

and (3) for the second time: a. completely soaking the organic foam template dried in the first step c in the calcium-phosphorus sol-gel precursor solution, keeping for 10min, vacuumizing the organic foam template in the soaking process, pumping out air in gaps of the template, and then taking out the organic foam template; b. centrifuging the organic foam template taken out in the step a at the rotating speed of 200rpm for 120 seconds, and removing redundant impregnation liquid on the organic foam template; c. drying the centrifuged organic foam template at 120 ℃ for 1 h;

and thirdly: a. completely soaking the organic foam template dried in the second step c in the calcium-silica sol-gel precursor solution, keeping for 10min, vacuumizing the organic foam template in the soaking process, pumping out air in the gaps of the template, and then taking out the organic foam template; b. centrifuging the organic foam template taken out in the step a at the rotating speed of 200rpm for 120 seconds, and removing redundant impregnation liquid on the organic foam template; c. drying the centrifuged organic foam template at 120 ℃ for 1h to obtain a compound;

and (3) sintering: and (4) placing the compound dried for the third time in an air atmosphere, and sintering at 1300 ℃ for 6h to obtain the calcium-silicon/calcium-phosphorus/calcium-silicon composite bioactive ceramic support.

Example 3

roughening the surface of the template: soaking the organic foam template in a sodium hydroxide solution with the concentration of 12% for 6h, then respectively cleaning the organic foam template for 2 times by using absolute ethyl alcohol and clear water, and finally drying the organic foam template at 70 ℃ for later use;

for the first time: a. completely soaking the template subjected to surface roughening treatment in a calcium-silica sol-gel precursor solution for 2min, vacuumizing the organic foam template in the soaking process, pumping out air in gaps of the template, and taking out the template; b. centrifuging the organic foam template taken out in the step a at the rotating speed of 1000rpm for 30 seconds, and removing redundant impregnation liquid on the organic foam template; c. drying the centrifuged organic foam template at 60 ℃ for 6 hours;

and (3) for the second time: a. completely soaking the organic foam template dried in the first step c in the calcium-phosphorus sol-gel precursor solution, keeping for 2min, vacuumizing the organic foam template in the soaking process, pumping out air in gaps of the template, and then taking out the organic foam template; b. centrifuging the organic foam template taken out in the step a at the rotating speed of 1000rpm for 30 seconds, and removing redundant impregnation liquid on the organic foam template; c. drying the centrifuged organic foam template at 60 ℃ for 6 hours;

and thirdly: a. completely soaking the organic foam template dried in the second step c in the calcium-silica sol-gel precursor solution, keeping for 2min, vacuumizing the organic foam template in the soaking process, pumping out air in the gaps of the template, and then taking out the organic foam template; b. centrifuging the organic foam template taken out in the step a at the rotating speed of 1000rpm for 30 seconds, and removing redundant impregnation liquid on the organic foam template; c. drying the centrifuged organic foam template at 60 ℃ for 6 hours to obtain a compound;

and (3) sintering: and (3) placing the compound dried for the third time in an air atmosphere, and sintering at 800 ℃ for 1h to obtain the calcium-silicon/calcium-phosphorus/calcium-silicon composite bioactive ceramic scaffold.

Example 4

Example 4 differs from example 1 in that: in example 4, alternate impregnations were performed 2 times, and the rest was identical to example 1.

Example 5

Example 5 differs from example 1 in that: in example 5, alternate impregnations were performed 5 times, and the rest was identical to example 1.

Example 6

Example 6 differs from example 1 in that: in example 6, the sequence of the dipping solutions in the three dipping steps is different, namely, the first dipping step is carried out in the calcium-phosphorus sol-gel precursor solution, the second dipping step is carried out in the calcium-phosphorus sol-gel precursor solution, and the third dipping step is carried out in the calcium-phosphorus sol-gel precursor solution, so as to obtain the calcium-phosphorus/calcium-phosphorus composite ceramic stent, and the rest steps are consistent with those in example 1.

Example 7

Example 7 differs from example 1 in that: the rotation speed of the centrifugation in step a of example 7 was 50rpm, the centrifugation time was 240s, and the rest was the same as that of example 1.

Example 8

Example 8 differs from example 1 in that: the rotation speed of the centrifugation in step a of example 8 was 3000rpm, the centrifugation time was 15s, and the rest was the same as that of example 1.

Example 9

Example 9 differs from example 1 in that: the calcium-phosphorus sol-gel precursor solution and the calcium-silica sol-gel precursor solution in example 9 were the same as those in example 1 except that the prepared product in preparation example two was used.

Example 10

Example 10 differs from example 1 in that: the calcium-phosphorus sol-gel precursor solution and the calcium-silica sol-gel precursor solution in example 10 were the same as those in example 1 except that the prepared product in preparation example three was used.

Example 11

Example 11 differs from example 1 in that: the calcium-phosphorus sol-gel precursor solution and the calcium-silica sol-gel precursor solution in example 11 were the same as those obtained in preparation example four, except that they were the same as those in example 1.

Example 12

obtaining a steeping fluid: taking the suspension slurry prepared in the fifth preparation example;

dipping with a dipping solution: the template after the surface roughening treatment is repeatedly dipped in the suspension slurry prepared in preparation example five for 3 times, and the specific operation is as follows:

a. completely soaking the template subjected to surface roughening treatment in the suspension slurry, keeping for 5min, vacuumizing the organic foam template in the soaking process, pumping out air in gaps of the template, and then taking out the template; b. centrifuging the organic foam template taken out in the step a at the rotating speed of 1000rpm for 60 seconds, and removing redundant impregnation liquid on the organic foam template; c. drying the centrifuged organic foam template at 60 ℃ for 3 h;

repeating the steps a, b and c for three times to obtain a compound;

and (3) sintering: and (3) placing the dried compound after repeating the steps for three times in an air atmosphere, and sintering the compound for 2 hours at 1000 ℃ to obtain the composite bioactive ceramic bracket.

Example 13

Example 13 differs from example 12 in that: in example 13, the impregnation was repeated 2 times, and the rest was the same as in example 12.

Example 14

Example 14 differs from example 12 in that: in example 14, the impregnation was repeated 5 times, and the rest was the same as in example 12.

Example 15

Example 15 differs from example 12 in that: in example 15, the rotation speed of the centrifugation in step a was 200rpm, the centrifugation time was 240s, and the rest was the same as in example 12.

Example 16

Example 16 differs from example 12 in that: in example 16, the rotation speed of the centrifugation in step a was 3000rpm, the centrifugation time was 15s, and the rest was the same as in example 12.

Example 17

Example 17 differs from example 12 in that: in example 17, the suspension slurry used was the product obtained in preparation example six, and the rest was the same as in example 12.

Example 18

Example 18 differs from example 12 in that: in example 18, the product obtained in preparation example seven was used as a suspension slurry, and the rest was the same as in example 12.

Example 19

Example 19 differs from example 12 in that: in example 19, the product obtained in preparation example eight was used as a suspension slurry, and the rest was the same as in example 12.

Comparative example 1

Comparative example 1 differs from example 1 in that: comparative example 1, the impregnation in the calcium silica sol-gel precursor solution prepared in preparation example one was repeated 3 times, and the rest was identical to example 1.

Comparative example 2

Comparative example 2 differs from example 1 in that: in comparative example 2, the impregnation of the calcium-phosphorus sol-gel precursor solution prepared in preparation example one was repeated 3 times, and the rest was the same as in example 1.

Comparative example 3

Comparative example 3 differs from example 1 in that: in comparative example 3, the sintering temperature was 500 ℃ and the sintering time was 10 hours, and the rest was the same as in example 1.

Comparative example 4

Comparative example 4 differs from example 1 in that: in comparative example 4, the sintering temperature was 1500 ℃ and the sintering time was 0.5h, the rest being identical to example 1.

Comparative example 5

Comparative example 5 differs from example 1 in that: in comparative example 5, the sintering temperature in the sintering step was 1500 ℃ and the sintering time was 0.5h, and the rest was the same as in example 1.

Comparative example 6

Comparative example 6 differs from example 1 in that: in the comparative example 6, the template is a porous carbon template, the porous carbon template is prepared by heating the wood biological tissue template to 500 ℃ at a temperature rise rate of 15 ℃/min in a vacuum environment and carbonizing for 2-5 hours, and the rest is consistent with that of the template in the example 1.

Performance test

The performance of the ceramic stent samples prepared in examples 1 to 19 and comparative examples 1 to 6 was examined.

1) Observing the bracket appearance images of the samples of each embodiment and the comparative example under a microscope, and observing the through hole condition of each bracket;

2) 3 samples in each embodiment or comparative example are taken as a group, each group of samples are respectively soaked in the same phosphate buffer solution, the phosphate buffer solution is replaced every day, and after 8 weeks, the average weight loss rate of each sample is respectively calculated, so that the degradation rate of each sample can be simulated, namely the degradation rate of each sample is equal to the weight of the sample after weight loss divided by the original weight of the sample;

3) respectively evaluating the strength of the samples of each example and each comparative example, wherein the higher the fraction is, the better the strength of the sample is;

4) bone marrow mesenchymal stem cell (BMSC) culture

The samples prepared in each example and comparative example were immersed in a culture solution for one day, bone marrow mesenchymal stem cells (BMSC) were cultured using the extract thereof, and after two weeks of cell differentiation, alizarin was used to stain the cells, and red aggregates were observed (in order to meet the requirements of the drawings, gray aggregates of each picture were treated in fig. 3, and the treated gray aggregates were equivalent to the untreated red aggregates), and the more red (gray in fig. 3) aggregates, the better the osteogenic activity of the samples was.

Table 1 table for testing performance of each sample

As can be seen from Table 1, the degradation rate of the composite ceramic scaffold prepared according to the formulation and the preparation method of the present invention is between the degradation rates of comparative example 1 and comparative example 2, and the degradation rate is neither too fast nor too slow, and the degradation rate is proper, and the strength is also proper, thereby demonstrating that the composite ceramic scaffold prepared according to the present invention has excellent performance. According to the data of each embodiment and each proportion, the proportion of the impregnation liquid (the calcium-silica sol-gel precursor solution, the calcium-phosphorus sol-gel precursor solution and the suspension slurry), the sintering conditions (temperature and time), the impregnation process (impregnation times, centrifugal rate and the like) and the like all influence the performance of the composite ceramic support prepared by the invention.

As can be seen from A, B and C in FIG. 2, the composite ceramic scaffold prepared by the present invention has a porous structure, a continuous through pore structure and a rough ceramic pore wall structure, and the pore wall has micropores, etc.; the ceramic support prepared by the method has the aperture of 30-400 mu m and the porosity of over 60-90 percent through measurement, statistics and other finishing.

Comparing the graphs of the results of the osteogenic activity tests of the samples in fig. 3, the distribution of calcium nodules (gray agglomerates indicate calcium nodules) in each graph of the samples is different, and the more calcium nodules in the graph indicate the better osteogenic activity of the corresponding composite ceramic scaffold; among them, the calcium nodules were the most abundant in example 11, and thus it is known that the incorporation of specific dopant ions into the impregnation fluid can improve the osteogenic activity of the composite ceramic scaffold, and thus can improve the bone conduction and osteoinduction properties of the composite ceramic scaffold; by observing the distribution of calcium nodules in comparative example 2 and the remaining examples, it can be seen that the osteogenic activity of the composite ceramic scaffold is superior to that of the single-phase ceramic scaffold.

The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.

Claims

1. A preparation method of a composite bioactive ceramic bracket for bone regeneration and repair is characterized by comprising the following steps: the preparation method comprises the following steps:

2. The method for preparing the composite bioactive ceramic scaffold for bone regeneration repair according to claim 1, wherein the method comprises the following steps: in the step of dipping with the dipping solution, when dipping is carried out alternately with the dipping solution, a calcium silica sol-gel precursor solution and a calcium phosphate sol-gel precursor solution are adopted.

3. The method for preparing the composite bioactive ceramic scaffold for bone regeneration repair according to claim 1, wherein the method comprises the following steps: the calcium-silica sol-gel precursor solution and the calcium-phosphorus sol-gel precursor solution both comprise precursor substances containing bioactive ions, which are required for preparing the target ceramic scaffold, and the precursor substances comprise at least two of calcium source substances, silicon source substances and phosphorus source substances.

4. The method for preparing the composite bioactive ceramic scaffold for bone regeneration repair according to claim 1, wherein the method comprises the following steps: the calcium-silicon sol-gel precursor solution, the calcium-phosphorus sol-gel precursor solution and the suspension slurry respectively further comprise doping ions, and the doping ions are selected from at least one of magnesium, zinc, manganese and strontium ions.

5. The method for preparing the composite bioactive ceramic scaffold for bone regeneration repair according to claim 1, wherein the method comprises the following steps: when the repeated impregnation or the alternate impregnation is carried out, each impregnation comprises the following steps:

6. The method for preparing the composite bioactive ceramic scaffold for bone regeneration repair according to claim 3, wherein the method comprises the following steps: the preparation of the calcium silica sol-gel precursor solution comprises the following steps:

adding a calcium source substance into a volatile organic solvent, preparing a salt alcohol solution, adding a silicon source substance after the salt alcohol solution is completely dissolved, and stirring to obtain a calcium-silica sol-gel precursor solution;

the concentration of the saline alcohol solution is 20-60 wt%;

the silicon source substance accounts for 20-60 wt% of the calcium-silica sol-gel precursor solution.

7. The method for preparing the composite bioactive ceramic scaffold for bone regeneration repair according to claim 3, wherein the method comprises the following steps: in the calcium-phosphorus sol-gel precursor solution, the molar ratio of calcium to phosphorus is 1.2-1.8.

8. The method for preparing the composite bioactive ceramic scaffold for bone regeneration repair according to claim 1, wherein the method comprises the following steps: the preparation of the suspension slurry comprises the following steps:

adding the biological ceramic nano powder into the sol-gel precursor solution, and fully mixing and dispersing to obtain uniformly dispersed suspension slurry;

the biological ceramic nano powder accounts for 20-80 wt% of the suspension slurry;

the biological ceramic nano powder is selected from at least one of hydroxyapatite, beta-tricalcium phosphate, whitlockite or clay.

9. The preparation method of the composite bioactive ceramic scaffold for bone regeneration and repair according to any one of claims 1 to 8, wherein the preparation method comprises the following steps: the aperture range of the organic foam template is 50-500 mu m.

10. The utility model provides a bone regeneration restores and uses compound bioactive ceramic support which characterized in that: the ceramic scaffold is prepared by the preparation method of the ceramic scaffold for bone regeneration and repair according to any one of claims 1 to 9.