WO2018072679A1

WO2018072679A1 - Biomimetic biomineralized artificial bone repair material and preparation method therefor and use thereof

Info

Publication number: WO2018072679A1
Application number: PCT/CN2017/106513
Authority: WO
Inventors: 张自强; 张以河; 陈飞旭
Original assignee: 张自强
Priority date: 2016-10-17
Filing date: 2017-10-17
Publication date: 2018-04-26
Also published as: CN107952115B; ZA201903065B; CN107952115A

Abstract

Disclosed are a biomimetic biomineralized artificial bone repair material and a preparation method therefor and the use thereof. The material comprises a crosslinked collagen matrix and a biomineralized collagen/hydroxyapatite composite powder dispersed in the matrix. The mass ratio of the collagen matrix to the biomineralized collagen/hydroxyapatite composite powder is 9 : 1 - 1 : 9. The material solves the problems of ready collapsibility, fast degradation speed and poor mechanical properties of materials, and realizes the biomimetic construction of nano-hydroxyapatite and collagen.

Description

Biomimetic biomineralized artificial bone repairing material and preparation method and application thereof

The present application is based on the application of the CN application number 201610902145.X, the filing date of which is filed on October 17, 2016, and the priority of which is hereby incorporated by reference.

Technical field

The invention relates to a biomimetic biomineralized artificial bone and a preparation method and application thereof, and belongs to the field of biomedical materials.

Background technique

The statements herein merely provide background information related to the present application and do not necessarily constitute prior art.

In clinical cases, bone defects are mainly caused by trauma, infection, and tumor. In response to this situation, various alternative materials for bone repair have been prepared by various methods or routes, and certain effects have been achieved.

Bone implant materials can be roughly divided into autologous bone, allogeneic bone, xenogenic bone, and synthetic materials. Autologous bone grafts are almost limited by the limitations of their own donors; allogeneic bones have better osteogenic effects, but there are also risks of rejection and viral infection, and due to limited sources, it is difficult to achieve a large number of clinically Use; xenogeneic bone is rarely used due to large rejection. Therefore, artificially synthesized bone repair materials with good biological activity and similar to natural bone components have emerged. This material can provide a microenvironment that contributes to the adhesion, proliferation and function of osteoblasts and is similar to natural bone. It can not only be used directly as a bone defect repair material, but also a good bone tissue engineering carrier material, which has opened up a broad prospect for the development of bone tissue engineering.

Synthetic bone materials are currently the hotspots of research, and can be divided into metal alloy materials, inorganic ceramic materials, polymer materials and composite materials containing metal/inorganic ceramics/polymer organic composites. In terms of metal materials, materials mainly used in the past are stainless steel, cobalt alloys, titanium alloys, niobium alloys, memory alloys and the like to replace gold. Since ordinary stainless steel is more susceptible to corrosion as an iron-based alloy, it is now mostly used to repair stainless steel containing molybdenum (Mo) bone, which is more resistant to biochemical corrosion than ordinary stainless steel. The excellent mechanical properties of metal materials can meet the demand, but it is too high to cause stress shielding. In addition, metal materials are inevitably subject to corrosion in biochemical environments, and toxic metal ions are another major problem in metal-based biomaterials. In terms of ceramic materials, it mainly includes several types of inorganic ceramic materials such as calcium sulfate filler, inert bioceramic, bioactive ceramics and absorbable ceramics. The composition of general inorganic ceramic materials is not toxic, but the strength and biological activity of the materials do not seem to have both. Generally, materials with good bioactivity have poor mechanical properties, and materials with good mechanical properties are not biologically active. In terms of polymer materials, polymer materials applied in orthopedics can be roughly classified into two types, namely, natural polymer materials and synthetic polymer materials. Orthopaedic polymer materials are generally not toxic, but their biological activity is low, and the strength of the material is also far from the bone tissue. Therefore, people have developed many new jobs by using the advantages and disadvantages of these materials. Arts and methods have synthesized a variety of composite materials, and materials with different properties are combined in a clever way, in order to meet the different applications. New processes such as nanocomposite, gradient composite, 3D printing, etc. have gradually become the mainstream of orthopedic repair materials, such as L-polylactic acid/hydroxyapatite whisker/collagen liquid crystal scaffold (Wu Di, L-polylactic acid/hydroxyapatite crystal) Preparation and characterization of the whisker/collagen liquid crystal scaffold [D]. Jinan University, 2014.).

At home and abroad, research on bone scaffolds mainly focuses on composite scaffolds of hydroxyapatite and collagen. The inventors realized that although CN1562387A, CN101229392A, CN101417145A, etc. have produced various types of collagen/hydroxyapatite composite bone scaffolds, Such materials have the problems of loose structure, poor strength, very weak toughness, too fast degradation rate, and can not be well matched with bone growth rate, making it difficult to become a good bone tissue engineering support. The inventors know that the three-dimensional scaffold spatial structure is the basis of bone repair materials and bone conduction properties. In recent years, researchers have improved collagen-based nano-hydroxyapatite composites by biomimetic synthesis, cross-linking treatment, and introduction of the third equivalent method. The performance, but there are still problems such as large brittleness, directional dispersal of hydroxyapatite microcrystals, poor adhesion with collagen, etc., and these stents are formed by compaction without forming a porous form. Not conducive to cell adhesion and proliferation. The inventors have realized that although CN102205150A, CN103785059A, CN102240415A use polylactic acid or its copolymer as a matrix to prepare a three-dimensional void structure of bone material, although it has good mechanical properties, the hydrophilic property of the material is poor, and the degradation products will continue. Produces acidic substances, resulting in poor biocompatibility. The inventors have realized that CN105457097A, CN101554493A are thoroughly mixed with anti-separation agents, dispersants and/or modifiers to obtain nano-hydroxyapatite/collagen scaffolds, but anti-seizure agents, dispersants and/or modifiers in scaffolds. Components that belong to non-natural bone structures affect material structure and osteogenesis.

In summary, the inventors have realized that there is a need in the art for a bone repair scaffold material having a good biomimetic effect in a three-dimensional structure, but it has not been effectively satisfied for a long time. Therefore, it is urgent to develop a new bone repair scaffold material to meet the reality. need.

Summary of the invention

The present application discloses a biomimetic biomineralized artificial bone repair material, which is a three-dimensional porous scaffold material, which comprises a crosslinked collagen matrix and a biomineralized collagen/hydroxyapatite composite powder wrapped in the matrix. The mass ratio of the collagen matrix to the biomineralized collagen/hydroxyapatite composite powder is from 9:1 to 1:9.

The present application further discloses a method for preparing the biomimetic artificial bone repairing material, the method comprising the following steps:

(a) providing a biomineralized collagen/hydroxyapatite composite powder and an aqueous collagen solution;

(b) adding the biomineralized collagen/hydroxyapatite composite powder to the collagen aqueous solution to form a mixture a system, wherein the mass ratio of the biomineralized collagen/hydroxyapatite composite powder to the collagen in the collagen aqueous solution is 9 to 1:1 to 9;

(c) adjusting the pH of the mixed system obtained in the step (b) to 7 to 9, and then stirring to form a mineralized collagen composite sol;

(d) freeze-drying the mineralized collagen composite sol to obtain a mineralized collagen composite porous material;

(e) cross-linking the obtained mineralized collagen composite porous material to obtain the biomimetic mineralized artificial bone repairing material.

The application further discloses the application of the biomimetic mineralized artificial bone repair material.

DRAWINGS

Figure 1 is a flow chart of Embodiments 1 to 7;

2 is an electron micrograph of the product obtained in Example 1;

3 and 4 are XRD comparison diagrams in the ninth embodiment.

detailed description

The technical solutions of the present application are described in detail below with reference to the specific embodiments and the accompanying drawings. The scope of the application. In the examples, each of the original reagent materials is commercially available, and the experimental methods not specifying the specific conditions are conventional methods and conventional conditions well known in the art, or in accordance with the conditions recommended by the instrument manufacturer.

The present application discloses a biomimetic biomineralized artificial bone repair material, which is a three-dimensional porous scaffold material, which comprises a crosslinked collagen matrix and a biomineralized collagen/hydroxyapatite composite powder wrapped in the matrix. The mass ratio of the collagen matrix to the biomineralized collagen/hydroxyapatite composite powder is 9:1 to 1:9, for example, 1 to 5:5 to 9, and for example, 3 to 5:5 to 7. .

By controlling the mass ratio of the collagen matrix to the composite powder in the range of 3 to 5:5 to 7, the mechanical properties, flexibility, and porosity of the material of the present application are excellently balanced, and the practical application requirements are more satisfied.

The bionic biomineralized artificial bone repairing material described in the present application is a collagen-based biomimetic mineralized artificial bone, which solves the long-term desire to provide a three-dimensional bone repair scaffold material mainly composed of collagen and hydroxyapatite, but has not been The technical problems achieved can meet the needs of artificial bone and tissue suture in GTR or GBR clinical operation, including hydroxyapatite and pure collagen, which are the main components of human natural tissue, non-toxic, and do not affect the material. Structure and osteogenic effect, with good biocompatibility. Due to the existence of cross-linked collagen matrix, the problems of material collapse, excessive degradation rate and poor mechanical properties are solved, and the biomimetic construction of nano-hydroxyapatite and collagen is realized.

The word "comprising" as used in the present application includes "substantially including", and the term "comprising" is used to mean that the present application does not specifically limit the present application without prejudice to the prior art contribution to the present application. In particular, the term "comprising" means consisting of the listed starting materials.

The bionic biomineralized artificial bone of the present application has a good three-dimensional structure, the mature collagen fibers of the triple helix are arranged in an orderly manner, and the biomineralized collagen/hydroxyapatite composite powder is closely attached to the collagen fiber, and is fully filled with Between the collagen fibers, there is a collagen-hydroxyapatite-collagen tertiary composite structure, which is a combination of multiple dimensions, so that the biomineralized collagen/hydroxyapatite composite powder can be stabilized in the three-dimensional porous scaffold structure and is not easy to fall off. , the stability of the three-dimensional support structure is increased, thereby improving the mechanical properties of the material and being not easily damaged.

The biomineralized collagen/hydroxyapatite composite powder described in the present application is prepared by biomineralization process, and controls hydroxyapatite at the molecular level through the interaction of collagen organic macromolecules and inorganic ions at the interface. Crystallization and growth of the inorganic mineral phase, so that the collagen/hydroxyapatite composite powder has a special hierarchical structure and assembly method, which can be prepared according to the existing method, for example, by the step (1) in the specific embodiment of CN1106861C~ (4) It is prepared, which is equivalent to the calcium phosphate salt dry powder in the case, and those skilled in the art know that the order of addition of phosphate and calcium ions has no substantial effect on biomineralization.

The collagen/biomineralized hydroxyapatite composite powder described in the present application is prepared by biomineralization of collagen, and hydroxyapatite is formed by orderly crystal growth of collagen on the molecular structure of collagen using collagen as a template. Collagen and hydroxyapatite are molecularly oriented. Through biomimetic biomineralization, the molecules re-order into an ordered structure with a more consistent and broader orientation of collagen fibers, resulting in high mechanical strength of the artificial bone.

In some embodiments, the biomimetic mineralized artificial bone repair material has a pore diameter of 10 to 500 μm and a porosity of 50% to 97%.

In some embodiments, the mass fraction of hydroxyapatite in the biomineralized collagen/hydroxyapatite composite powder is from 50 to 99% by weight. In some embodiments it is 80 to 99 wt%. The mass fraction of hydroxyapatite in the composite powder is controlled within the range of 80 to 99 wt%, so that the particle size of the composite powder becomes smaller and the dispersion is more uniform in the collagen matrix.

In some embodiments, the XRD pattern of the biomimetic mineralized artificial bone repair material is substantially as shown in any of col-HA in FIG. 4 or bionic bone 19, bionic bone 37 or bionic bone 55 in FIG. As is apparent from FIG. 3 or FIG. 4, in some embodiments of the present application, the biomimetic mineralized artificial bone repair material has a high degree of similarity to natural bone, particularly bionic bone 37 or bionic bone 55.

In some embodiments, the collagen matrix or the collagen in the biomineralized collagen/hydroxyapatite composite powder is a native type I collagen of an atelopeptide and/or a recombinant human type I collagen of an atelopeptide. . In some embodiments, the natural type I collagen can extract skin and/or Achilles tendon of an animal (eg, pig, cow, sheep, horse, etc.), and is subjected to an enzymatic process (which may further include dialysis purification, etc.). The resulting depeptide peptide collagen.

In another aspect, the present application discloses a preparation method of the biomimetic mineralized artificial bone repairing material, wherein the method comprises the following steps:

(a) providing a biomineralized collagen/hydroxyapatite composite powder and an aqueous collagen solution; the mass fraction of collagen in the aqueous collagen solution is 0.8% to 5% in some embodiments;

(b) adding the biomineralized collagen/hydroxyapatite composite powder to the aqueous collagen solution to form a mixed system, wherein the biomineralized collagen/hydroxyapatite composite powder and the collagen aqueous solution are The mass ratio of collagen is 9 to 1:1 to 9; in some embodiments, 1 to 5: 5 to 9; in some embodiments, 3 to 5: 5 to 7;

(c) adjusting the pH of the mixed system obtained in step (b) is 7 to 9, followed by stirring to form a mineralized collagen composite sol; in some embodiments, the pH of the resulting mixed system is adjusted to 7 to 9 using NaOH, KOH or ammonia;

Optionally, the mineralized collagen composite sol obtained in the step (c) is pre-frozen to obtain a mineralized collagen jelly, and then the freeze-drying is performed;

In the present application, the resulting biomimetic mineralized artificial bone repair material can be cut into a desired shape and packaged aseptically.

In some embodiments, the crosslinking in step (e) comprises physical crosslinking and/or chemical crosslinking.

In some embodiments, the physical crosslinking includes, but is not limited to, one or more of ultraviolet radiation, thermal dehydrogenation, and radiation sterilization crosslinking.

In some embodiments, the chemical crosslinking includes, but is not limited to, the use of carbodiimide, diamine, epoxy compound, hydroxysuccinimide, diphenyl phosphate (DPPA), glutaraldehyde, formaldehyde, B. One or more of aldehyde acid and genipin are crosslinked, and after using a chemical crosslinking agent, the residual crosslinking agent is removed by an elution procedure.

In some embodiments, the biomineralized collagen/hydroxyapatite composite powder described herein is prepared as follows:

(i) placing collagen in an acid to form an acidic aqueous solution of collagen; the mass fraction of collagen in the acidic aqueous solution is, for example, 0.01% to 2%, and the pH is 2.5 to 5.5;

(ii) slowly adding an aqueous solution containing calcium ions to the acidic aqueous solution of the collagen; in an acidic aqueous solution of the collagen, the amount of calcium ions added is, for example, 0.01 to 0.1 mol per gram of collagen;

(iii) adding a phosphoric acid solution to the solution obtained in step (ii), stirring and mixing to form a mixed system; the phosphoric acid solution is added in an amount such that the molar ratio of calcium ions to phosphate ions in the system is, for example, 1.5 to 2;

(iv) adjusting the pH of the system obtained in the step (iii) to 7 to 10 to obtain a white suspension;

(v) The white suspension obtained in the step (iii) is allowed to stand, for example, at 15 to 25 ° C for 22 to 26 hours, filtered, and the filter cake is freeze-dried, for example, sufficiently frozen at -50 to -20 ° C. Drying for 24 to 48 hours, followed by grinding the prepared dry powder is biomineralized collagen / hydroxyapatite composite powder;

In some embodiments, the acid in step (i) includes, but is not limited to, one or more of sulfuric acid, hydrochloric acid, nitric acid, acetic acid, propionic acid, and citric acid;

In some embodiments, the calcium ion-containing aqueous solution described in step (ii) is formed using one or more of CaCl ₂ , Ca(NO ₃ ) _{2 ,} and CaCO ₃ .

The above method for preparing biomineralized collagen/hydroxyapatite in the present application can effectively control the precipitation effect of the powder, and is more advantageous for obtaining a composite powder having a smaller particle diameter than the method known to the inventors, thereby making the obtained composite powder Disperse more evenly in the collagen matrix.

In some embodiments, when pH is adjusted in step (iv), when pH = 5-6, the mixed system begins to precipitate, and when pH = 7-10, a white suspension appears in the mixed system.

According to a specific embodiment of the present application, the method for preparing the biomimetic mineralized artificial bone repairing material of the present application comprises the following steps:

(S1) Preparation of a biomineralized collagen/hydroxyapatite composite powder comprising:

(S1-1) dissolving collagen in an acid solution to prepare an acidic aqueous solution of collagen; in some embodiments, the mass fraction of collagen in the acidic aqueous solution is 0.01% to 2%, and the pH is 2.5 to 5.5. ;

(S1-2) continuously stirring the solution obtained in the step (S1-1), slowly adding an aqueous solution containing calcium ions; in some embodiments, the amount of calcium ions added per gram of collagen in the acidic aqueous solution of collagen is 0.01 to 0.1 mol;

(S1-3) continuously stirring the solution obtained in the step (S1-2), adding a phosphoric acid solution, and stirring and mixing to form a mixed system; in some embodiments, the amount of the phosphoric acid solution added depends on the calcium ion in the step (S1-2). The amount of addition, so that the molar ratio of calcium ions to phosphate ions reaches 1.5 to 2;

(S1-4) continuously stirring the solution obtained in the step (S1-3), adjusting the pH of the mixed system to 7 to 10 to obtain a white suspension;

(S1-5) The mixed system obtained in the step (S1-4) is allowed to stand, and in some embodiments, it is allowed to stand at 15 to 25 ° C for 22 to 26 hours, filtered, and the filter cake is freeze-dried, in some embodiments. Fully freeze-drying at -50 to -20 ° C for 24 to 48 hours, followed by grinding to obtain a dry powder which is a biomineralized collagen/hydroxyapatite composite powder;

(S2) a composite of collagen and biomineralized collagen/hydroxyapatite composite powder, comprising:

(S2-1) dissolving collagen in purified water to prepare an aqueous solution of collagen; in some embodiments, the mass fraction of collagen in the aqueous collagen solution is 0.8% to 5%;

(S2-2) continuously stirring the solution obtained in the step (S2-1), adding the biomineralized collagen/hydroxyapatite composite powder obtained in the step (S1-5), stirring and mixing to form a mixed system, biomineralized collagen/ The ratio of the amount of the hydroxyapatite composite powder added to the amount of collagen protein in the aqueous collagen solution obtained in the step (S2-1) is from 9:1 to 1:9, and in some embodiments, from 1 to 5:5 to 9, In some embodiments, it is 3 to 5: 5 to 7;

(S2-3) continuously stirring the solution obtained in the step (S2-2), adjusting the pH of the mixed system to 7 to 9;

(S2-4) The mixed system obtained in the step (S2-3) is allowed to stand and stirred to form a mineralized collagen composite solution; in some embodiments, it is allowed to stand at 15 to 25 ° C for 10 to 48 hours, and stirred for 5 to 30. Minutes to make it fully mixed to form a mineralized collagen composite solution;

(S3) Preparation of biomimetic biomineralized artificial bone repair material, comprising:

(S3-1) pre-freezing the mineralized collagen composite solution obtained in the step (S2-4), and in some embodiments, pre-freezing at 1 to 5 ° C for 3 to 24 hours to obtain a mineralized collagen jelly;

(S3-2) freeze-drying the mineralized collagen of step (S3-1), and in some embodiments, performing sufficient freeze-drying at -50 to -20 ° C for 22 to 26 hours to obtain a mineralized collagen composite porous material;

(S3-3) The biomimetic collagen composite porous material is crosslinked by physical and/or chemical methods to obtain the biomimetic mineralized artificial bone repair material.

The method described in the present application has a simple production process, is easy to be mass-produced, and has low production cost, and is very beneficial for reducing the medical cost of the patient.

The technical means of the present application can be combined with each other to achieve further effects.

In still another aspect, the present application discloses the use of the biomimetic mineralized artificial bone repair material or the biomimetic biomineralized artificial bone repair material prepared in the present application for preparing an implant at a location supporting a bone defect of a human or animal.

When the bone repair material of the present application is placed as an implant at a bone defect position, it is used as a three-dimensional stent for bone formation, bone regeneration, bone repair or bone replacement, or for repair and regeneration of bone defects such as bones and extraction sockets.

The application has the following beneficial effects:

(1) The biomimetic artificial bone has a collagen-hydroxyapatite-collagen tertiary composite structure, the triple-helix mature collagen fibers are arranged in an orderly manner, and the biomineralized collagen/hydroxyapatite composite powder is closely arranged. Attached to collagen fibers and fully filled between collagen fibers, it has a collagen-hydroxyapatite-collagen tertiary composite structure, which is a combination of multiple dimensions, enabling biomineralized collagen/hydroxyapatite composite powder to Stabilized in the three-dimensional porous scaffold structure, so that the hydroxyapatite crystal is not easy to fall off, and the stability of the hydroxyapatite in the three-dimensional scaffold structure is increased.

(2) The biggest feature of the biomimetic artificial bone is the ordered three-dimensional interconnected structure of collagen and hydroxyapatite. The interconnected pore structure facilitates the communication of the blood vessels deep into the material to ensure the growth of the material. The nutrient supply of deep tissues; in addition to the combination of materials and implanted beds, the porous structure is the growth of the body's bone tissue, the formation of mechanical interlocks, and the combination of implant materials, creating conditions for retention; porous surfaces are beneficial for blood vessels And the soft tissue grows in, and the pores communicate with each other and are the prerequisites for the inward growth of the bone. Interconnecting is beneficial to the flow of cells and body fluids in the body, tissue metabolism, bone growth into the micropores and a large number of external epiphyses to form a solid "biological fixation." "Micropores constitute a large surface area for bone sedimentation Provide a good matrix.

(3) The biomimetic artificial bone is composed of hydroxyapatite and pure collagen, and does not use polylactic acid, chitosan, chitin, anti-killing agent, templating agent, dispersing agent, modifier, etc., and does not affect Material structure and osteogenic effect, non-toxic, with good biocompatibility; and made with the use of the terminal peptide collagen, no immunogenicity; achieved simultaneous biomimetic composition and structure.

(4) Biomineralized collagen/hydroxyapatite composite powder is mostly nano-scale; nano-sized hydroxyapatite particles are more common than ordinary hydroxy-phosphorus due to the unique properties of nano-materials such as surface effect, small size effect and quantum effect. Graystone has stronger biological activity. The artificial bone made of nano-sized hydroxyapatite particles is white or yellowish-fibrous. The surface is smooth and opaque. It has a porous three-dimensional network structure, which is beneficial to cell adhesion and growth.

(5) Through physical or chemical cross-linking, the mechanical properties of the material are greatly improved, it has strong anti-degradation effect, is not easy to collapse, and realizes the biomimetic construction of nano-hydroxyapatite and collagen.

(6) The production process is simple, easy to mass-produce, and the production cost is low, which is very beneficial to reduce the medical cost of the patient.

The reagents and instruments in the following examples and comparative examples are as follows:

Hydroxyapatite (Beijing Yusheng Biotechnology Co., Ltd.), type I collagen (Beijing Yusheng Biotechnology Co., Ltd.), NaOH (analytical grade), and the like.

FT/IR-6800 FT-IR spectrometer (Jasco, Japan), D8 Focus X-ray diffractometer (Bruker, Germany), HP-500 digital display force gauge (Yueqing Aidebao Instrument Co., Ltd.) , GLZ-2 vacuum freeze dryer (Shanghai Pudong Freeze Drying Equipment Co., Ltd.), DZF-6050AF vacuum drying oven (Tianjin Gongxing Laboratory Instrument Co., Ltd.), 2XZ-2 rotary vane vacuum pump (Shanghai Double Goose Refrigeration) Equipment Co., Ltd.).

Example 1

In the present embodiment, the preparation of the bionic biomineralized artificial bone is carried out by using an acid solution using hydrochloric acid, a calcium ion solution using a CaCl ₂ aqueous solution, an alkaline solution using an aqueous NaOH solution, and an ultraviolet irradiation physical crosslinking method. FIG. 1 is a flow chart showing a method for preparing a biomimetic biomineralized artificial bone of the present application.

According to the steps shown in Figure 1, the preparation method of the biomimetic biomineralized artificial bone is:

Step S1 (preparation of biomineralized collagen/hydroxyapatite composite powder) specifically includes:

Step S1-1 (dissolved collagen): 2g of collagen is dissolved in 1000mL of a concentration of 0.001mol / L of hydrochloric acid solution, formulated into an acid solution of collagen, wherein the collagen concentration is 0.2%, the pH of the solution is 3;

Step S1-2 (adding calcium ions): continuously stirring the solution obtained in the step S1-1, slowly adding 200 mL of a 0.2 mol/L CaCl ₂ aqueous solution;

Step S1-3 (adding phosphoric acid): continuously stirring the solution obtained in the step S1-2, adding 400 mL of a phosphoric acid solution having a concentration of 0.06 mol/L, stirring and mixing to form a mixed system, and the amount of the phosphoric acid solution added depends on the step S1- 2 the amount of calcium ions added, so that the molar ratio of calcium ions to phosphate ions reaches 1.67;

Step S1-4 (adjusting the pH): continuously stirring the solution obtained in the step S1-3, slowly adding a NaOH solution having a concentration of 0.03 mol/L to the mixed system pH=7 to 10, and when the pH is 5-6, the mixed system starts. Precipitation occurs, when the pH is 7-10, a white suspension appears in the mixed system;

Step S1-5 (filtering, freeze-drying, and grinding to obtain a composite powder): The mixed system obtained in the step S1-4 is allowed to stand at 15 to 25 ° C for 24 hours, filtered, and the filter cake is sufficiently freeze-dried at -40 ° C. After hours, the dry powder obtained by the subsequent grinding is a biomineralized collagen/hydroxyapatite composite powder.

Step S2 (composite of collagen and biomineralized collagen/hydroxyapatite composite powder) specifically includes:

Step S2-1 (dissolved collagen): 3 g of collagen is dissolved in 97 mL of purified water to prepare a solution of collagen, wherein the collagen concentration is 3%;

Step S2-2 (blending): continuously stirring the solution obtained in the step S2-1, adding 3 g of the biomineralized collagen/hydroxyapatite composite powder prepared in the step S1-5, stirring and mixing to form a mixed system;

Step S2-3 (adjust pH): continue to stir the solution obtained in step S2-2, slowly add 0.01mol / L aqueous NaOH solution to the mixed system pH = 9;

Step S2-4 (Standing, stirring): The mixed system obtained in the step S2-3 is allowed to stand at 25 ° C for 24 hours, poured into a blender and stirred for 15 minutes to be thoroughly mixed, and the mixed system becomes a white emulsion to form a ore. Collagen complex solution;

Step S3 (preparation of mineralized collagen composite porous material) specifically includes:

Step S3-1 (filling, pre-freezing): taking the mineralized collagen composite solution obtained in step S2-4 into a rectangular mold and flattening, pre-freezing at 4 ° C for 3 hours to obtain mineralized collagen jelly;

Step S3-2 (freeze-drying): the mineralized collagen gelatinized in step S3-1 is freeze-dried at -50 ° C for 24 hours to obtain a mineralized collagen composite porous material;

Step S3-3 (crosslinking): crosslinking the mineralized collagen composite porous material by ultraviolet irradiation, and the ultraviolet irradiation condition is: ultraviolet light with a wavelength of 254 nm, and irradiation with 10 W for 24 hours;

Step S3-4 (cutting, aseptic packaging, sterilization): The cross-linked material is cut into a desired shape, and then sterilized and used after being aseptically packaged.

The biomimetic artificial bone (numbered as bionic bone 55) has a three-layer composite structure of collagen-hydroxyapatite-collagen, and adopts a block-shaped material with regular shape, and measures the length, width, height and weight, respectively, and is calculated. The apparent density is 0.13 g/cm ³ and the porosity is 65%. The electron micrograph is shown in Fig. 2.

Example 2

In the present embodiment, the biomimetic artificial bone is prepared by using hydrochloric acid, a calcium ion solution using a CaCl ₂ aqueous solution, an alkaline solution using an aqueous NaOH solution, and glutaraldehyde for chemical crosslinking.

The mineralized collagen composite porous material is prepared according to steps S1-1 to S3-2 in Example 1, and then, the mineralized collagen composite porous material is chemically crosslinked using glutaraldehyde, specifically, the ore obtained in step S3-2. The collagen composite porous material is immersed in 0.05 wt% of glutaraldehyde in anhydrous ethanol for 48 hours for crosslinking; then the crosslinked mineralized collagen composite porous material is taken out and placed in a chromatography column to flow pure The water was washed for 48 hours to remove residual cross-linking agent; the cross-linked mineralized collagen composite porous material was vacuum dried at 110 ° C for 48 hours to obtain the bionic biomineralized artificial bone of the present example. The bionic biomineralized artificial bone of the present embodiment was tested to have an apparent density of 0.24 g/cm ³ and a porosity of 85%.

Example 3

In the present embodiment, the preparation of the biomimetic biomineralized artificial bone is carried out by using an aqueous solution of nitric acid in an acid solution, an aqueous solution of Ca(NO ₃ ) _{2 in a} calcium ion solution, and an aqueous solution of KOH in an alkaline solution, and physically crosslinking using a thermal dehydrogenation method.

The biomimetic biomineralized artificial bone was prepared according to the substantially same procedure as in Example 1, except that the acid solution in the step S1-1 was replaced with 1000 mL of a 0.001 mol/L aqueous solution of nitric acid, and the calcium ion solution in the step S1-2 was 20 mL. The Ca(NO ₃ ) ₂ aqueous solution having a concentration of 0.1 mol/L is substituted for the CaCl ₂ aqueous solution, and the alkaline aqueous solution in the step S1-4 and the step S2-3 is replaced with the NaOH aqueous solution having a concentration of 0.01 mol/L, after the step S3-2. The mineralized collagen composite porous material is physically crosslinked by a method of thermal dehydrogenation, and the parameters of the method for thermal dehydrogenation are a temperature of 130 ° C and a vacuum degree of -0.1 MPa, and the remaining steps are the same as in the first embodiment. The biomimetic biomineralized artificial bone prepared in the present example was tested to have an apparent density of 0.15 g/cm ³ and a porosity of 70%.

Example 4

In the present embodiment, the biomimetic biomineral artificial bone is prepared, and the calcium ion aqueous solution is CaCO ₃ and physically crosslinked by a thermal dehydrogenation method.

The biomimetic biomineralized artificial bone is prepared according to the substantially same procedure as in the embodiment 1, except that the calcium ion solution in the step S1-2 is CaCO ₃ particles, and after the step S3-2, the method for thermally dehydrogenating the mineralized collagen composite porous material is used. The parameters of the method for thermal dehydrogenation were as follows: temperature 130 ° C, vacuum degree -0.1 MPa, and the remaining steps were the same as in Example 1. The bionic biomineralized artificial bone of the present embodiment was tested to have an apparent density of 0.14 g/cm ³ and a porosity of 72%.

Example 5

In the present embodiment, the biomimetic artificial bone is prepared by using an aqueous solution of acetic acid in an acid solution, an aqueous solution of CaCl _{2 in} an aqueous solution of calcium ions, and an aqueous solution of Na ₂ CO _{3 in} an aqueous alkaline solution, which is chemically crosslinked using formaldehyde.

Preparing the biomimetic biomineralized artificial bone according to the substantially same procedure as in Example 1, except that the acid solution in the step S1-1 is replaced by a 1000 mL concentration of 0.01 mol/L aqueous acetic acid solution instead of the hydrochloric acid solution, in steps S1-4 and S2-3. The alkaline solution is adjusted to pH with a 0.5 mol/L Na ₂ CO ₃ aqueous solution instead of the NaOH aqueous solution, and after the step S3-2, the mineralized collagen composite porous material is chemically crosslinked using 0.025 wt% of formaldehyde anhydrous ethanol solution. The remaining steps are the same as in the first embodiment. The bionic biomineralized artificial bone of the present embodiment was tested to have an apparent density of 0.28 g/cm ³ and a porosity of 67%.

Example 6

In the present embodiment, the preparation of the biomimetic biomineralized artificial bone is carried out by using an aqueous solution of acetic acid as an aqueous solution, an aqueous solution of calcium ions as CaCl ₂ , an aqueous alkaline solution using KOH, and chemical crosslinking using formaldehyde.

Preparing the biomimetic biomineralized artificial bone according to the substantially same procedure as in Example 1, except that the acid solution in the step S1-1 is replaced with 1000 mL of 0.01 mol/L acetic acid instead of the hydrochloric acid solution, and the alkali in the step S1-4 and the step S2-3. The solution was replaced by KOH with a concentration of 0.02 mol/L. After step S3-2, the mineralized collagen composite porous material was chemically crosslinked using a 0.025 wt% formaldehyde anhydrous ethanol solution, and the other steps were the same as in Example 1. The bionic biomineralized artificial bone of the present embodiment was tested to have an apparent density of 0.31 g/cm ³ and a porosity of 66%.

Example 7

In the present embodiment, the preparation of the biomimetic biomineralized artificial bone is carried out by using an aqueous solution of citric acid as an acid solution, an aqueous solution of CaSO _{4 in} an aqueous solution of calcium ions, and an aqueous solution of ammonia in an alkaline solution, and chemical crosslinking using genipin.

Preparing biomimetic biomineralized artificial bone according to substantially the same procedure as in Example 1, except that the acid solution in step S1-1 is replaced by 1000 mL of 0.01 mol/L citric acid instead of hydrochloric acid solution, in steps S1-4 and S2-3. The alkaline solution was replaced with aqueous ammonia in the aqueous solution of NaOH. After the step S3-2, the mineralized collagen composite porous material was chemically crosslinked using a 0.5 wt% aqueous solution of genipin, and the remaining steps were the same as in Example 1. The bionic biomineralized artificial bone of the present embodiment was tested to have an apparent density of 0.25 g/cm ³ and a porosity of 74%.

Example 8

This embodiment is the same as the first embodiment except that the step S2-1 and the step S2-2 are different from the first embodiment; the specific steps of the steps S2-1 and S2-2 are:

Step S2-1: Dissolving 1 g of collagen in 99 mL of purified water to prepare a solution of collagen, wherein the collagen concentration is 1%;

Step S2-2 (blending): continuously stirring the solution obtained in the step S2-1, adding 9 g of the biomineralized collagen/hydroxyapatite composite powder obtained in the step S1-5, stirring and mixing to form a mixed system;

The biomimetic biomineralized artificial bone prepared in this example is numbered as bionic bone 19.

Example 9

This embodiment is the same as Embodiment 1 except that Step S2-1 and Step S2-2 are different from Embodiment 1; The specific steps of S2-1 and S2-2 are:

Step S2-1: Dissolving 3 g of collagen in 97 mL of purified water to prepare a solution of collagen, wherein the collagen concentration is 3%;

Step S2-2 (blending): continuously stirring the solution obtained in the step S2-1, adding 7 g of the biomineralized collagen/hydroxyapatite composite powder prepared in the step S1-5, stirring and mixing to form a mixed system;

The biomimetic biomineralized artificial bone prepared in this example is numbered as bionic bone 37.

The crystal phase structure and crystallinity of biomimetic biomineralized artificial bone were studied by Focus X-ray diffraction (XRD).

3 is an XRD pattern of natural bone (porcine bone), hydroxyapatite (HA), and a sample prepared in Example 1 (Col-HA). Comparing the two maps of HA and Col-HA in Fig. 3, it is found that the diffraction peak of the biomimetic artificial bone is consistent with the diffraction peak spectrum of the standard hydroxyapatite crystal, indicating that the inorganic phase in the biomimetic artificial bone is mainly hydroxyl. apatite. Comparing the two maps of Col-HA and pig bone in Figure 3, it is found that the positions and numbers of the diffraction peaks of the biomimetic artificial bone and the natural bone are consistent, indicating that the inorganic phase in the composite is similar to the natural bone inorganic phase.

Figure 4 is an XRD pattern of collagen, hydroxyapatite, bionic bone 19, bionic bone 37, bionic bone 55 and natural rabbit bone. From Figure 4, the ratio of biomineralized collagen/hydroxyapatite composite powder is At 3:7 and 5:5, the crystal phase structure and crystallinity of natural bone are more similar, and the bionic effect is more obvious.

In the present application, the stability of the prepared biomimetic bone 19, bionic bone 37 and bionic bone 55 in the prepared biomimetic bone 19 was observed by immersion shaking in physiological saline to observe the stability of the hydroxyapatite. The specific steps are as follows:

1 g of bionic bone was placed in a test tube, 10 ml of physiological saline was added, and the mixture was allowed to stand at room temperature of 15 to 25 ° C for 24 hours, and shaken for 1 minute using a vortex mixer. After standing for 5 minutes, the white precipitate was observed at the bottom of the test tube, and a white precipitate was observed. Decomposed for hydroxyapatite. The experimental results showed that there was no white precipitate in bionic bone 55 and released bone 37, indicating that no hydroxyapatite was shed, and bone 19 was released, with a slight white precipitate. After drying calculation, the hydroxyapatite shedding rate was less than 10%.

It is to be noted that the above embodiments are only used to explain the implementation process and features of the present application, and do not limit the technical solutions of the present application. Although the present application is described in detail with reference to the above embodiments, those skilled in the art should understand that: Modifications or equivalent substitutions of the present application may be made without departing from the spirit and scope of the invention.

Claims

A biomimetic biomineralized artificial bone repair material, the material being a three-dimensional porous scaffold material comprising a crosslinked collagen matrix and a biomineralized collagen/hydroxyapatite composite powder encased in the matrix, the collagen The mass ratio of the matrix to the biomineralized collagen/hydroxyapatite composite powder is 9 to 1:1 to 9.
The biomimetic mineralized artificial bone repairing material according to claim 1, wherein a mass ratio of the collagen matrix to the biomineralized collagen/hydroxyapatite composite powder is from 1 to 5:5 to 9.
The biomimetic mineralized artificial bone repairing material according to claim 1, wherein a mass ratio of the collagen matrix to the biomineralized collagen/hydroxyapatite composite powder is from 3 to 5:5 to 7.
The biomimetic mineralized artificial bone repairing material according to any one of claims 1 to 3, wherein the biomimetic mineralized artificial bone repairing material has a pore diameter of 10 to 500 μm and a porosity of 50%. 97%.
The biomimetic mineralized artificial bone repairing material according to any one of claims 1 to 4, wherein a mass fraction of the hydroxyapatite in the biomineralized collagen/hydroxyapatite composite powder is 50 to 99% by weight. .
The biomimetic mineralized artificial bone repairing material according to any one of claims 1 to 5, wherein a mass fraction of the hydroxyapatite in the biomineralized collagen/hydroxyapatite composite powder is 80 to 99% by weight. .
The biomimetic mineralized artificial bone repairing material according to any one of claims 1 to 6, wherein the XRD pattern of the biomimetic mineralized artificial bone repairing material is substantially as shown in Figure 3 in col-HA or in Figure 4 Bone 19, bionic bone 37 or bionic bone 55 is shown.
The biomimetic mineralized artificial bone repairing material according to any one of claims 1 to 7, wherein the collagen matrix or the collagen in the biomineralized collagen/hydroxyapatite composite powder is an endopeptide Recombinant human type I collagen with native type I collagen and/or no terminal peptide.
The biomimetic mineralized artificial bone repairing material according to claim 8, wherein the natural type I collagen is an exopeptide collagen obtained by extracting an animal's skin and/or Achilles tendon and undergoing an enzymatic hydrolysis process. protein.
The method for preparing a biomimetic mineralized artificial bone repairing material according to any one of claims 1 to 9, the method comprising the steps of:

(a) providing a biomineralized collagen/hydroxyapatite composite powder and an aqueous collagen solution;

(b) adding the biomineralized collagen/hydroxyapatite composite powder to the aqueous collagen solution to form a mixed system, wherein the biomineralized collagen/hydroxyapatite composite powder and the collagen aqueous solution are The mass ratio of collagen is 9 to 1:1 to 9;

(c) adjusting the pH of the mixed system obtained in the step (b) to 7 to 9, and then stirring to form a mineralized collagen composite sol;

(d) freeze-drying the mineralized collagen composite sol to obtain a mineralized collagen composite porous material;

Optionally, the mineralized collagen composite sol obtained in the step (c) is pre-frozen to obtain a mineralized collagen jelly, and then the freeze-drying is performed;

(e) cross-linking the obtained mineralized collagen composite porous material to obtain the biomimetic mineralized artificial bone repairing material.
The method according to claim 10, wherein in step (a), the mass fraction of collagen in the aqueous collagen solution is, for example, 0.8% to 5%.
The method according to any one of claims 10 to 11, wherein in step (b), the mass ratio of the biomineralized collagen/hydroxyapatite composite powder to the collagen in the collagen aqueous solution is 1 to 5: 5 to 9, for example, 3 to 5: 5 to 7.
The method according to any one of claims 10 to 12, wherein the pH of the resulting mixed system is adjusted to 7 to 9 by using NaOH, KOH or aqueous ammonia in the step (c).
The method according to any one of claims 10 to 13, wherein the crosslinking in step (e) comprises physical crosslinking and/or chemical crosslinking.
The method of claim 14, wherein the physical crosslinking comprises one or more of ultraviolet radiation, thermal dehydrogenation, and irradiation sterilization crosslinking.
The method of claim 14 wherein

The chemical crosslinking includes the use of one or more of carbodiimide, diamine, epoxy compound, hydroxysuccinimide, diphenyl phosphate, glutaraldehyde, formaldehyde, glyoxylic acid, and genipin. After cross-linking, using a chemical cross-linking agent, the residual cross-linking agent is removed by an elution procedure.
The method according to any one of claims 10 to 16, wherein the biomineralized collagen/hydroxyapatite composite powder is prepared as follows:

(i) placing collagen in an acid to form an acidic aqueous solution of collagen; the mass fraction of collagen in the acidic aqueous solution is, for example, 0.01% to 2%, and the pH is 2.5 to 5.5;

(ii) slowly adding an aqueous solution containing calcium ions to the acidic aqueous solution of the collagen; in an acidic aqueous solution of the collagen, the amount of calcium ions added is, for example, 0.01 to 0.1 mol per gram of collagen;

(iii) adding a phosphoric acid solution to the solution obtained in step (ii), stirring and mixing to form a mixed system; the phosphoric acid solution is added in an amount such that the molar ratio of calcium ions to phosphate ions in the system is, for example, 1.5 to 2;

(iv) adjusting the pH of the system obtained in the step (iii) to 7 to 10 to obtain a white suspension;

(v) The white suspension obtained in the step (iii) is allowed to stand, for example, at 15 to 25 ° C for 22 to 26 hours, filtered, and the filter cake is freeze-dried, for example, sufficiently frozen at -50 to -20 ° C. Drying for 24 to 48 hours, followed by grinding the prepared dry powder is biomineralized collagen / hydroxyapatite composite powder;

The acid in the step (i) includes, for example, one or more of sulfuric acid, hydrochloric acid, nitric acid, acetic acid, propionic acid, and citric acid;

The aqueous solution containing calcium ions described in the step (ii) is formed, for example, by one or more of CaCl 2 , Ca(NO 3 ) 2 and CaCO 3 .
The method of claim 17 wherein said method comprises the steps of:

(S1) Preparation of a biomineralized collagen/hydroxyapatite composite powder comprising:

(S1-1) dissolving collagen in an acid solution to prepare an acidic aqueous solution of collagen; the mass fraction of collagen in the acidic aqueous solution is, for example, 0.01% to 2%, and the pH is 2.5 to 5.5;

(S1-2) continuously stirring the solution obtained in the step (S1-1), slowly adding an aqueous solution containing calcium ions; in an acidic aqueous solution of collagen, the amount of calcium ions added is, for example, 0.01 to 0.1 mol per gram of collagen;

(S1-3) continuously stirring the solution obtained in the step (S1-2), adding a phosphoric acid solution, stirring and mixing to form a mixed system; the amount of the phosphoric acid solution added depends on the amount of calcium ions added in the step (S1-2), so that the calcium The molar ratio of ions to phosphate ions is, for example, 1.5 to 2;

(S1-4) continuously stirring the solution obtained in the step (S1-3), adjusting the pH of the mixed system to 7 to 10 to obtain a white suspension;

(S1-5) The mixed system obtained in the step (S1-4) is allowed to stand, for example, at 15 to 25 ° C for 22 to 26 hours, filtered, and the filter cake is freeze-dried, for example, at -50 to -20 ° C. Fully freeze-dried for 24 to 48 hours, and then the dried powder obtained by grinding is a biomineralized collagen/hydroxyapatite composite powder;

(S2) a composite of collagen and biomineralized collagen/hydroxyapatite composite powder, comprising:

(S2-1) dissolving collagen in purified water to prepare an aqueous solution of collagen; the mass fraction of collagen in the aqueous collagen solution is, for example, 0.8% to 5%;

(S2-2) continuously stirring the solution obtained in the step (S2-1), adding the biomineralized collagen/hydroxyapatite composite powder obtained in the step (S1-5), stirring and mixing to form a mixed system, biomineralized collagen/ The ratio of the amount of the hydroxyapatite composite powder to the amount of collagen protein in the aqueous collagen solution obtained in the step (S2-1) is from 9:1 to 1:9, for example, from 1 to 5:5 to 9, and for example, 3 to 5:5~7;

(S2-3) continuously stirring the solution obtained in the step (S2-2), adjusting the pH of the mixed system to 7 to 9;

(S2-4) The mixed system obtained in the step (S2-3) is allowed to stand and stirred to form a mineralized collagen composite solution; for example, it is allowed to stand at 15 to 25 ° C for 10 to 48 hours, and stirred for 5 to 30 minutes to be thoroughly mixed. Evenly, forming a mineralized collagen composite solution;

(S3) Preparation of biomimetic biomineralized artificial bone repair material, comprising:

(S3-1) pre-freezing the mineralized collagen composite solution obtained in the step (S2-4), for example, pre-freezing at 1 to 5 ° C for 3 to 24 hours to obtain a mineralized collagen jelly;

(S3-2) freeze-drying the mineralized collagen of step (S3-1), for example, fully frozen at -50 to -20 °C Drying for 22 to 26 hours to obtain a mineralized collagen composite porous material;

(S3-3) The biomimetic collagen composite porous material is crosslinked by physical and/or chemical methods to obtain the biomimetic mineralized artificial bone repair material.
The bionic biomineralized artificial bone repairing material according to any one of claims 1 to 9 or the biomimetic biomineralized artificial bone repairing material prepared according to any one of claims 10 to 18 for preparing bone defects supporting human or animal Application in the implant at the location.