CN119367619A

CN119367619A - A high-strength artificial bone repair material and preparation method thereof

Info

Publication number: CN119367619A
Application number: CN202411597662.1A
Authority: CN
Inventors: 崔嵬; 洪东立; 陈进庆; 芮轩文
Original assignee: Changzhou Banglai Medical Technology Co ltd
Current assignee: Changzhou Banglai Medical Technology Co ltd
Priority date: 2024-11-08
Filing date: 2024-11-08
Publication date: 2025-01-28

Abstract

The invention discloses a high-strength artificial bone repair material and a preparation method thereof. The preparation method takes collagen and calcium phosphate as raw materials, and is formed into an artificial bone repair material through the following step S100 or step S200, wherein the step S100 comprises the steps of mixing a collagen solution and calcium phosphate, adding a cross-linking agent to obtain collagen hydrogel, compressing and removing free water in the hydrogel to reduce the volume of the hydrogel, and freeze-drying and forming, and the step S200 comprises the steps of uniformly mixing collagen and calcium phosphate to prepare calcium phosphate collagen composite particles, weighing the collagen composite particles and calcium phosphate powder, adding the collagen composite particles and the calcium phosphate powder into the collagen solution, adding the cross-linking agent to crosslink, and freeze-drying and forming. The preparation method obviously enhances the mechanical strength of the artificial bone repair material on the premise of ensuring excellent degradation performance and bioactivity.

Description

High-strength artificial bone repair material and preparation method thereof

Technical Field

The invention relates to a high-strength artificial bone repair material and a preparation method thereof, in particular to a high-strength collagen artificial bone repair material and a preparation method thereof.

Background

As a bone repair raw material, the tricalcium phosphate material has better degradation performance, can be chemically combined with bone after being implanted into a body, but still lacks sufficient biological activity, and cannot further promote the formation of new bone. Some researchers increase the bioactivity of calcium phosphate materials by compounding collagen molecules, which are important components of human bones, have excellent osteoinductive properties, and induce proliferation and differentiation of bone cells (see literature ：Jiang Q.S.,Wang L.R.and et al.Canine ACL reconstruction with an injectable hydroxyapatite/collagen paste for accelerated healing of tendon-bone interface.Bioact.Mater.2023,20:1-15).

However, the mechanical strength of collagen sponge is low, the compressive strength is generally not higher than 10kPa (see ：Yunoki S.,Ikoma T.and et al.Development of collagen condensation method to improve mechanical strength of tissue engineering scaffolds.Mater.Charact.2010,61:907-911.). cancellous bone strength of about 2-5MPa, the strength of collagen sponge is greatly different therefrom, though, by compounding with materials such as hydroxyapatite, the compression strength of the collagen sponge can be obviously improved by compression molding, but compression densification can lead to the reduction of the wettability of the collagen product to blood and prevent crawling and transportation of cells and nutrients, so that development of a collagen sponge porous bone repair material with the compression strength similar to that of cancellous bone is urgently needed.

Disclosure of Invention

The invention aims to provide a high-strength artificial bone repair material and a preparation method thereof, which obviously enhance the mechanical strength of the artificial bone repair material on the premise of ensuring excellent degradation performance and bioactivity.

The invention adopts the following technical scheme:

A preparation method of an artificial bone repair material takes collagen and calcium phosphate as raw materials, and the artificial bone repair material is formed through the following step S100 or step S200;

Step S100 comprises mixing collagen solution and calcium phosphate, adding a cross-linking agent to obtain collagen hydrogel, compressing to remove free water in the hydrogel to reduce the volume of the hydrogel, and freeze-drying to form;

Step S200 comprises the steps of uniformly mixing collagen and calcium phosphate to prepare calcium phosphate collagen composite particles, weighing the collagen composite particles and calcium phosphate powder, adding the collagen composite particles and the calcium phosphate powder into a collagen solution, adding a cross-linking agent for cross-linking, and freeze-drying and forming.

In a preferred embodiment, the freeze-drying molding comprises primary freeze-drying molding and secondary freeze-drying molding, the composite material after the primary freeze-drying molding is immersed in a cross-linking agent solution for secondary chemical cross-linking, and the composite material after the secondary chemical cross-linking is cleaned and then subjected to secondary freeze-drying molding, so that the high-strength artificial bone repair material is obtained.

In a more preferred embodiment, the secondary chemical crosslinking time is 6-48 hours, the cleaning is performed by placing the mixture into pure water for stirring and cleaning at 200-600 rpm for 6-24 hours, the crosslinking agent in the crosslinking agent solution comprises one or more of genipin, glutaraldehyde and carbodiimide, and the concentration of the crosslinking agent in the crosslinking agent solution is 0.05-5wt%.

In a preferred embodiment, in step S100, a cross-linking agent is added, freezing and cross-linking are performed at-40 to-20 ℃ for 0.5 to 7 days, and then the collagen white water gel is obtained after the temperature is restored to the room temperature, and the collagen gel is placed into a compression mold for compression and dehydration, wherein the volume of the collagen gel is compressed to 10 to 70 percent of the initial volume.

In a specific and preferred embodiment, step S100 specifically includes:

s101, weighing collagen, adding the collagen into an acid solution to obtain a collagen acid solution, adding calcium phosphate powder, uniformly mixing, adding a cross-linking agent, uniformly stirring to obtain slurry, injecting the slurry into a forming die, freezing for cross-linking, demolding, and recovering to room temperature to obtain collagen hydrogel;

step S102, placing the collagen hydrogel into a compression mold for compression dehydration, and then performing freeze drying to obtain a preliminarily molded composite material;

and step S103, immersing the preliminarily formed composite material in a cross-linking agent solution, carrying out secondary chemical cross-linking, cleaning the composite material subjected to secondary chemical cross-linking by pure water, and carrying out secondary freeze drying forming to obtain the high-strength artificial bone repair material.

In a more preferred embodiment, in step S101, the weight ratio of the collagen to the calcium phosphate powder is 0.05-1, the weight percentage of the collagen in the collagen acid solution is 5-10%, the concentration of the acid solution is 0.2-10 wt%, and the concentration of the crosslinking agent in the slurry is 10-500 ppm.

In a preferred embodiment, in step S200, calcium phosphate powder is added into an acid solution of collagen, and mixed uniformly, freeze-dried and mechanically crushed to obtain calcium phosphate collagen composite particles, wherein the mesh number of the composite particles is 20-300.

In a more preferred embodiment, in step S200, the collagen composite particles and the calcium phosphate powder are added into a collagen acid solution, and after being uniformly mixed, a cross-linking agent is added to be uniformly mixed to prepare a slurry, and the slurry is freeze-dried.

In another specific and preferred embodiment, step S200 specifically includes:

step S201, weighing collagen, adding the collagen into an acid solution to obtain a collagen acid solution, adding calcium phosphate powder, uniformly mixing, injecting the slurry into a forming die, and freeze-drying to obtain calcium phosphate collagen composite sponge, and obtaining calcium phosphate collagen composite particles by a mechanical crushing mode;

Step S202, weighing the calcium phosphate collagen composite particles, and adding the calcium phosphate collagen composite particles into an acid solution to obtain acidified calcium phosphate collagen;

Step S203, weighing collagen, adding the collagen into an acid solution to obtain a collagen acid solution, adding calcium phosphate powder and the calcium phosphate collagen acidified in the step S202, uniformly mixing, adding a cross-linking agent, uniformly stirring, injecting the slurry into a forming die, and freeze-drying to obtain a primary formed composite material;

And S204, immersing the preliminarily formed composite material in a cross-linking agent solution, carrying out secondary chemical cross-linking, cleaning the composite material subjected to secondary chemical cross-linking by pure water, and carrying out secondary freeze drying forming to obtain the high-strength artificial bone repair material.

In a more preferred embodiment, in the steps S201 and S203, the weight ratio of the collagen to the calcium phosphate powder is 0.05-1, the weight percentage of the collagen in the collagen acid solution is 5-10%, the concentration of the acid solution is 0.2-10 wt%, the weight ratio of the calcium phosphate collagen composite particles to the acid solution is 0.5-3, the weight ratio of the collagen to the acidified calcium phosphate collagen is 0.03-0.2, and the concentration of the chemical cross-linking agent in the slurry is 10-500 ppm.

In a preferred embodiment, the collagen comprises one or more selected from the group consisting of type I collagen, type II collagen and recombinant collagen, the weight average molecular weight is 50000-300000Da, the acid solution comprises hydrochloric acid solution or acetic acid solution, the calcium phosphate comprises one or more selected from the group consisting of tricalcium phosphate, magnesium-containing tricalcium phosphate, hydroxyapatite and octacalcium phosphate, and the cross-linking agent comprises genipin and/or glutaraldehyde.

The principle of improving the mechanical strength of the artificial bone repair material is that the densification of a product is realized by regulating the crosslinking state of collagen hydrogel and slowly compressing and removing free water, the inventor discovers that partial free water can appear in the collagen hydrogel in a specific stage of the crosslinking process, the collagen hydrogel can be dehydrated in a compressing mode and the density of the product can be improved, the rest of bound water can be redistributed in the compressing process to help the fixation of compressed shape in the freeze drying process, or the collagen and calcium phosphate composite material can be mechanically crushed to obtain composite particles, the composite particles are added into a mixed solution of the collagen and the calcium phosphate after surface acid treatment and activation, and the surface of the solid composite particles is dissolved and is compounded with protein molecules in the solution under the action of a crosslinking agent to form the high-density collagen/tricalcium phosphate composite product.

The invention also adopts the following technical scheme:

an artificial bone repair material prepared according to the above-described preparation method.

In a preferred embodiment, the artificial bone repair material has a porosity of 20-80% and a compressive strength of 0.5-2 MPa.

Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:

The artificial bone repair material prepared by the method has the advantages that the compactness is remarkably improved, the compressive strength is improved by 10-50 times, the degradation time after being implanted into a human body can be effectively prolonged, and the new bone growth rate can be updated and matched.

Drawings

In order to more clearly illustrate the technical solutions of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

Fig. 1 is a photograph of an artificial bone repair material prepared in example 1.

Fig. 2 is a microscopic morphology of the artificial bone repair material prepared in example 1.

Fig. 3 is a bar graph showing compressive strength of the artificial bone repair materials prepared in example 1 and comparative example 1.

Fig. 4 is a photograph of an anti-enzymatic test (30 days, 37 ℃) of the artificial bone repair materials prepared in example 1 and example 3.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the attached drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.

The traditional bone repair material mainly comprises collagen and hydroxyapatite powder, wherein the content of the hydroxyapatite is about 55%, the collagen powder and the hydroxyapatite are blended by the process, and then the mixture is subjected to hot press molding at 90-130 ℃, and the product has obvious defects in the aspects of degradation performance (difficult degradation of the hydroxyapatite in vivo), pore structure (compact hot press molding) and the like.

The traditional bone repair material preparation technology also comprises a collagen/hydroxyapatite composite freeze-drying forming method and a porous tricalcium phosphate ceramic surface perfusion collagen freeze-drying technology. The content of nano hydroxyapatite powder used in the collagen/hydroxyapatite composite freeze-drying forming method is 20-80%, higher ceramic phase content cannot be realized, and hydroxyapatite is difficult to degrade in vivo. The main body of the porous tricalcium phosphate ceramic surface impregnated collagen freeze-drying technology is sintered calcium phosphate ceramic, the toughness of the matrix is insufficient, and the special shaping technology of the collagen sponge cannot be achieved.

The applicant also provides an improved artificial bone repair material, which adopts tricalcium phosphate powder raw material, has higher bioactivity, and fully degrades in vivo magnesium-containing tricalcium phosphate material and collagen molecules to form a new generation of degradable artificial bone repair material with high bioactivity. However, in the scheme, the dissolution concentration of the bovine type I collagen in the acid solution is difficult to exceed 10mg/mL, and the compactness and mechanical strength of the collagen/tricalcium phosphate composite material are severely restricted.

The dissolution concentration of bovine type I collagen in an acid solution is difficult to exceed 10mg/mL, so that the addition amount of the collagen/tricalcium phosphate composite material obtained after freeze drying is severely restricted. The degradation time of collagen molecules in vivo is generally shorter, the degradation time of bovine type I collagen (about 300 kDa) with higher molecular weight is difficult to exceed three months, and the regeneration and repair time of bone defects is generally more than 6 months. Increasing the density of collagen sponge can effectively prolong the degradation time of collagen sponge, but the solubility of collagen material is limited.

In view of the above problems and phenomena, the inventors innovatively found a metastable state of collagen in a chemical crosslinking process, free water in the collagen can be removed by extrusion in the metastable state, and the residual bound water can be transformed and distributed in the collagen sponge so that the collagen sponge remodels a crosslinked network, thereby achieving the aim of compressing and densifying a sponge network structure. The prepared surface-acidified calcium phosphate collagen composite particles and calcium phosphate can be added into collagen solution, so that the compactness of the artificial bone repair material is greatly improved, the compressive strength of the material is obviously enhanced, and the degradation time of the bone repair material in vivo is prolonged.

Further, compared with the traditional collagen compacting and forming mode, the high-strength collagen artificial bone repair material formed by two times has high crosslinking degree, and the in-vivo stability is greatly enhanced.

The first preparation method of the artificial bone repair material of the embodiment comprises the steps of mixing collagen solution with calcium phosphate, adding a chemical crosslinking agent for freezing and crosslinking, recovering to room temperature to obtain collagen hydrogel, slowly compressing to remove free moisture and reduce the volume of the hydrogel, and finally freeze-drying and molding.

The first preparation method specifically comprises the following steps:

(1-1) weighing collagen, adding the collagen into an acid solution to obtain a collagen acid solution, adding calcium phosphate powder, and uniformly mixing. Then adding the cross-linking agent and stirring uniformly. The slurry is injected into a forming die and slightly oscillated until the liquid level is flat. And (3) putting the mould into a freezing refrigerator for freezing and crosslinking, and then demoulding and recovering to room temperature to obtain the collagen hydrogel.

(1-2) Placing the frozen crosslinked collagen hydrogel into a compression mold for compression dehydration, and then freeze-drying to obtain the preliminarily molded high-strength collagen sponge.

(1-3) Immersing the preliminarily formed high-strength collagen sponge in a cross-linking agent solution for the second chemical cross-linking. And (3) putting the crosslinked composite material into pure water, stirring and cleaning, and then freeze-drying and forming to obtain the high-strength collagen artificial bone repair material.

The step (1-1) comprises the steps of weighing collagen and an acid solution by using an analytical balance, adding the collagen into the acid solution, and stirring for 10-30 minutes by using a tetrafluoro stirring paddle at 200-600 rpm to completely dissolve the collagen. And weighing the calcium phosphate powder by using an analytical balance, adding all the calcium phosphate powder into the collagen acid solution, and continuously stirring for 5-20 minutes until the calcium phosphate powder is uniformly mixed to obtain mixed slurry. And then adding the cross-linking agent into the mixed slurry, and stirring for 1-5 minutes. And injecting the mixed slurry into a mold, and oscillating the mold to level the slurry level. Crosslinking for 0.5-7 days at a low temperature in a freezing refrigerator at-40 to-20 ℃, demolding and recovering to room temperature to obtain the collagen hydrogel.

The collagen used in step (1-1) includes, but is not limited to, type I collagen, type II collagen, recombinant collagen, etc., and has a weight average molecular weight of 50000-300000Da. Acid solutions include, but are not limited to, hydrochloric acid solutions, acetic acid solutions, and the like. The calcium phosphate powder used includes, but is not limited to, tricalcium phosphate, magnesium-containing tricalcium phosphate, hydroxyapatite, octacalcium phosphate, and the like. Crosslinking agents include, but are not limited to, genipin, glutaraldehyde, and the like. The weight ratio of the weighed collagen to the calcium phosphate powder is 0.05-1, the weight percentage of the collagen in the collagen acid solution is 5-10%, the acid concentration in the acid solution is 0.2-10wt%, and the concentration of the crosslinking agent in the mixed slurry is 10-500 ppm.

The process of the step (1-2) comprises the steps of putting the collagen hydrogel obtained by freezing and crosslinking into a compression mold for compression and dehydration, and then freezing and drying to obtain the preliminarily molded high-strength collagen sponge. The volume of the collagen hydrogel is compressed to 10-70% of the initial volume, preferably 15-50% of the initial volume, and further 20-35% of the initial submission.

The step (1-3) comprises the specific steps of immersing the high-strength collagen sponge in a cross-linking agent solution, and carrying out secondary chemical cross-linking for 6-48 hours. And (3) placing the crosslinked collagen sponge into pure water at 200-600 rpm, stirring and cleaning for 6-24 hours, and then performing freeze drying treatment to obtain the high-strength collagen artificial bone repair material. Wherein the cross-linking agent comprises, but is not limited to, genipin, glutaraldehyde, carbodiimide and the like, and the concentration is 0.05-5wt%.

In the preparation method, the discovery that the collagen sponge is in a metastable state in the chemical crosslinking process is utilized, free water in the collagen can be removed by extrusion in the metastable state, and the residual bound water can be transformed and distributed in the sponge so that the collagen sponge remodels a crosslinked network, thereby achieving the aim of compression densification of a sponge network structure. In addition, the high-strength collagen artificial bone repair material formed by two times has high crosslinking degree, and the in-vivo stability is greatly enhanced. Compared with the collagen sponge prepared by the conventional preparation method, the collagen sponge has the advantages that the density is remarkably improved, the compressive strength is improved by 10-50 times, the degradation time after the collagen sponge is implanted into a human body can be effectively prolonged, and the collagen sponge can be more matched with the growth rate of new bones.

The second preparation method of the artificial bone repair material of the embodiment comprises the steps of mixing a collagen solution with calcium phosphate, freeze-drying, mechanically crushing to obtain calcium phosphate collagen composite particles, acidizing the calcium phosphate collagen composite particles, adding the calcium phosphate collagen composite particles into the mixed solution of collagen and calcium phosphate, adding a cross-linking agent for low-temperature cross-linking, and finally freeze-drying to form the artificial bone repair material.

The second preparation method specifically comprises the following steps:

And (2-1) weighing the collagen, adding the collagen into an acid solution to obtain a collagen acid solution, and adding calcium phosphate powder for uniform mixing. And (3) injecting the slurry into a forming die, performing freeze drying to obtain the calcium phosphate collagen composite sponge, and obtaining the calcium phosphate collagen composite powder by a mechanical crushing mode.

(2-2) Weighing the calcium phosphate collagen composite particles, and adding the calcium phosphate collagen composite particles into an acid solution to obtain acidified calcium phosphate collagen;

And (2-3) weighing the collagen and adding the collagen into an acid solution to obtain a collagen acid solution, sequentially adding the acidified calcium phosphate collagen and calcium phosphate powder, uniformly mixing, adding a cross-linking agent, and uniformly stirring. The slurry is injected into a forming die and slightly oscillated until the liquid level is flat. And freeze-drying to obtain the preliminarily molded high-strength collagen artificial bone repair material.

(2-4) Immersing the preliminarily formed high-strength collagen artificial bone repair material in a cross-linking agent solution for a second chemical cross-linking. And (3) putting the crosslinked composite material into pure water, stirring and cleaning, and then freeze-drying and forming to obtain the high-strength collagen artificial bone repair material.

The step (2-1) comprises the steps of weighing collagen and an acid solution by using an analytical balance, adding collagen sponge into the acid solution, and stirring for 10-30 minutes by using a tetrafluoro stirring paddle at 200-600 rpm to completely dissolve. And weighing the calcium phosphate powder by using an analytical balance, adding the calcium phosphate powder into the collagen acid solution, and continuously stirring for 5-20 minutes until the calcium phosphate powder is uniformly mixed. And then freezing for 2-6 hours in a refrigerator at-20 to-40 ℃ and then freeze-drying to obtain the calcium phosphate collagen composite sponge. Crushing the calcium phosphate collagen composite sponge by a granulator and obtaining composite material particles with 20-300 meshes.

The collagen used in step (2-1) includes, but is not limited to, type I collagen, type II collagen, recombinant collagen, etc., and has a weight average molecular weight of 50000-300000Da. Acid solutions include, but are not limited to, hydrochloric acid solutions, acetic acid solutions, and the like. The calcium phosphate powder used includes, but is not limited to, tricalcium phosphate, magnesium-containing tricalcium phosphate, hydroxyapatite, octacalcium phosphate, and the like. Wherein the mass ratio of the collagen to the calcium phosphate powder is 0.05-1, the mass percentage of the collagen in the collagen acid solution is 5-10%, and the acid concentration in the acid solution is 0.2-10wt%.

In the step (2-2), the calcium phosphate collagen composite particles are weighed by using an analytical balance and acidified by adding an acid solution, so that the crosslinking activity of collagen molecules on the surface of the calcium phosphate collagen composite powder is increased. The acid solution comprises, but is not limited to, hydrochloric acid solution and acetic acid solution, wherein the concentration of the acid solution is 0.2-10wt%, and the mass ratio of the calcium phosphate collagen composite particles to the acid solution is 0.5-3.

The step (2-3) comprises the steps of weighing collagen and an acid solution by using an analytical balance, adding the collagen into the acid solution, and stirring for 10-30 minutes by using a tetrafluoro stirring paddle at 200-600 rpm to completely dissolve the collagen. And then weighing the calcium phosphate collagen composite particles and the calcium phosphate powder, adding the collagen acid solution, and continuously stirring for 5-20 minutes until the mixture is uniformly mixed. And then adding the cross-linking agent into the mixed slurry, and stirring for 1-5 minutes. And injecting the mixed slurry into a mold, and oscillating the mold to level the slurry level. Freezing for 2-6 hours in a refrigerator at the temperature of minus 20 to minus 40 ℃ and freeze-drying to obtain the preliminarily molded high-strength collagen artificial bone repair material.

In the step (2-3), the mass ratio of the collagen to the calcium phosphate powder is 0.05-1, the mass ratio of the collagen to the calcium phosphate collagen composite particles is 0.03-0.2, the mass percentage of the collagen in the collagen acid solution is 5-10%, the concentration of the acid solution is 0.2-10wt%, and the concentration of the crosslinking agent in the slurry is 10-500 ppm.

The step (2-4) comprises the steps of soaking the preliminarily molded high-strength collagen artificial bone repair material in a cross-linking agent solution, performing secondary chemical cross-linking for 6-48 hours, putting into pure water for stirring and cleaning for 6-24 hours at 200-600 rpm, and then performing freeze-drying treatment to obtain the high-strength collagen artificial bone repair material. Wherein the cross-linking agent comprises, but is not limited to, genipin, glutaraldehyde, carbodiimide and the like, and the concentration is 0.05-5wt%.

In the second preparation method, the prepared surface-acidified calcium phosphate collagen composite particles are added into the mixed solution of collagen and calcium phosphate, so that the compactness of the artificial bone repair material is greatly improved, and the compressive strength of the material is remarkably enhanced. In addition, the high-strength collagen artificial bone repair material formed by two times has high crosslinking degree, and the in-vivo stability is greatly enhanced.

Preferably, the calcium phosphate powder used in the above steps is selected from tricalcium phosphate powder containing magnesium or not containing magnesium. The tricalcium phosphate powder material is prepared by dissolving calcium salt and magnesium salt in water to obtain a first solution, wherein the concentration of the calcium salt is 1-100 g/L, and the concentration of the magnesium salt is 0.4-40 g/L. Further, the molar ratio of calcium ions to magnesium ions is 6-10, phosphate is dissolved in water, pH is regulated to 9.5-10.5 by using alkali liquor (preferably ammonia water or sodium hydroxide) to obtain a second solution, the concentration of the phosphate is 1-100 g/L, the first solution and the second solution with equal volumes are heated to 60-95 ℃ and then mixed, wet chemical reaction is carried out under normal pressure at 60-95 ℃ and under stirring conditions, wherein the stirring speed is 100-500 rpm, the molar ratio of calcium ions to phosphate ions is 1.1-1.5, the suspension after the wet chemical reaction is centrifugally separated to obtain precipitate, the precipitate after washing is centrifugally washed by water for 4-8 times, the washed precipitate is added into water with the mass of 5-20 times, stirring is uniformly carried out to form slurry, the slurry is processed into porous tricalcium phosphate powder by using a spray dryer, the air inlet temperature of the spray dryer is 240-300 ℃, the air outlet temperature is 120-150 ℃, and the feeding speed is 30-200 mL/min.

The calcium salt is selected from one or more of anhydrous calcium chloride, calcium chloride dihydrate and calcium nitrate tetrahydrate, the magnesium salt is selected from magnesium chloride hexahydrate and/or magnesium nitrate hexahydrate, and the phosphate is selected from one or more of disodium hydrogen phosphate, diammonium hydrogen phosphate and sodium hydrogen phosphate. The microcosmic porous tricalcium phosphate powder has a porous structure, wherein the magnesium element with specific content shows better bioactivity, and can effectively promote new bone formation. Compared with hydroxyapatite, the porous tricalcium phosphate powder material has better degradation performance, so that the porous tricalcium phosphate powder material has remarkable advantages in application of bone repair materials.

The artificial bone repair material prepared by the method solves the following problems:

(1) Insufficient density and compressive strength

And the density of the composite material is improved to 2-8 times by compression dehydration in the crosslinking process. Creatively discovers a metastable state of the collagen sponge in the chemical crosslinking process, free water in the collagen can be removed by extrusion under the metastable state, and the residual bound water can be transformed and distributed in the sponge so that the collagen sponge remodels a crosslinked network, thereby achieving the aim of compressing and densifying the sponge network structure. The surface-acidified calcium phosphate collagen composite powder prepared in advance can be added into a mixed collagen solution, and under the action of a cross-linking agent, the surface of the solid composite particles is dissolved and is compounded with collagen molecules in the solution to form a high-density collagen/calcium phosphate composite product, so that the compactness of the artificial bone repair material is greatly improved, and the compressive strength of the material is remarkably enhanced.

(2) Too fast degradation time

By greatly increasing the density of the collagen/tricalcium phosphate composite material, the effective extension of the degradation time in vivo is realized. Compared with the traditional sponge compaction molding mode, the high-strength collagen artificial bone repair material formed by two times has high crosslinking degree, and the in-vivo stability is greatly enhanced.

The porosity of the prepared high-strength collagen artificial bone repair material is 20-80%, the compressive strength is 0.5-2 mpa, the compressive strength is improved by 10-50 times, the degradation time after being implanted into a human body can be effectively prolonged, and the high-strength collagen artificial bone repair material can be more matched with the growth rate of new bones.

The magnesium-containing tricalcium phosphate powder used in the following examples and comparative examples was prepared by the following steps:

Weighing and dissolving 900g of anhydrous calcium chloride and 270g of magnesium chloride hexahydrate, dissolving in 10L of water to obtain a solution A, weighing 830g of diammonium phosphate, dissolving in 10L of water, and regulating the pH value of the solution to 10.0 by using ammonia water to obtain a solution B.

Heating and mixing, namely heating the solution A and the solution B to 70 ℃ and mixing, and continuously stirring at a rotating speed of 100rpm to form a suspension.

Wet chemical reaction the mixed suspension was heated at 70 ℃ and stirred at 100rpm for 12 hours.

And (3) centrifugal cleaning, namely centrifuging the suspension subjected to wet chemical reaction at 2000rpm for 3 minutes to obtain a precipitate, and adding water for centrifugal cleaning for 5 times.

Spray drying, namely adding the cleaned precipitate into 10L of water, and stirring uniformly to form slurry. Setting the air inlet temperature of a spray dryer to be 250 ℃, setting the air outlet temperature to be 120 ℃, and spray-drying the slurry at the speed of 50mL/min to obtain the final tricalcium phosphate powder, wherein the particle size distribution is in the range of 1-20 mu m.

Example 1

Preparation of calcium phosphate collagen composite particles 1g of bovine type I collagen and 10g of acetic acid solution having a concentration of 1wt% were weighed using an analytical balance, and collagen sponge was added to the acetic acid solution and stirred for 30 minutes to complete dissolution using a tetrafluoro stirrer at 500 rpm. 5.5g of magnesium-containing tricalcium phosphate powder was weighed using an analytical balance, and the magnesium-containing tricalcium phosphate powder was added to the collagen solution in its entirety, followed by continuous stirring for 10 minutes until uniformly mixed. Then, the calcium phosphate collagen composite sponge is obtained by freeze-drying after being frozen in a refrigerator at-40 ℃ for 3 hours. Crushing the calcium phosphate collagen composite sponge by a granulator and obtaining powder particles with 20-300 meshes.

Weighing and dissolving, namely weighing 0.3g of bovine type I collagen and 30g of 1wt% acetic acid solution by using an analytical balance, adding collagen sponge into the acetic acid solution, and stirring for 30 minutes by using a tetrafluoro stirring paddle at 500rpm to completely dissolve. And 6g of calcium phosphate collagen composite particles are weighed, added into 3g of 1wt% acetic acid solution for acidification, and added into collagen acid solution after acidification.

The mixing configuration was such that 1.5g of magnesium-containing tricalcium phosphate was added and stirred for 10 minutes to complete dissolution using a tetrafluoro-stirrer at 500 rpm. To the mixed solution was added 3ml of a 1wt.% glutaraldehyde solution. Stirring was carried out for 1 minute using a tetrafluoro stirrer at 500 rpm.

Injection molding, namely injecting the mixed slurry into a mold, and oscillating the mold to level the slurry liquid surface. Freezing in a refrigerator at-40 ℃ for 6 hours, and then freeze-drying to obtain the preliminarily molded high-strength collagen artificial bone repair material.

Crosslinking cleaning, namely immersing the composite material in 1wt.% glutaraldehyde solution for 12 hours, putting the composite material into pure water, stirring and cleaning at 400rpm for 12 hours.

And freeze drying, namely putting the cleaned sample into a refrigerator at the temperature of-40 ℃ to freeze for 6 hours, and then freeze drying for 48 hours to finally obtain the high-strength collagen artificial bone repair material (the appearance photo is shown in figure 1).

The microscopic morphology of the prepared high-strength collagen artificial bone repair material is detected as shown in figure 2. The compressive strength was measured to be 1560kPa, as shown on the right side in FIG. 3. After soaking for 30 days at 37 ℃ with 120 mug/mL type I collagenase, the high-strength collagen artificial bone repair material subjected to the two-time crosslinking in the embodiment has no obvious degradation and has higher biological stability (see the left side of fig. 4).

Example 2

Weighing and dissolving, namely weighing 1g of bovine type I collagen and 100g of 1wt% acetic acid solution by using an analytical balance, adding collagen sponge into the acetic acid solution, and stirring for 30 minutes to complete dissolution by using a tetrafluoro stirring paddle at 400 rpm.

The mixing was carried out by adding 6g of tricalcium phosphate containing magnesium and stirring at 400rpm using a tetrafluoro stirring paddle for 10 minutes to complete dissolution. To the mixed solution was added 1mL of a 1wt.% glutaraldehyde solution. Stirring was carried out for 1 minute using a tetrafluoro stirrer at 400 rpm.

Freezing and crosslinking, namely injecting the mixed slurry into a mold, and oscillating the mold to level the liquid level of the slurry. And freezing in a refrigerator at-20 ℃ for 7 days, taking out and recovering to room temperature to obtain the collagen sponge hydrogel.

Compression dehydration, namely placing the collagen hydrogel obtained by freezing and crosslinking into a compression mold to be compressed to 30% of the initial height, reducing the weight to 30% of the initial height after dehydration, and freeze-drying to obtain the collagen sponge material.

Crosslinking cleaning, namely immersing the collagen sponge in a glutaraldehyde solution with the concentration of 1wt.% for 6 hours, putting the collagen sponge into pure water, stirring and cleaning at 400rpm for 6 hours.

And freeze-drying, namely putting the cleaned sample into a refrigerator at the temperature of-40 ℃ for freezing for 3 hours, and then freeze-drying for 36 hours to finally obtain the high-strength collagen artificial bone repair material, wherein the compressive strength is measured to be 420kPa.

Example 3

Injection molding, namely injecting the mixed slurry into a mold, and oscillating the mold to level the slurry liquid surface. Freezing in a refrigerator at-40 ℃ for 6 hours, and freeze-drying to obtain the artificial bone repair material. The single crosslinked artificial bone repair material of this example showed partial degradation after soaking for 30 days with 120 μg/mL type I collagenase at 37 ℃ (see right side of fig. 4).

Comparative example

Weighing and dissolving, namely weighing 0.5g of bovine type I collagen sponge by using an analytical balance, weighing 4.5g of tricalcium phosphate powder containing magnesium, weighing 40g of 1wt.% acetic acid solution by using the balance, and adding the collagen sponge into the solution completely. Stirring was carried out for 30 minutes using a tetrafluoro stirrer at 400 rpm.

The mixing configuration was such that the entire magnesium-containing tricalcium phosphate powder was added to the collagen solution, and stirred at 400rpm using a tetrafluoro stirring paddle for 10 minutes. To the mixed solution was added 0.5ml of a 1wt.% glutaraldehyde solution. Stirring was carried out for 1 minute using a tetrafluoro stirrer at 400 rpm.

Injection molding, namely injecting the mixed slurry into a mold, and oscillating the mold to level the slurry level. And (3) crosslinking for 1 hour at the temperature of-4 ℃, then putting the die into a low-temperature storage cabinet at the temperature of-40 ℃ for 4 hours, demolding, and taking out the preliminarily molded composite material.

Crosslinking cleaning, namely immersing the composite material in a glutaraldehyde solution with the concentration of 1wt.% for 6 hours, putting the composite material into pure water, stirring and cleaning at 400rpm for 6 hours.

And freeze drying, namely putting the cleaned sample into a refrigerator at the temperature of-40 ℃ to freeze for 6 hours, and then freeze drying for 36 hours to finally obtain the high-strength collagen artificial bone repair material.

The compressive strength of the prepared artificial bone repair material was measured to be 34kPa, which is far lower than that of example 1, as shown on the left side of fig. 3.

As used in this specification and in the claims, the terms "comprises" and "comprising" merely indicate that the steps and elements are explicitly identified, and do not constitute an exclusive list, as other steps or elements may be included in a method or apparatus. The term "and/or" as used herein includes any combination of one or more of the associated listed items.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. If a definition used herein contradicts or is inconsistent with a definition set forth in other publications, the definition used herein should prevail.

The above-described embodiments are provided for illustrating the technical concept and features of the present invention, and are intended to be preferred embodiments for those skilled in the art to understand the present invention and implement the same according to the present invention, not to limit the scope of the present invention. All equivalent changes or modifications made according to the principles of the present invention should be construed to be included within the scope of the present invention.

Claims

1. The preparation method of the artificial bone repair material is characterized in that collagen and calcium phosphate are used as raw materials, and the artificial bone repair material is formed through the following step S100 or step S200;

2. The method according to claim 1, wherein the freeze-drying molding comprises a primary freeze-drying molding and a secondary freeze-drying molding, wherein the composite material after the primary freeze-drying molding is immersed in a cross-linking agent solution for secondary chemical cross-linking, and the composite material after the secondary chemical cross-linking is washed and then subjected to secondary freeze-drying molding, so as to obtain the high-strength artificial bone repair material.

3. The method according to claim 2, wherein the secondary chemical crosslinking is performed for 6 to 48 hours, the washing is performed by stirring and washing in pure water at 200 to 600rpm for 6 to 24 hours, the crosslinking agent in the crosslinking agent solution comprises one or more selected from genipin, glutaraldehyde and carbodiimide, and the concentration of the crosslinking agent in the crosslinking agent solution is 0.05 to 5wt%.

4. The preparation method according to claim 1, wherein in the step S100, the collagen white water gel is obtained after the cross-linking agent is added and frozen and cross-linked for 0.5 to 7 days at-40 to-20 ℃ and the temperature is restored to the room temperature, and the collagen hydrogel is placed into a compression mold for compression dehydration, wherein the volume of the collagen hydrogel is compressed to 10 to 70% of the initial volume.

5. The method according to claim 4, wherein step S100 specifically comprises:

6. The preparation method of claim 5, wherein in step S101, the weight ratio of collagen to calcium phosphate powder is 0.05-1, the weight percentage of collagen in the collagen acid solution is 5-10%, the concentration of the acid solution is 0.2-10 wt%, and the concentration of the crosslinking agent in the slurry is 10-500 ppm.

7. The preparation method of claim 1, wherein in step S200, calcium phosphate powder is added into an acid solution of collagen, and the mixture is uniformly mixed, freeze-dried and mechanically crushed to obtain calcium phosphate collagen composite particles, wherein the mesh number of the composite particles is 20-300.

8. The method according to claim 7, wherein in step S200, the collagen composite particles and the calcium phosphate powder are added into a collagen acid solution, and the slurry is prepared by adding a crosslinking agent after mixing uniformly, and freeze-drying.

9. The method according to claim 8, wherein step S200 specifically comprises:

10. The preparation method according to claim 9, wherein in the steps S201 and S203, the weight ratio of collagen to calcium phosphate powder is 0.05-1, the weight percentage of collagen in the collagen acid solution is 5-10%, the concentration of the acid solution is 0.2-10%, the weight ratio of the calcium phosphate collagen composite particles to the acid solution is 0.5-3, the weight ratio of collagen to the acidified calcium phosphate collagen is 0.03-0.2, and the concentration of the chemical cross-linking agent in the slurry is 10-500 ppm.

11. The preparation method according to any one of claims 1 to 9, wherein the collagen comprises one or more selected from the group consisting of type I collagen, type II collagen and recombinant collagen, the weight average molecular weight is 50000-300000 da, the acid solution comprises a hydrochloric acid solution or an acetic acid solution, the calcium phosphate comprises one or more selected from the group consisting of tricalcium phosphate, magnesium-containing tricalcium phosphate, hydroxyapatite and octacalcium phosphate, and the crosslinking agent comprises genipin and/or glutaraldehyde.

12. An artificial bone repair material, characterized in that it is produced according to the production method as claimed in any one of claims 1 to 11.