CN115177787B

CN115177787B - 3D printing composite bone repair material and preparation method and application thereof

Info

Publication number: CN115177787B
Application number: CN202210910869.4A
Authority: CN
Inventors: 何志敏; 宋天喜; 朱金亮; 崔云; 胡艳丽; 胡刚; 仇志烨; 吴晶晶
Original assignee: Beijing Aojing Health Technology Co ltd; Aojing Medical Technology Co ltd
Current assignee: Aojing Medical Technology Co ltd; Weifang Aojing Health Technology Co ltd
Priority date: 2022-07-29
Filing date: 2022-07-29
Publication date: 2023-07-28
Anticipated expiration: 2042-07-29
Also published as: CN115177787A

Abstract

The invention provides a 3D printing composite bone repair material, a preparation method and application thereof, which are applied to the technical field of bone repair materials, wherein the 3D printing composite bone repair material comprises an inner layer material and/or an outer layer material; the inner layer material and the outer layer material are obtained by 3D printing by taking the first biological ink and the second biological ink as raw materials; the first biological ink is prepared from mineralized collagen porous calcium phosphate composite material and high polymer solution; the second biological ink is prepared from mineralized collagen porous calcium phosphate composite material, bioactive material and a first solvent. The 3D printing composite bone repair material provided by the invention can be customized and printed according to the actual bone injury condition.

Description

3D printing composite bone repair material and preparation method and application thereof

Technical Field

The invention relates to the technical field of bone repair materials, in particular to a 3D printing composite bone repair material, and a preparation method and application thereof.

Background

The existing implant materials for bone defect treatment have insufficient bone inducibility, the degradation speed cannot be matched with the growth speed of bone cells, and the customized material only has single strength and structure, so that the bone damage condition of the simultaneous existence of cortical bone and cancellous bone defects is difficult to meet, and the difficult problems of personalized customization and the like cannot be realized according to the actual condition of the bone defects, so that development of a composite bone repair material with different strengths and structures is urgently needed to meet the requirements of novel bone repair materials of bone damage under different conditions.

Disclosure of Invention

In order to solve one or more technical problems in the prior art, the invention provides a 3D printing composite bone repair material, a preparation method and application thereof, wherein the composite bone repair material with different strength and structure can be prepared through 3D printing according to actual bone defect conditions, and customized printing can be realized according to different requirements.

The present invention provides in a first aspect a 3D printed composite bone repair material comprising an inner layer material and/or an outer layer material;

the inner layer material and the outer layer material are obtained by 3D printing by taking the first biological ink and the second biological ink as raw materials;

the first biological ink is prepared from mineralized collagen porous calcium phosphate composite material and high polymer solution;

the second biological ink is prepared from mineralized collagen porous calcium phosphate composite material, bioactive material and a first solvent.

Preferably, in the inner layer material, the mass ratio of the first bio-ink to the second bio-ink is 1 (1.5-9).

Preferably, in the outer layer material, the mass ratio of the first bio-ink to the second bio-ink is (1.5 to 9): 1.

Preferably, the high polymer solution is prepared by dissolving a high polymer in a second solvent;

the dosage ratio of the high polymer to the second solvent is 1g (5-12) mL;

in the first biological ink, the mass ratio of the mineralized collagen porous calcium phosphate composite material to the polymer is 1 (1.5-9).

Preferably, the high polymer is one or more of polylactic acid, polycaprolactone and polyvinyl alcohol;

the second solvent is one or more of 1, 4-dioxane, acetone, chloroform and dichloromethane, preferably 1, 4-dioxane.

Preferably, in the second biological ink, the mineralized collagen porous calcium phosphate composite material accounts for 80 to 99.5 percent of the total mass of the mineralized collagen porous calcium phosphate composite material and the bioactive material, and the total mass of the bioactive material accounts for 0.5 to 20 percent, preferably 1 to 6 percent;

in the second biological ink, the total mass of the mineralized collagen porous calcium phosphate composite material and the bioactive material and the volume ratio of the first solvent are 1g (10-20) mL.

Preferably, the bioactive material is one of concentrated growth factor, transforming growth factor-beta, bone morphogenic protein;

The first solvent is purified water, normal saline or sterilized water for injection.

Preferably, the compressive strength of the outer layer material is 10-20 MPa, and the porosity is 30-70%;

the compressive strength of the inner layer material is 0.8-2 MPa, and the porosity is 50-90%.

The present invention provides in a second aspect a method for preparing the 3D printed composite bone repair material according to the first aspect, the method comprising the steps of:

s1, adding a mineralized collagen porous calcium phosphate composite material into a high polymer solution, and performing first stirring treatment to obtain first biological ink;

s2, mixing the mineralized collagen porous calcium phosphate composite material, the bioactive material and the first solvent, and performing second stirring treatment to obtain second biological ink;

s3, constructing a three-dimensional simulation model according to the structural characteristics of the bone tissue defect part, adjusting the dosage of the first biological ink and the second biological ink, and forming an inner layer material through 3D printing; and/or

Adjusting the dosage of the first biological ink and the second biological ink, and forming an outer layer material through 3D printing;

s4, freeze-drying, resolving and removing solvent residues and sterilizing the inner layer material and/or the outer layer material prepared in the step S3 to obtain the 3D printing composite bone repair material comprising the inner layer material and/or the outer layer material.

Preferably, in step S1, the stirring speed of the first stirring treatment is 100-200 r/min, and the stirring time is 2-8 h.

Preferably, in step S2, the stirring speed of the second stirring treatment is 100-200 r/min, and the stirring time is 2-8 h.

The invention provides in a third aspect the use of a 3D printed composite bone repair material according to the first aspect in bone repair.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) The 3D printing composite bone repair material provided by the invention has an outer layer material similar to the structure and strength of the bionic cortical bone of the autologous bone and/or an inner layer material similar to the structure and strength of the bionic cancellous bone of the autologous bone, and can be customized and printed according to the actual bone injury condition.

(2) In some preferred embodiments of the present invention, the inner layer material has a low content of the first bio-ink and a high content of the second bio-ink, and is in a loose porous structure; the outer layer material has high content of the first biological ink and low content of the second biological ink, and is of a compact structure; the composite bone repair material with the inner layer material with the bionic cancellous bone structure and strength similar to the autologous bone and/or the outer layer material with the bionic cortical bone structure and strength similar to the autologous bone is prepared by regulating and controlling the mass ratio of the first biological ink to the second biological ink, and has excellent osteogenesis induction activity, can rapidly induce blood vessel to grow into and regenerate new bone tissue, has excellent mechanical support strength, and can meet the bone grafting requirement of a load bearing part.

(3) In some specific embodiments, according to the actual bone defect condition, the proportion of the first biological ink and the second biological ink is adjusted, and an inner layer material with a structure and strength similar to that of the bionic cancellous bone of the autologous bone is obtained by printing; and/or adjusting the proportion of the first biological ink and the second biological ink, and printing to obtain an outer layer material with the structure and strength similar to those of the autologous bone bionic cortical bone; for the bone injury condition of the cortical bone and the cancellous bone defect, the composite bone repair material with the inner layer material and the outer layer material can be printed according to the requirement; for the case of bone injury with only cortical bone defects, the bone repair material with only outer layer material can be printed as needed; for the bone injury condition with only cancellous bone defect, the bone repair material with only inner layer material can be printed according to the requirement; custom printing may be implemented according to different requirements.

Drawings

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

FIG. 1 is a flow chart of a method for preparing a 3D printed composite bone repair material provided by the invention;

FIG. 2 is a chart of skin and subcutaneous group pathology after 8 weeks of surgery in the placebo group;

FIG. 3 is a view showing the pathology of the skin and subcutaneous tissue after 8 weeks of surgery (implantation of the composite bone repair material provided in example 1 of the present invention) in the experimental group;

FIG. 4 is a graph of thymus histopathology 8 weeks after surgery in the placebo group;

FIG. 5 is a diagram showing the pathology of thymus tissue after 8 weeks of surgery (implantation of the composite bone repair material provided in example 1 of the present invention) in the experimental group;

FIG. 6 is a diagram of spleen tissue pathology 8 weeks after surgery in the placebo group;

FIG. 7 is a view showing the pathology of spleen tissue after 8 weeks of surgery (implantation of the composite bone repair material provided in example 1 of the present invention) in the experimental group;

FIG. 8 is a chart showing skin and subcutaneous tissue pathology 26 weeks after surgery in the placebo group;

FIG. 9 is a view showing the pathology of the skin and subcutaneous tissue after 26 weeks of surgery (implantation of the composite bone repair material provided in example 1 of the present invention) in the experimental group;

FIG. 10 is a graph of thymus histopathology 26 weeks after surgery in the placebo group;

FIG. 11 is a diagram showing the pathology of thymus tissue after 26 weeks of surgery (implantation of the composite bone repair material provided in example 1 of the present invention) in the experimental group;

FIG. 12 is a view of spleen tissue pathology 26 weeks after surgery in the placebo group;

FIG. 13 is a view showing the pathology of spleen tissue after 26 weeks of surgery (implantation of the composite bone repair material provided in example 1 of the present invention) in the experimental group;

it should be noted that the implant in the figure is the composite bone repair material prepared in example 1 of the present invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments, and all other embodiments obtained by those skilled in the art without making any inventive effort based on the embodiments of the present invention are within the scope of protection of the present invention.

The composite bone repair material comprises an inner layer material and/or an outer layer material, wherein the inner layer material is a bionic cancellous bone material with a structure and strength similar to those of an autologous bone bionic cancellous bone, and the outer layer material is a bionic cortical bone material with a structure and strength similar to those of the autologous bone bionic cortical bone.

The mineralized collagen porous calcium phosphate composite material adopted by the invention is prepared by adopting a method in a patent CN 108553691A; the specific method comprises the following steps: (1) preparation of biphasic calcium phosphate porous particles: the preparation component comprises a first calcium salt, polyvinyl alcohol and PMMA microspheres, wherein the mass ratio is (0.1-0.5): 3:2, the first calcium salt consists of hydroxyapatite and beta-calcium phosphate according to the mass ratio of 5:1-1:3, and the preparation method comprises the following steps: (a) Preparing polyvinyl alcohol solution with the concentration of 0.2-0.5 g/mL; (b) preparing a biphasic phosphate suspension; adding hydroxyapatite and beta-calcium phosphate into PBS solution to form suspension; (c) Adding the polyvinyl alcohol solution into the biphase phosphate suspension, stirring for 0.5-1 hour, adding PMMA microspheres, and stirring for 0.5-1 hour to obtain a sintering base solution; (d) Placing the sintering base solution into sintering equipment for sintering to obtain a sintering material; the sintering comprises the following stages: the first stage: the temperature rising rate is 5-10 ℃/min, the target temperature is 400-800 ℃, and the constant temperature time is 300-350 min; and a second stage: the temperature rising rate is 5-10 ℃/min, the target temperature is 1000-1200 ℃, and the constant temperature time is 180-200 min; and a third stage: stopping heating the sintering equipment, and naturally cooling to room temperature; (e) pulverizing the sintered material; (f) sieving; (2) preparation of mineralized collagen porous calcium phosphate composite material: adding the biphasic calcium phosphate porous particles into a collagen acid solution with the concentration of 1-5 mg/mL, stirring and mixing for 2-3 hours to prepare a suspension with the concentration of 10-30 wt%; continuously stirring the suspension, and dropwise adding a solution containing calcium ions, wherein the adding amount of the calcium ions is 0.01-0.15 mol of the calcium ions added per gram of collagen; dropwise adding a solution containing phosphate ions, wherein the molar ratio of the phosphate ions to the calcium ions is Ca/P=0.8-1.8; continuously stirring, dropwise adding NaOH solution until the pH value of the mixed system is 7-8, when the pH value reaches 5-6, precipitating the mixed system, and when the pH value reaches 7, forming white suspension in the mixed system; and standing the obtained mixed system for 24-96 hours, separating out precipitate, washing out impurity ions, then freeze-drying, and grinding to obtain the mineralized collagen porous calcium phosphate composite material with the mineralized collagen content of 2-25 wt%.

In some preferred embodiments of the present invention, the second bio-ink is prepared by dispersing the mineralized collagen porous calcium phosphate composite material in the first solvent, and then adding the bioactive material to avoid a decrease in the bioactivity of the bioactive material due to too long stirring time.

The 3D printing composite bone repair material provided by the invention has an outer layer material similar to the structure and strength of the bionic cortical bone of the autologous bone and/or an inner layer material similar to the structure and strength of the bionic cancellous bone of the autologous bone, and can be customized and printed according to the actual bone injury condition.

According to some preferred embodiments, the mass ratio of the first bio-ink and the second bio-ink in the inner layer material is 1 (1.5-9) (e.g., may be 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, or 1:9).

The inner layer material has low content of the first biological ink and high content of the second biological ink, and is of a loose porous structure; meanwhile, the inner layer material prepared by controlling the mass ratio of the first biological ink to the second biological ink in the range has a structure and strength similar to that of an autologous bone bionic cancellous bone, so that the osteogenesis induction activity of the bone material is improved, and the vascular ingrowth and the regeneration of new bone tissues can be rapidly induced.

According to some preferred embodiments, the mass ratio of the first bio-ink to the second bio-ink in the outer layer material is (1.5-9): 1 (e.g., may be 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or 9:1).

The first biological ink in the outer layer material has high content, and the second biological ink has low content and is of a compact structure; meanwhile, the outer layer material prepared by controlling the mass ratio of the first biological ink to the second biological ink in the range has a structure and strength similar to that of an autologous bone bionic cortical bone, has excellent mechanical supporting strength, and can meet the bone grafting requirement of a load bearing part.

According to some preferred embodiments, the polymer solution is formulated from a polymer dissolved in a second solvent;

the ratio of the amount of the polymer to the second solvent is 1g (5-12) mL (e.g., 1g:5mL, 1g:6mL, 1g:7mL, 1g:8mL, 1g:9mL, 1g:10mL, 1g:11mL, or 1g:12 mL);

in some specific embodiments, the polymer is added into the second solvent, and the polymer and the second solvent are fully mixed by stirring to prepare a polymer solution; wherein the stirring speed is 150-400 r/min, and the stirring time is 24-72 hours.

In the first bio-ink, the mass ratio of the mineralized collagen porous calcium phosphate composite to the polymer is 1 (1.5-9) (e.g., may be 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, or 1:9).

The mass ratio of the high polymer to the mineralized collagen porous calcium phosphate composite material in the first biological ink is controlled in the range, and the prepared composite bone repair material has excellent biological performance and mechanical performance; the mineralized collagen porous calcium phosphate composite material has high content, and the obtained material has good biological performance but poor mechanical performance; the mineralized collagen porous calcium phosphate composite material has low content, and the obtained composite bone repair material has poor biological performance; in addition, the amount of the second solvent should be controlled within the above range, and if the amount of the second solvent is too small, the mineralized collagen porous calcium phosphate composite material cannot be uniformly dispersed; if the amount of the second solvent is too large, the second solvent volatilizes during the freeze-drying process, which results in a high porosity of the finally prepared composite bone repair material, resulting in a decrease in mechanical strength.

According to some preferred embodiments, the high polymer is one or more of polylactic acid, polycaprolactone, polyvinyl alcohol;

According to some preferred embodiments, in the second bio-ink, the mineralized collagen porous calcium phosphate composite is 80 to 99.5% (e.g., may be 80%, 82%, 84%, 85%, 86%, 88%, 90%, 92%, 95%, 96%, or 99.5%) of the total mass of the mineralized collagen porous calcium phosphate composite and the bioactive material, and the bioactive material is 0.5 to 20% (e.g., may be 0.5%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, or 20%), preferably 1 to 6% (e.g., may be 1%, 2%, 3%, 4%, 5%, or 6%) of the total mass;

in the second bio-ink, the total mass of the mineralized collagen porous calcium phosphate composite to bioactive material and the volume ratio of the first solvent is 1g (10-20) mL (e.g., may be 1g:10mL, 1g:11mL, 1g:12mL, 1g:13mL, 1g:14mL, 1g:15mL, 1g:16mL, 1g:17mL, 1g:18mL, 1g:19mL, or 1g:20 mL).

In the second biological ink, the mass ratio of the bioactive material to the mineralized collagen porous calcium phosphate composite material is controlled in the range, so that the cost can be controlled, and the prepared composite bone repair material has excellent biological performance and mechanical performance; in addition, the amount of the first solvent should be controlled within the above range, and if the amount of the first solvent is too small, the mineralized collagen porous calcium phosphate composite material cannot be uniformly dispersed; if the amount of the first solvent is too large, the first solvent volatilizes during the freeze-drying process, which results in a high porosity of the finally prepared composite bone repair material, resulting in a decrease in mechanical strength.

According to some preferred embodiments, the bioactive material is one of Concentrated Growth Factor (CGF), transforming growth factor- β (TGF- β), bone Morphogenic Protein (BMP);

According to some preferred embodiments, the outer layer material has a compressive strength of 10 to 20MPa (e.g., may be 10MPa, 11MPa, 12MPa, 13MPa, 14MPa, 15MPa, 16MPa, 17MPa, 18MPa, 19MPa, or 20 MPa) and a porosity of 30 to 70% (e.g., may be 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%);

the compressive strength of the inner layer material is 0.8 to 2MPa (for example, may be 0.8MPa, 1MPa, 1.2MPa, 1.3MPa, 1.4MPa, 1.5MPa, 1.6MPa, 1.7MPa, 1.8MPa, 1.9MPa or 2 MPa), and the porosity is 50 to 90% (for example, may be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%).

The present invention provides in a second aspect a method for preparing the 3D printed composite bone repair material according to the first aspect, characterized in that the method comprises the steps of:

It should be noted that the 3D printed composite bone repair material obtained by final printing may include only the inner layer material, or may include only the outer layer material, or may include both the inner layer material and the outer layer material; when the finally printed 3D printing composite bone repair material simultaneously comprises an inner layer material and an outer layer material, adjusting the dosage of the first biological ink and the second biological ink, forming the inner layer material through 3D printing, and then adjusting the dosage of the first biological ink and the second biological ink, forming the outer layer material on the surface of the inner layer material, so as to prepare the high-grade bionic composite bone repair material with a compact/loose two-phase composite structure, and having the strength support and the osteogenesis induction activity.

In some specific embodiments, the first bio-ink is prepared by controlling the usage amount of the mineralized collagen porous calcium phosphate composite material, the high polymer and the second solvent in the high polymer solution, and then the second bio-ink is prepared by controlling the usage amount of the mineralized collagen porous calcium phosphate composite material, the bioactive material and the first solvent; and finally, acquiring data materials of the defect part by CT tomography according to the structural characteristics of the defect parts of different bone tissues, introducing the data materials into computer aided design software for three-dimensional reconstruction of the defect part, constructing a three-dimensional simulation model, and printing by a 3D printer to obtain the composite bone repair material.

In some specific embodiments, according to the actual bone defect condition, the proportion of the first biological ink and the second biological ink is adjusted, and an inner layer material with a structure and strength similar to that of the bionic cancellous bone of the autologous bone is obtained by printing; and/or adjusting the proportion of the first biological ink and the second biological ink, and printing to obtain an outer layer material with the structure and strength similar to those of the autologous bone bionic cortical bone; for the bone injury condition of the cortical bone and the cancellous bone defect, the composite bone repair material with the inner layer material and the outer layer material can be printed according to the requirement; for the case of bone injury with only cortical bone defects, the bone repair material with only outer layer material can be printed as needed; for the bone injury condition with only cancellous bone defect, the bone repair material with only inner layer material can be printed according to the requirement; custom printing may be implemented according to different requirements.

According to some preferred embodiments, in step S1, the stirring speed of the first stirring process is 100-200 r/min (for example, may be 100r/min, 110r/min, 120r/min, 130r/min, 140r/min, 150r/min, 160r/min, 170r/min, 180r/min, 190r/min or 200 r/min), and the stirring time is 2-8 h (for example, may be 2h, 3h, 4h, 5h, 6h, 7h or 8 h).

According to some preferred embodiments, in step S2, the stirring speed of the second stirring process is 100-200 r/min (for example, may be 100r/min, 110r/min, 120r/min, 130r/min, 140r/min, 150r/min, 160r/min, 170r/min, 180r/min, 190r/min or 200 r/min), and the stirring time is 2-8 h (for example, may be 2h, 3h, 4h, 5h, 6h, 7h or 8 h).

In some specific embodiments of the invention, the freeze-drying comprises a pre-freezing stage, a first sublimation stage, a second sublimation stage and a cooling stage, wherein the process conditions of each stage are as follows:

pre-freezing: keeping the temperature at-22 to-18 ℃ for 230-250 min;

the first sublimation stage comprises two heating steps, which are respectively:

the temperature rising rate is 1-2 ℃/min, the target temperature is-12 to-8 ℃, and the constant temperature is 230-250 min;

the temperature rising rate is 1-2 ℃/min, the target temperature is-2 ℃, and the constant temperature time is 170-190 min;

The second sublimation stage comprises six temperature rising steps, which are respectively as follows:

the temperature rising rate is 1-2 ℃/min, the target temperature is 8-12 ℃, and the constant temperature time is 170-190 min;

the temperature rising rate is 1-2 ℃/min, the target temperature is 18-22 ℃, and the constant temperature time is 170-190 min;

the temperature rising rate is 1-2 ℃/min, the target temperature is 28-32 ℃, and the constant temperature time is 110-130 min;

the temperature rising rate is 1-2 ℃/min, the target temperature is 38-42 ℃, and the constant temperature time is 110-130 min;

the temperature rising rate is 1-2 ℃/min, the target temperature is 48-52 ℃, and the constant temperature time is 110-130 min;

the temperature rising rate is 1-2 ℃/min, the target temperature is 53-57 ℃, and the constant temperature time is 110-130 min;

and (3) a cooling stage: cooling to room temperature for 110-130 min.

In some embodiments of the present invention, the steps of resolving and removing solvent residues are: placing the trimmed material in a culture dish, placing in a vacuum drying oven for vacuum analysis, setting the temperature to 50+/-5 ℃, and analyzing for at least 72 hours; detecting solvent residues; if the solvent is not qualified, repeating the analysis process until the residual limit value is qualified (the solvent residual quantity is less than or equal to 0.01%).

In other embodiments of the present invention, the step of resolving and removing solvent residues comprises: placing the sample into a beaker, and completely immersing the sample in alcohol for at least 2 hours; ultrasonic cleaning, pouring out cleaning liquid after 10min, and repeating ultrasonic for 1 time; the centrifugal machine loads the filter bag, and is centrifuged once (5 seconds), and the rotating speed of the centrifugal machine is defaulted to 3000r/min; drying for at least 8 hours by using a vacuum drying oven or a forced air drying oven at a set temperature of 50 ℃; detecting solvent residues; if the residual limit value is not qualified, repeating the analysis process until the residual limit value is qualified. (solvent residue was 0.01% or less).

In some embodiments, the method further comprises the step of trimming the material after the solvent residue is resolved.

In some embodiments of the invention, the sterilization is performed by cobalt 60 irradiation, and the dosage control range is 15-25KGy.

In order to more clearly illustrate the technical scheme and advantages of the present invention, the present invention will be further described below with reference to examples.

The materials and the reagents in the invention can be obtained by direct purchase or self-synthesis in the market, and the specific model is not limited.

The related performance tests in the examples and comparative examples of the present invention were carried out by referring to the following methods:

porosity test: the reference standard is GB/T1966-1996

Sample materials (prepared in examples and comparative examples)Is trimmed to a regular shape with a blade, the dry mass W1 of the sample is precisely weighed (to the nearest 0.01 g), the apparent size of the sample is measured by a universal or special gauge (to the nearest 0.1 mm), and the sample volume V (cm) is calculated ³ ). Placing the dried sample in a boiling container containing isopropanol solvent, filling clean gauze at the bottom of the sample and container, boiling for 2h (the water surface should be kept above the sample by more than 50mm in the boiling process), cooling to room temperature after boiling, taking out the saturated sample from the liquid, wiping off the liquid attached to the surface of the sample by using the multilayer gauze saturated with isopropanol, and rapidly weighing the mass W2 (accurate to 0.01 g) of the saturated sample in the air. Sample porosity q, q= (W2-W1)/(0.7863 ×v) was calculated as follows, where 0.7863 is the density of isopropyl alcohol, and three samples were measured in parallel, taking the average of the porosities.

Compressive strength test: the measurements were carried out by the method specified in reference standard GB 23101.1-20084.4

The cylindrical samples (the bone repair materials prepared in the examples and the comparative examples) are used for axial loading, the height h and the diameter of the samples are in accordance with the size ratio of 1.5 h/d to 2.0, the samples are placed into a material mechanical testing machine for compression test, the compression rate is 0.5mm/min, and the average value of loading force recorded at the moment when the stress begins to drop is measured.

Cytotoxicity test: the materials of the example and the comparative example are subjected to irradiation sterilization, the materials of the example and the comparative example are leached according to the proportion of 0.2g/mL by taking a cell culture medium containing 10% calf serum as a leaching medium, and the materials are inoculated into a 96-well culture plate after cell counting and placed into CO ₂ Culturing in incubator at 37 deg.c for 24 hr, and eliminating culture liquid; fresh cell culture solution is added into blank control group, sample leaching solutions of the example and the comparative example are respectively added into experimental group, and the mixture is placed into CO ₂ Culturing in incubator for 24 hr, observing cell morphology with microscope, adding MTT solution with mass concentration of 5g/L into each well, culturing for 4 hr, discarding liquid in the well, adding 200 μLDMSO, measuring absorbance at 570nm and 650nm wavelength of enzyme-labeled instrument, calculating relative increment rate,

Relative increment rate = (experimental group absorbance mean/blank group absorbance mean) ×100%.

Biological evaluation: in order to verify the safety of the material, the implantation amount of the mouse is calculated according to the weight of the mouse by 10 times of the maximum use amount of the human body, the operation is carried out on the mouse, and the composite bone repair material prepared by the invention is subcutaneously implanted at the back of the mouse to be used as an experimental group; the blank group performs sham surgery (same surgery but without composite bone repair material) on mice for control; and periodically analyzing the pathological conditions of skin, subcutaneous tissue, thymus tissue and spleen tissue of the experimental group and the control group.

Example 1

Preparing a 3D printing composite bone repair material comprising an inner layer material and an outer layer material:

s1, dissolving polylactic acid in 1, 4-dioxane, and stirring for 48 hours at a stirring speed of 300r/min to obtain a high polymer solution, wherein the dosage ratio of the polylactic acid to the 1, 4-dioxane is 1g to 8mL;

s2, adding the mineralized collagen porous calcium phosphate composite material into a high polymer solution, and stirring for 4 hours at a stirring speed of 150r/min to obtain first biological ink, wherein the mass ratio of the mineralized collagen porous calcium phosphate composite material to the high polymer is 3:7;

S3, mixing the mineralized collagen porous calcium phosphate composite material, the concentrated growth factor and the purified water, and stirring for 3 hours at a stirring speed of 120r/min to obtain second biological ink, wherein the mineralized collagen porous calcium phosphate composite material accounts for 97% of the total mass of the mineralized collagen porous calcium phosphate composite material and the concentrated growth factor, the concentrated growth factor accounts for 3% of the total mass, and the volume ratio of the total mass of the mineralized collagen porous calcium phosphate composite material and the concentrated growth factor to the purified water is 1 g/12 mL;

s4, constructing a three-dimensional simulation model according to the structural characteristics of the bone tissue defect part, adjusting the mass ratio of the first biological ink to the second biological ink to be 2:8, and forming an inner layer material through 3D printing;

adjusting the mass ratio of the first biological ink to the second biological ink to be 8:2, and forming an outer layer material on the surface of the inner layer material through 3D printing;

and S5, freeze-drying, analyzing and removing solvent residues and sterilizing the material prepared in the step S4 to obtain the 3D printing composite bone repair material comprising the inner layer material and the outer layer material.

The porosity and compressive strength data of the composite bone repair material prepared in example 1 are shown in table 1.

The relative increment rates of the composite bone repair material prepared in example 1 are shown in table 2.

Example 2

s1, dissolving polycaprolactone in 1, 4-dioxane, and stirring for 48 hours at a stirring speed of 150r/min to obtain a high polymer solution, wherein the dosage ratio of the polycaprolactone to the 1, 4-dioxane is 1g to 5mL;

s2, adding the mineralized collagen porous calcium phosphate composite material into a high polymer solution, and stirring for 2 hours at a stirring speed of 100r/min to obtain first biological ink, wherein the mass ratio of the mineralized collagen porous calcium phosphate composite material to the high polymer is 1:1.5;

s3, mixing the mineralized collagen porous calcium phosphate composite material, the transforming growth factor-beta and the physiological saline, and stirring for 3 hours at a stirring speed of 120r/min to obtain second biological ink, wherein the mineralized collagen porous calcium phosphate composite material accounts for 80% of the total mass of the mineralized collagen porous calcium phosphate composite material and the transforming growth factor-beta, the transforming growth factor-beta accounts for 20% of the total mass of the mineralized collagen porous calcium phosphate composite material, and the volume ratio of the total mass of the mineralized collagen porous calcium phosphate composite material and the transforming growth factor-beta to the physiological saline is 1g to 10mL;

S4, constructing a three-dimensional simulation model according to the structural characteristics of the bone tissue defect part, adjusting the mass ratio of the first biological ink to the second biological ink to be 1:1.5, and then forming an inner layer material through 3D printing;

adjusting the mass ratio of the first biological ink to the second biological ink to be 3:1, and forming an outer layer material on the surface of the inner layer material through 3D printing;

The porosity and compressive strength data of the composite bone repair material prepared in example 2 are shown in table 1.

The relative increment rates of the composite bone repair material prepared in example 2 are shown in Table 2.

Example 3

s1, dissolving polyvinyl alcohol in 1, 4-dioxane, and stirring for 48 hours at a stirring speed of 300r/min to obtain a high polymer solution, wherein the dosage ratio of the polyvinyl alcohol to the 1, 4-dioxane is 1g to 12mL;

s2, adding the mineralized collagen porous calcium phosphate composite material into a high polymer solution, and stirring for 8 hours at a stirring speed of 200r/min to obtain first biological ink, wherein the mass ratio of the mineralized collagen porous calcium phosphate composite material to the high polymer is 1:9;

S3, mixing the mineralized collagen porous calcium phosphate composite material, the bone morphogenetic protein and the sterilized water for injection, and stirring for 3 hours at a stirring speed of 120r/min to obtain second biological ink, wherein the mineralized collagen porous calcium phosphate composite material accounts for 99.5% of the total mass of the mineralized collagen porous calcium phosphate composite material and the bone morphogenetic protein, the bone morphogenetic protein accounts for 0.5% of the total mass of the mineralized collagen porous calcium phosphate composite material and the bone morphogenetic protein, and the volume ratio of the sterilized water for injection is 1g to 20mL;

s4, constructing a three-dimensional simulation model according to the structural characteristics of the bone tissue defect part, adjusting the mass ratio of the first biological ink to the second biological ink to be 1:9, and forming an inner layer material through 3D printing;

adjusting the mass ratio of the first biological ink to the second biological ink to be 7:1, and forming an outer layer material on the surface of the inner layer material through 3D printing;

The porosity and compressive strength data of the composite bone repair material prepared in example 3 are shown in table 1.

The relative increment rates of the composite bone repair material prepared in example 3 are shown in Table 2.

Example 4

s1, dissolving polylactic acid in chloroform, and stirring for 48 hours at a stirring speed of 400r/min to obtain a high polymer solution, wherein the dosage ratio of the polylactic acid to the chloroform is 1g to 12mL;

s3, mixing the mineralized collagen porous calcium phosphate composite material, the concentrated growth factor and the purified water, and stirring for 3 hours at a stirring speed of 120r/min to obtain second biological ink, wherein the mineralized collagen porous calcium phosphate composite material accounts for 99.5% of the total mass of the mineralized collagen porous calcium phosphate composite material and the concentrated growth factor, the concentrated growth factor accounts for 0.5% of the total mass, and the volume ratio of the mineralized collagen porous calcium phosphate composite material to the concentrated growth factor to the purified water is 1 g/15 mL;

According to the structural characteristics of the bone tissue defect part, a three-dimensional simulation model is constructed, the mass ratio of the first biological ink to the second biological ink is adjusted to be 9:1, and then an outer layer material is formed on the surface of the inner layer material through 3D printing;

The porosity and compressive strength data of the composite bone repair material prepared in example 4 are shown in table 1.

The relative increment rates of the composite bone repair material prepared in example 4 are shown in table 2.

Example 5

Preparing a 3D printed composite bone repair material comprising only an inner layer material:

s1, dissolving polycaprolactone in acetone, and stirring for 48 hours at a stirring speed of 400r/min to obtain a high polymer solution, wherein the dosage ratio of the polycaprolactone to the acetone is 1 g/8 mL;

s2, adding the mineralized collagen porous calcium phosphate composite material into a high polymer solution, and stirring for 8 hours at a stirring speed of 200r/min to obtain first biological ink, wherein the mass ratio of the mineralized collagen porous calcium phosphate composite material to the high polymer is 1:7;

s3, mixing the mineralized collagen porous calcium phosphate composite material, the transforming growth factor-beta and purified water, and stirring for 3 hours at a stirring speed of 120r/min to obtain second biological ink, wherein the mineralized collagen porous calcium phosphate composite material accounts for 99.5% of the total mass, the transforming growth factor-beta accounts for 0.5% of the total mass, and the volume ratio of the mineralized collagen porous calcium phosphate composite material to the transforming growth factor-beta to the purified water is 1g to 15mL;

S4, constructing a three-dimensional simulation model according to the structural characteristics of the bone tissue defect part, adjusting the mass ratio of the first biological ink to the second biological ink to be 1:6, and forming an inner layer material through 3D printing;

s5, performing freeze drying, analysis and solvent residue removal on the inner layer material prepared in the step S4, and sterilizing to obtain the 3D printing composite bone repair material comprising only the inner layer material.

The porosity and compressive strength data of the composite bone repair material prepared in example 5 are shown in table 1.

The relative increment rates of the composite bone repair material prepared in example 5 are shown in table 2.

Example 6

s1, dissolving polycaprolactone in acetone, and stirring for 48 hours at a stirring speed of 400r/min to obtain a high polymer solution, wherein the dosage ratio of the polycaprolactone to the acetone is 1g to 12mL;

s2, adding the mineralized collagen porous calcium phosphate composite material into a high polymer solution, and stirring for 8 hours at a stirring speed of 200r/min to obtain first biological ink, wherein the mass ratio of the mineralized collagen porous calcium phosphate composite material to the high polymer is 1:8;

s3, mixing the mineralized collagen porous calcium phosphate composite material, bone morphogenetic protein and normal saline, and stirring for 3 hours at a stirring speed of 120r/min to obtain second biological ink, wherein the mineralized collagen porous calcium phosphate composite material accounts for 80% of the total mass of the mineralized collagen porous calcium phosphate composite material and the bone morphogenetic protein, the bone morphogenetic protein accounts for 20% of the total mass, and the volume ratio of the mineralized collagen porous calcium phosphate composite material to the bone morphogenetic protein to the normal saline is 1 g/10 mL;

S4, constructing a three-dimensional simulation model according to the structural characteristics of the bone tissue defect part, adjusting the mass ratio of the first biological ink to the second biological ink to be 1:7, and forming an inner layer material through 3D printing;

The porosity and compressive strength data of the composite bone repair material prepared in example 6 are shown in table 1.

The relative increment rates of the composite bone repair material prepared in example 6 are shown in Table 2.

Example 7

Preparing a 3D printed composite bone repair material comprising only an outer layer material:

s1, dissolving polylactic acid in 1, 4-dioxane, and stirring for 48 hours at a stirring speed of 400r/min to obtain a high polymer solution, wherein the dosage ratio of the polylactic acid to the 1, 4-dioxane is 1g to 5mL;

s3, mixing the mineralized collagen porous calcium phosphate composite material, bone morphogenetic protein and purified water, and stirring for 3 hours at a stirring speed of 120r/min to obtain second biological ink, wherein the mineralized collagen porous calcium phosphate composite material accounts for 80% of the total mass of the mineralized collagen porous calcium phosphate composite material and the concentrated growth factor, the concentrated growth factor accounts for 20% of the total mass, and the volume ratio of the mineralized collagen porous calcium phosphate composite material to the concentrated growth factor to the purified water is 1g to 10mL;

S4, constructing a three-dimensional simulation model according to the structural characteristics of the bone tissue defect part, adjusting the mass ratio of the first biological ink to the second biological ink to be 5:1, and forming an outer layer material through 3D printing;

and S5, performing freeze drying, analysis and solvent residue removal on the outer layer material prepared in the step S4, and sterilizing to obtain the 3D printing composite bone repair material only comprising the outer layer material.

The porosity and compressive strength data of the composite bone repair material prepared in example 7 are shown in table 1.

The relative increment rates of the composite bone repair material prepared in example 7 are shown in Table 2.

Example 8

s2, adding the mineralized collagen porous calcium phosphate composite material into a high polymer solution, and stirring for 8 hours at a stirring speed of 200r/min to obtain first biological ink, wherein the mass ratio of the mineralized collagen porous calcium phosphate composite material to the high polymer is 1:5;

s3, mixing the mineralized collagen porous calcium phosphate composite material, the transforming growth factor-beta and the purified water, and stirring for 3 hours at a stirring speed of 120r/min to obtain second biological ink, wherein the mineralized collagen porous calcium phosphate composite material accounts for 95% of the total mass of the mineralized collagen porous calcium phosphate composite material and the transforming growth factor-beta, the transforming growth factor-beta accounts for 5% of the total mass of the mineralized collagen porous calcium phosphate composite material and the transforming growth factor-beta, and the volume ratio of the mineralized collagen porous calcium phosphate composite material to the purified water is 1g to 15mL;

S4, constructing a three-dimensional simulation model according to the structural characteristics of the bone tissue defect part, adjusting the mass ratio of the first biological ink to the second biological ink to be 8:1, and forming an outer layer material through 3D printing;

The porosity and compressive strength data of the composite bone repair material prepared in example 8 are shown in table 1.

The relative increment rates of the composite bone repair material prepared in example 8 are shown in Table 2.

The following comparative examples and the preparation methods of the raw materials of the mineralized gum in table 2 of the present invention were: dropwise adding a solution containing calcium ions into 1mg/mL of collagen acid solution, wherein the addition amount of the calcium ions is 0.01mol of calcium ions added into each gram of collagen; dropwise adding a solution containing phosphate ions, wherein the molar ratio of the phosphate ions to the calcium ions is Ca/P=1.5; continuously stirring, dropwise adding NaOH solution until the pH value of the mixed system is 7-8, when the pH value reaches 5-6, precipitating the mixed system, and when the pH value reaches 7, forming white suspension in the mixed system; and standing the obtained mixed system for 24 hours, separating out precipitate, washing out impurity ions, then freeze-drying, and grinding to obtain the mineralized collagen material.

Comparative example 1

Substantially the same as in example 1, except that: the mass ratio of the first biological ink to the second biological ink in the inner layer material is 1:10, and the mass ratio of the first biological ink to the second biological ink in the outer layer material is 10:1.

The porosity and compressive strength data of the composite bone repair material prepared in comparative example 1 are shown in table 1.

The relative increment rate of the composite bone repair material prepared in comparative example 1 is shown in table 2.

The mass ratio of the first biological ink to the second biological ink in the inner layer material is too small, so that the strength of the inner layer material of the prepared composite bone repair material is too low, the mass ratio of the first biological ink to the second biological ink in the outer layer material is too large, the content of the bioactive material of the outer layer material is relatively low, and the relative increment rate is reduced.

Comparative example 2

Substantially the same as in example 5, except that: the mass ratio of the first biological ink to the second biological ink in the inner layer material is 1:11.

The porosity and compressive strength data of the composite bone repair material prepared in comparative example 2 are shown in table 1.

The relative increment rate of the composite bone repair material prepared in comparative example 2 is shown in table 2.

Because the mass ratio of the first biological ink to the second biological ink is too small, the strength of the inner layer material of the prepared composite bone repair material is too low, and the prepared composite bone repair material is not good in molding and has collapse phenomenon.

Comparative example 3

Substantially the same as in example 5, except that: the mineralized collagen porous calcium phosphate composite material in the first biological ink and the mineralized collagen porous calcium phosphate composite material in the second biological ink are replaced by the mineralized collagen material prepared by the method.

The porosity and compressive strength data of the composite bone repair material prepared in comparative example 3 are shown in table 1.

The relative increment rate of the composite bone repair material prepared in comparative example 3 is shown in table 2.

The strength of the mineralized collagen material is lower than that of the mineralized collagen porous calcium phosphate composite material, so that the prepared composite bone repair material has poor mechanical property and low compressive strength, but the biological activity of the mineralized collagen material is equivalent to that of the mineralized collagen porous calcium phosphate composite material, and the biological activity (relative increment rate) of the prepared composite bone repair material is basically consistent.

Comparative example 4

Substantially the same as in example 7, except that: the mass ratio of the first biological ink to the second biological ink in the outer layer material is 11:1.

The porosity and compressive strength data of the composite bone repair material prepared in comparative example 4 are shown in table 1.

The relative increment rate of the composite bone repair material prepared in comparative example 4 is shown in table 2.

Because the mass ratio of the first biological ink to the second biological ink is too large, the content of the bioactive material is low, the mechanical property (compressive strength) of the prepared composite bone repair material is improved, but the biological property (relative increment rate) is reduced.

Comparative example 5

Substantially the same as in example 7, except that: the mineralized collagen porous calcium phosphate composite material in the first biological ink and the mineralized collagen porous calcium phosphate composite material in the second biological ink are replaced by the mineralized collagen material prepared by the method.

The porosity and compressive strength data of the composite bone repair material prepared in comparative example 5 are shown in table 1.

The relative increment rate of the composite bone repair material prepared in comparative example 5 is shown in table 2.

The biological activity of the mineralized collagen material is equivalent to that of the mineralized collagen porous calcium phosphate composite material, and the biological activity (relative increment rate) of the prepared composite bone repair material is equivalent; however, the strength of the mineralized collagen material is lower than that of the mineralized collagen porous calcium phosphate composite material, and the prepared composite bone repair material has poor mechanical property and low compressive strength.

TABLE 1

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It should be noted that "-" in the table indicates that this data is not present.

TABLE 2

In conclusion, the composite bone repair material prepared by the invention has excellent mechanical property and biological property.

As can be seen from fig. 2, after 8 weeks of the operation, skin and subcutaneous tissues of the blank group were seen as epidermis, dermal connective tissue, subcutaneous adipose tissue and one layer of thin adipose membrane musculature, skin appendages such as hair follicles, hair roots and sebaceous glands were seen in the dermis and subcutaneous adipose tissue, and no abnormality was seen, pigmentation was seen in the epidermis and hair follicles, showing approximately normal skin, subcutaneous tissue and striated muscle tissue; as can be seen from fig. 3, after 8 weeks of surgery, the deep portion of the diaphragmatic muscle in the subcutaneous tissue in the skin and subcutaneous tissue sections of the experimental group was seen to have a nearly circular cystic structure, the capsule wall was a thin layer of fibrous connective tissue in which blue stained implants were seen, and the implants were divided into small pieces of varying sizes by the proliferated fibrous connective tissue and capillaries, showing approximately normal skin, subcutaneous tissue and striated muscle tissue.

As can be seen from fig. 4, 8 weeks after the operation, the thymus of the blank group was covered with a thin film, the leaflet structure was visible, and the tissues were not changed by atrophy, hyperplasia, hypertrophy, etc., most of the thymus cortex: the medullary ratio is about 2:1, showing that the cortical and medullary areas are approximately in the normal range; the cortex is composed of a large number of lymphocytes, epithelial-like cells and a small number of macrophages, the lymphocytes are larger and densely arranged, a large number of lymphocytes can see clear nucleoli, some nucleoli are smaller, some nucleoli are not clear, the medulla can see more epithelial-like cells, the lymphocytes are different in size, the cells are loose than the cortex, the medulla can see individually scattered macrophages which engulf apoptotic lymphocytes, fewer in the cortex, thymus bodies are not seen, epithelial-free areas in thymus are not seen, and lymphocytosis and reduction are not seen; as can be seen from fig. 5, 8 weeks after the operation, the thymus of the experimental group was substantially identical to that of the blank control group, and no lesions were seen.

As can be seen from fig. 6, after 8 weeks of operation, the spleen of the blank group was coated with fiber, the white marrow, the marginal zone, and the red marrow structure were clearly seen; the white marrow is mainly composed of lymphocyte dense with central arteriole visible in the center, and lymphocyte dense in periarterial cell intrathecal lymphoid tissue, while lymphocyte follicular is not obvious, central germinal center is not obvious, and is composed of more naive lymphoblastic cells; the peripheral area is visible outside the white marrow, the red marrow is clear, the intramedullary canal is filled with red blood cells, the red marrow and the white marrow are visible to scatter in deposited ferrioxacin, the size and/or the number of the spleen white intramedullary lymphatic follicular and periarterial cell sheaths are reduced, and the lymphocytes are reduced; the extramedullary hematopoiesis of red marrow is increased, and three lines of cells of red line, grain line and megakaryocyte line are all visible, and are most common; as can be seen from fig. 7, the spleens of the experimental group were substantially identical to the blank group after 8 weeks of operation, and no lesions were seen.

As can be seen from fig. 8, 26 weeks after the operation, the skin and subcutaneous tissue of the blank group were seen as epidermis, dermal connective tissue, subcutaneous adipose tissue and one of the thin layers of adipose membrane muscle striated muscle tissue; skin appendages such as hair follicles, hair roots, sebaceous glands and the like are visible in dermis and subcutaneous adipose tissue, and no abnormality is seen; the epidermis and hair follicles are seen with pigmentation, and locally with slight inflammatory cell infiltration, showing approximately normal skin, subcutaneous tissue and striated muscle tissue; as can be seen from fig. 9, the deep fat membranous striated muscle in subcutaneous tissue in the skin and subcutaneous tissue sections of the experimental group after 26 weeks of surgery was seen to have a nearly circular blue-stained implant, a thinner capsule wall consisting of proliferating fibroblasts and collagen fibers was seen to be outside the implant, and some proliferated capillaries were seen, fibrous connective tissue grew into the implant, and no abnormality was seen.

As can be seen from fig. 10, 26 weeks after the operation, the thymus of the blank group was covered with a thin envelope, and the leaflet structure was visible, and the tissues were not changed by atrophy, hyperplasia, hypertrophy, and the like, and most of thymus cortex: the medullary area ratio is about 2:1, showing that the cortical and medullary areas are approximately in the normal range; the cortex is composed of a large number of lymphocytes, epithelial-like cells and a small number of macrophages, the lymphocytes are large and densely arranged, clear nucleoli are visible for many lymphocytes, some nucleoli are smaller, and some nucleoli are unclear; the medulla can see more epithelial cells, the sizes of lymphocytes are different, the cells are loose compared with the cortex, the medulla can see individual scattered macrophages phagocytizing apoptotic lymphocytes, fewer in the cortex, the thymus body is not seen, the epithelial-free area in the thymus is seen, and the lymphocytosis are not seen; as can be seen from fig. 11, the spleens of the experimental group were substantially identical to the blank group after 26 weeks of operation, and no lesions were seen.

As can be seen from fig. 12, after 26 weeks of surgery, the spleen of the blank group was coated with fiber, the white marrow, the marginal zone, and the red marrow structure were clearly seen; the white marrow is mainly composed of lymphocyte dense with central arteriole visible in the center, and lymphocyte dense in periarterial cell intrathecal lymphoid tissue, while lymphocyte follicular is not obvious, central germinal center is not obvious, and is composed of more naive lymphoblastic cells; the outer visible border area of the white marrow, the red medullary cord, during which the medullary sinus is filled with red blood cells, the red marrow and the white marrow are visible to scatter in deposited ferrioxacin, the majority of the spleen white marrow is visible to have reduced size and/or reduced number of lymphatic follicles and periarterial cell sheaths, and lymphopenia; and the extramedullary hematopoiesis of red marrow is increased, and red cells, granular cells and megakaryocyte cells are all visible; as can be seen from fig. 13, the spleens of the experimental group were substantially identical to the blank group after 26 weeks of operation, and no lesions were seen.

It should be noted that in the description of embodiments of the invention, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying any particular importance unless expressly specified or limited otherwise.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A 3D printed composite bone repair material, characterized by:

the 3D printing composite bone repair material comprises an inner layer material and/or an outer layer material;

the first biological ink is prepared from mineralized collagen porous calcium phosphate composite material and high polymer solution; the high polymer solution is prepared by dissolving a high polymer in a second solvent; the mass ratio of the mineralized collagen porous calcium phosphate composite material to the polymer is 1 (1.5-9);

the second biological ink is prepared from mineralized collagen porous calcium phosphate composite material, bioactive material and a first solvent; based on the total mass of the mineralized collagen porous calcium phosphate composite material and the bioactive material, the mineralized collagen porous calcium phosphate composite material accounts for 80-99.5% of the total mass, and the bioactive material accounts for 0.5-20% of the total mass;

In the inner layer material, the mass ratio of the first biological ink to the second biological ink is 1 (1.5-9); and/or

In the outer layer material, the mass ratio of the first biological ink to the second biological ink is (1.5-9): 1.

2. The composite bone repair material of claim 1, wherein:

the dosage ratio of the high polymer to the second solvent is 1g (5-12 mL).

3. The composite bone repair material of claim 2, wherein:

the high polymer is one or more of polylactic acid, polycaprolactone and polyvinyl alcohol;

the second solvent is one or more of 1, 4-dioxane, acetone, chloroform and dichloromethane.

4. A composite bone repair material according to claim 3, wherein:

the second solvent is 1, 4-dioxane.

5. The composite bone repair material of claim 1, wherein:

in the second biological ink, the bioactive material accounts for 1-6% of the total mass;

the total mass of the mineralized collagen porous calcium phosphate composite material and the bioactive material and the volume ratio of the first solvent are 1g (10-20) mL.

6. The composite bone repair material according to claim 5, wherein:

The bioactive material is one of concentrated growth factor, transforming growth factor-beta and bone morphogenetic protein;

7. The composite bone repair material of claim 1, wherein:

the compressive strength of the outer layer material is 10-20 MPa, and the porosity is 30-70%;

8. A method of preparing a 3D printed composite bone repair material according to any one of claims 1-7, comprising the steps of:

s1, adding a mineralized collagen porous calcium phosphate composite material into a high polymer solution, and performing first stirring treatment to obtain first biological ink; the high polymer solution is prepared by dissolving a high polymer in a second solvent; the mass ratio of the mineralized collagen porous calcium phosphate composite material to the polymer is 1 (1.5-9);

s2, mixing the mineralized collagen porous calcium phosphate composite material, the bioactive material and the first solvent, and performing second stirring treatment to obtain second biological ink; based on the total mass of the mineralized collagen porous calcium phosphate composite material and the bioactive material, the mineralized collagen porous calcium phosphate composite material accounts for 80-99.5% of the total mass, and the bioactive material accounts for 0.5-20% of the total mass;

In the outer layer material, the mass ratio of the first biological ink to the second biological ink is (1.5-9) 1;

9. The method of manufacturing according to claim 8, wherein:

in the step S1, the stirring speed of the first stirring treatment is 100-200 r/min, and the stirring time is 2-8 h; and/or

In the step S2, the stirring speed of the second stirring treatment is 100-200 r/min, and the stirring time is 2-8 h.