Composite artificial bone based on 3D printing and preparation method thereof

Abstract

The invention discloses a composite artificial bone based on 3D printing and a preparation method thereof. The preparation method of the composite artificial bone comprises the following steps: (1) mixing polycaprolactone and calcium phosphate to prepare a wire material; (2) according to a preset model, the wire is used for printing and forming to obtain a 3D printing porous scaffold, and the porous scaffold is of a pore channel structure; (3) filling slurry containing collagen and calcium phosphate is injected into the pore channel structure of the porous scaffold, drying is performed, and the formed composite artificial bone is obtained; and (4) performing chemical crosslinking on the composite artificial bone and a crosslinking agent, and cleaning and drying after crosslinking to obtain a composite artificial bone product. The composite artificial bone has excellent mechanical strength, and can effectively promote the growth of early blood vessels and new bones in the treatment process of large bone defect and bone nonunion.

Description

Technical Field

[0001] The present invention relates to a composite artificial bone based on 3D printing and a preparation method thereof. Background Art

[0002] At present, common methods for treating bone defects include autologous bone transplantation, allogeneic bone transplantation, and artificial bone transplantation. Autologous bone transplantation is the "gold standard" in the field of bone repair. An important reason is that autologous bone contains a rich network of nerves and blood vessels. However, its source is limited and it is difficult to be widely used. Compared with autologous bone materials, existing artificial bone materials have poor blood infiltration, and it is difficult for blood vessels and new bone to penetrate the interior of the implant. Often, after the material is implanted into the defect site, it needs to grow slowly and layer by layer into the interior. In clinical application scenarios such as large segment bone defects, using artificial bone implants to repair bone defects is very likely to result in problems such as nonunion and bone nonunion. Therefore, at present, the repair of large segment bone defects can only rely on autologous bone.

[0003] To solve the healing problem of large segment bone defects, the team of the 175th Hospital of the Chinese People's Liberation Army used a mixed transplantation of autologous bone and composite bovine cancellous bone to repair 8 cases of large segment bone defects in the femur / tibia, and achieved good results (see the literature: Repair of large segment bone defects in long bones by autologous bone / composite bovine deproteinized bone mixed transplantation, Ding Zhenqi, et al., Journal of Bone and Joint Injury, 4: 313, 2000). Some researchers improved the bioactivity of artificial bone materials by compounding collagen molecules. Collagen is an important component of human bone and can induce the proliferation and differentiation of bone cells (see the 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). The hydrophilic property of collagen molecules can improve the blood infiltration of artificial bone and promote bone penetration growth. However, relying solely on collagen molecules to provide bioactivity is far from enough. For the scheme of compounding collagen and hydroxyapatite, in the process, collagen powder and hydroxyapatite are blended and injection molded, and its products have obvious deficiencies in terms of degradation performance (hydroxyapatite is difficult to degrade in the body), pore structure (the injection molding is relatively dense), mechanical strength, etc.

[0004] The technologies of composite collagen include the freeze-drying forming method of collagen / hydroxyapatite composite and the freeze-drying technology of collagen perfusion on the surface of porous tricalcium phosphate ceramics. Among them, the content of the nano-hydroxyapatite powder used in the freeze-drying forming method of collagen / hydroxyapatite composite is 20-80%, and a higher ceramic phase content cannot be achieved. Moreover, hydroxyapatite is difficult to degrade in the body. The main body of the freeze-drying technology of collagen perfusion on the surface of porous tricalcium phosphate ceramics is the sintered calcium phosphate ceramics, and the matrix toughness is insufficient, and the unique shaping technology of collagen sponge cannot be achieved.

[0005] At present, the biodegradable polymer materials used for 3D printing in biomedicine are mainly polylactic acid. The melt deposition molding (FDM) is used to prepare filamentous materials and form them in a layer-by-layer stacking manner. This molding method has high precision, low cost and strong repeatability in the printing process. However, the degradation products of polylactic acid are acidic, which is not conducive to new bone growth and has poor toughness. At the same time, hydroxyapatite is compounded in the 3D printing product. Its chemical composition is the same as the inorganic component in human bone, which has a promoting effect on the adhesion and proliferation of osteoblasts. However, it degrades slowly in the body and only forms new bone tissue on the surface, lacking the ability to induce bone formation.

[0006] Some researchers also have relevant achievements in binding collagen molecules to the surface of 3D printed polycaprolactone scaffolds. Specifically, the polycaprolactone material is 3D printed to form a porous scaffold, and then soaked successively with sodium hydroxide and collagen solution to obtain a scaffold material with collagen molecules grafted on the surface. This structure can improve the cell adhesion performance to a certain extent, but the surface collagen content is very low, and it is difficult to continuously promote new bone ingrowth, and the technology cannot be applied to the repair of large segment bone defects.

[0007] The above information disclosed in the background art section is only used to strengthen the understanding of the background of the present application. Therefore, it may include information that does not constitute the prior art known to those of ordinary skill in the art. Summary of the Invention

[0008] The purpose of the present invention is to provide a 3D printing-based composite artificial bone and its preparation method. The composite artificial bone has better mechanical strength and can effectively promote the early blood vessel and new bone growth in the treatment of large segment bone defects and nonunion.

[0009] One aspect of the present invention provides a preparation method of a composite artificial bone, including the following steps:

[0010] (1) Mix polycaprolactone and calcium phosphate and make them into filaments;

[0011] (2) According to a preset model, use the filaments to print and form a 3D printed porous scaffold, and the porous scaffold has a pore structure;

[0012] (3) Inject the filling slurry into the pore structure of the porous scaffold. The filling slurry contains collagen and calcium phosphate, and then dry it to obtain the formed composite artificial bone.

[0013] In a preferred embodiment, the preparation method further includes the following steps:

[0014] (4) Chemically crosslink the composite artificial bone and the crosslinking agent, wash it after crosslinking, and then dry it to obtain the composite artificial bone product.

[0015] In a preferred embodiment, in step (4), immerse the composite artificial bone in the crosslinking agent solution. The crosslinking agent includes one or a combination of more selected from genipin, glutaraldehyde, and carbodiimide, and the concentration of the crosslinking agent is 0.05 - 5 wt.%. More preferably, the concentration of the crosslinking agent is 1 - 3 wt.%. For example, the concentration of the crosslinking agent can be 1 wt.%, 2 wt.%, or 3 wt.%.

[0016] In a preferred embodiment, in steps (1) and (3), the calcium phosphate includes one or a combination of more selected from tricalcium phosphate, magnesium-containing tricalcium phosphate, and hydroxyapatite. The calcium phosphate is in powder form, and its d50 particle size is 500 nm - 50 μm. More preferably, the calcium phosphate is magnesium-containing tricalcium phosphate, and the particle size is 1 - 20 μm.

[0017] In a more preferred embodiment, in step (1), the content of calcium phosphate in the wire is 10 - 50 wt.%, the intrinsic viscosity of polycaprolactone is 0.5 - 1.2 dL / g. Add polycaprolactone and calcium phosphate powder into a mixer and heat and mix them to obtain a uniformly mixed polycaprolactone / calcium phosphate composite material. Extrude it through a screw extruder to obtain a wire with a diameter of 1 - 3 mm, and set the wire extrusion temperature to 70 - 120 °C. Further preferably, the content of calcium phosphate in the wire is 20 - 45 wt.%; even more preferably, the content of calcium phosphate in the wire is 30 - 45 wt.%; specifically, it is 33 - 43 wt.%.

[0018] In a preferred embodiment, in step (2), set the printing temperature to 120 - 250 °C, the printing layer height to 0.1 - 0.3 mm, the printing speed to 5 - 50 mm / s, the filling rate to 20 - 100%, and the wire diameter to 0.1 - 0.8 mm.

[0019] In a preferred embodiment, in step (3), collagen is added to an acid solution and stirred until completely dissolved. Then, calcium phosphate powder and a crosslinking agent are added in sequence and stirred evenly to obtain the mixed slurry. The porous scaffold is immersed in the mixed slurry, and vacuum filtration is carried out to make the internal pores of the porous scaffold filled with the mixed slurry. Among them, the mass ratio of collagen to calcium phosphate powder in the mixed slurry is 0.1 - 1, the concentration of the acid solution is 0.2 - 2 wt.%, the concentration of collagen is 5 - 30 mg / mL, the concentration of the crosslinking agent is 10 - 500 ppm, the vacuum degree of vacuum filtration is 0.060 - 0.095 MPa, the acid solution includes hydrochloric acid or acetic acid, and the crosslinking agent includes genipin and / or glutaraldehyde selected therefrom. More preferably, the mass ratio of collagen to calcium phosphate powder in the mixed slurry is 0.1 - 5, and further preferably 0.1 - 0.3.

[0020] In a preferred embodiment, the specific implementation of the preparation method is as follows:

[0021] Polycaprolactone and calcium phosphate powder are added to a mixer and heated and mixed. The heating temperature is 60 - 150 °C, and the mixing time is 0.5 - 12 hours. The obtained mixed material is put into a granulator and crushed to form particulate materials with a diameter of 0.2 - 5 mm. Then, it is added to the mixer again and mixed evenly. The heating temperature is 60 - 150 °C, and the mixing time is 0.5 - 1 hour to obtain a uniformly mixed polycaprolactone / calcium phosphate composite material, wherein the calcium phosphate content is 10 - 50 wt%. The polycaprolactone / calcium phosphate powder composite material is added to a screw extruder, and the wire extrusion temperature is set at 70 - 120 °C, and the wire diameter is 1 - 3 mm;

[0022] According to a preset model, 3D printed porous scaffolds are formed using the wire material, wherein the printing temperature is 120 - 250 °C, the printing layer height is 0.1 - 0.3 mm, the printing speed is 5 - 50 mm / s, the filling rate is 20 - 100%, and the wire extrusion diameter is 0.1 - 0.8 mm;

[0023] Collagen is added to the acid solution, and stirred with a tetrafluoro stirring paddle at 200 - 600 rpm for 1 - 10 minutes until completely dissolved. The calcium phosphate powder is added to the collagen solution and continuously stirred for 5 - 30 minutes. The crosslinking agent is added to the mixed slurry and stirred for 1 - 5 minutes. The porous scaffold is completely immersed in the mixed slurry, placed in a dryer, and vacuum filtered to make the internal pores completely filled with the mixed slurry. After freeze-drying, a preliminarily formed composite artificial bone is obtained. The mass ratio of collagen to calcium phosphate powder in the mixed slurry is 0.1 - 1;

[0024] The composite artificial bone is soaked in the crosslinking agent solution for 6 - 48 hours. After crosslinking, it is put into pure water and stirred and washed at 200 - 600 rpm for 6 - 24 hours, and then freeze-dried to obtain the composite artificial bone product.

[0025] The second aspect of the present invention provides a composite artificial bone, comprising a porous scaffold with a pore structure and a filling material injected into the pore structure. The porous scaffold comprises polycaprolactone and calcium phosphate, and the filling material comprises collagen and calcium phosphate.

[0026] In a preferred embodiment, the porous scaffold is a 3D printed porous scaffold, the porosity of the porous scaffold is 20-70%, the compressive strength is 10-15 Mpa, the filaments constituting the porous scaffold are composed of polycaprolactone and calcium phosphate, wherein the content of calcium phosphate is 10-50 wt.%, and the filament diameter is 0.1-0.8 mm; the mass ratio of collagen to calcium phosphate powder in the filling material is 0.1-1.

[0027] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0028] For the composite artificial bone of the present invention, a porous scaffold is printed and made with polycaprolactone / calcium phosphate as the raw material. By filling a slurry containing collagen and calcium phosphate into the internal pores of the porous scaffold, the mechanical strength of the prepared composite artificial bone can reach 5-20 MPa. After being implanted into the body for 1-3 months, the growth and penetration of blood vessels and new bone inside the scaffold can be achieved, which can effectively promote the early blood vessel growth during the treatment of large segment bone defects and non-union of bones. At the same time, the 3D printed polycaprolactone / calcium phosphate degrades after being implanted for 1-2 years, and can provide mechanical support for the defect site for 6-12 months, ultimately realizing bone reconstruction at the large segment bone defect and non-union site. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the present invention, the drawings required for describing the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0030] Figure 1 It is a photograph of the composite artificial bone prepared in Example 1 of the present invention.

[0031] Figure 2 It is a photograph of the liquid wettability test of the composite artificial bone prepared in Example 1 of the present invention.

[0032] Figure 3 It is a graph of the compressive strength test of the composite artificial bone prepared in Example 1 of the present invention before and after degradation in vitro.

[0033] Figure 4 It is a photograph of the Masson staining results of the composite artificial bone prepared in Example 1 of the present invention after being implanted into the femoral condyle of New Zealand rabbits for 1 and 3 months.

[0034] Figure 5 This is a photograph of the liquid wettability test of the porous scaffold prepared in the comparative example of the present invention. DETAILED DESCRIPTION

[0035] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0036] Compared with autologous bone materials, existing artificial bone materials have poor blood infiltration, and it is difficult for blood vessels and new bone to penetrate the interior of the implant. Often, after the material is implanted into the defect, it needs to grow slowly and layer by layer into the defect. In clinical application scenarios such as large bone defects, the use of artificial bone implants to repair bone defects is very likely to cause problems such as non-union and bone non-union. Therefore, the current repair of large bone defects can only rely on autologous bone.

[0037] Porous scaffolds can be constructed using 3D printing technology. Currently reported technologies include 3D printed products made of materials such as polylactic acid, polycaprolactone, and polyetheretherketone. However, porous scaffolds prepared by organic molecules generally have poor affinity with bone tissue, and it is difficult for new bone to grow into the internal pores. To address this problem, even if powder materials such as hydroxyapatite are compounded in polymer materials to increase biological activity, or hydrophilic groups such as collagen sponges and gelatin molecules are grafted on the surface of 3D printed scaffolds. However, it is still unable to solve the problems of poor blood infiltration and lack of blood vessel growth in orthopedic problems such as large bone defects and nonunions.

[0038] Therefore, the following implementation method forms a composite artificial bone product by constructing a porous scaffold of 3D printed polycaprolactone / calcium phosphate and infusing cross-linked collagen sponge. The collagen sponge part in the composite artificial bone is expected to degrade in 3-6 months, which can effectively promote the early blood vessel growth in the treatment of large bone defects and nonunion. At the same time, 3D printed polycaprolactone / calcium phosphate degrades after 1-2 years of implantation, and can provide mechanical support for the defect site in 6-12 months, and ultimately achieve bone reconstruction of large bone defects and nonunion sites. By compounding collagen sponge in the 3D printed scaffold, the blood infiltration of the material is significantly improved, which helps the cells in the blood to quickly penetrate the interior of the scaffold after implantation, and realizes the penetration and ingrowth of new bone and blood vessels in 1-3 months, which can effectively promote the early blood vessel growth in the treatment of large bone defects and nonunion.

[0039] The 3D printed scaffold of the following embodiments is a composite of polycaprolactone and calcium phosphate, and has better osteogenic activity than the pure polycaprolactone component. At the same time, there are also significant differences in the form of the internally compounded collagen. In the following embodiments, collagen and calcium phosphate are compounded, vacuum-injected into the polycaprolactone scaffold / calcium phosphate, and crosslinked into a sponge network, which penetrates each other with the porous 3D printed scaffold to form a composite artificial bone. The collagen sponge structure in the composite artificial bone plays a role in early filling of the implant, and the degradation time is 3-6 months, which can effectively guide new bone regeneration. If directly treated by soaking in a collagen solution, the collagen sponge mainly plays a role in surface modification, with a short action time and unable to guide blood vessels and new bone growth.

[0040] A preparation method of a 3D printed composite artificial bone includes the following steps:

[0041] (1) Weigh polycaprolactone and calcium phosphate and mix them evenly by melt blending, and then use a screw extruder to make filaments from the evenly mixed materials;

[0042] (2) According to a preset model, print the filaments into a 3D printed porous scaffold;

[0043] (3) Weigh a collagen sponge, dissolve it in an acid solution, mix it with calcium phosphate powder, and then add a crosslinking agent for crosslinking; then inject the mixed slurry into the 3D printed porous scaffold through a vacuum filtration process, and obtain a preliminarily formed composite artificial bone material after freeze-drying;

[0044] (4) Immerse the composite artificial bone in a chemical crosslinking agent solution for a second chemical crosslinking, put it into a cleaning agent and pure water for stirring and cleaning after crosslinking, and then freeze-dry and shape it to obtain a 3D printed composite artificial bone material.

[0045] The process of step (1) is specifically as follows: Add polycaprolactone and calcium phosphate powder into a melt blender for heating and mixing, the heating temperature is 60-150 °C, and the mixing time is 0.5-12 hours; put the obtained mixed material into a pelletizer for crushing to form granular materials with a diameter of 0.2-5 mm; then add them into the melt blender for mixing evenly again, the heating temperature is 60-150 °C, and the mixing time is 0.5-1 hour to obtain a uniformly mixed polycaprolactone / calcium phosphate composite material. Add the polycaprolactone / calcium phosphate powder composite material into a screw extruder, and set the wire extrusion temperature to 70-120 °C, and the wire diameter width to 1-3 mm.

[0046] In the polycaprolactone / calcium phosphate composite material in step (1), the calcium phosphate content is 10-50 wt%, and the intrinsic viscosity of the used polycaprolactone material is 0.5-1.2 dL / g. The used calcium phosphate powder materials include but are not limited to tricalcium phosphate, magnesium-containing tricalcium phosphate, hydroxyapatite, etc., and the d50 particle size of the calcium phosphate powder is 500 nm-50 μm.

[0047] In step (2), the printing temperature is 120 - 250 °C, the printing layer height is 0.1 - 0.3 mm, the printing speed is 5 - 50 mm / s, the filling rate is 20 - 100%, and the filament width is 0.1 - 0.8 mm.

[0048] The process of step (3) is as follows: Weigh collagen and add it to an acid solution. Stir with a tetrafluoro stirring paddle at 200 - 600 rpm for 1 - 10 minutes until completely dissolved. Add all the calcium phosphate powder to the collagen solution and continue stirring for 5 - 30 minutes. Add a chemical crosslinking agent to the mixed slurry and stir for 1 - 5 minutes. Immerse the 3D printed porous scaffold obtained in step (2) completely in the mixed slurry, place it in a dryer and perform vacuum pumping so that the internal pores are completely filled with the mixed slurry, and obtain a preliminarily formed composite artificial bone material after freeze-drying. The collagen used in step (3) includes but is not limited to type I collagen, type II collagen, and recombinant collagen, etc., with a molecular weight of 20,000 - 300,000 Da. The acid solution includes but is not limited to hydrochloric acid solution, acetic acid solution, etc. The calcium phosphate powder includes but is not limited to tricalcium phosphate, magnesium-containing tricalcium phosphate, hydroxyapatite, and octacalcium phosphate, etc. The acid solution includes but is not limited to hydrochloric acid solution, acetic acid solution, etc. The chemical crosslinking agent includes but is not limited to genipin, glutaraldehyde, etc. The mass ratio of collagen to calcium phosphate powder is 0.1 - 1, the concentration of the acid solution is 0.2 - 2 wt.%, the concentration of collagen is 5 - 30 mg / mL, and the concentration of the chemical crosslinking agent is 10 - 500 ppm. The vacuum degree of vacuum filtration is 0.060 - 0.095 MPa.

[0049] The process of step (4) is as follows: Immerse the preliminarily formed composite artificial bone material in a chemical crosslinking agent solution for 6 - 48 hours. After crosslinking, put it into pure water and stir and wash at 200 - 600 rpm for 6 - 24 hours, and obtain the composite artificial bone material after freeze-drying treatment. Among them, the chemical crosslinking agent includes but is not limited to genipin, glutaraldehyde, carbodiimide, etc., with a concentration of 0.05 - 5%.

[0050] The mechanical strength of the prepared composite artificial bone is 5 - 20 MPa, and after being implanted in the body for 1 - 3 months, the growth and penetration of blood vessels and new bone inside the scaffold can be achieved.

[0051] The following is specifically elaborated through examples.

[0052] The magnesium-containing tricalcium phosphate used in the examples and comparative examples was prepared according to the method in Example 1 disclosed in the applicant's patent CN117122734A, and the magnesium content was 1.9 wt.%.

[0053] Example 1

[0054] Mixing and Kneading: Weigh 300 g of polycaprolactone and 150 g of magnesium-containing tricalcium phosphate using an analytical balance, add them to a kneader, and mix at 100 °C for 1 hour. Take out the product, put it into a granulator to crush, and then add it back to the kneader and mix at 100 °C for 0.5 hour.

[0055] Pelletizing and Filament Making: Put the mixed material into a granulator to crush, and sieve to obtain mixed granular materials with a size of 0.2 - 5 mm. Then put them into a screw extruder to make filaments. The filament-making temperature is set at 90 °C, the filament-making speed is 1.5 m / min, and the average filament diameter is 2.85 mm.

[0056] Printing and Molding: Connect the prepared filaments to a 3D printer and import the digital model. The model filling rate is set at 40%, the printing temperature is set at 200 °C, the layer height is 0.2 mm, the printing speed is 10 mm / s, and the filament width is 0.4 mm. Print to obtain a 3D printed porous scaffold.

[0057] Weighing and Dissolving: Weigh 100 g of 1 wt% acetic acid solution using an analytical balance, add 1 g of bovine type I collagen sponge, and stir with a tetrafluoro stirring paddle at 400 rpm for 30 minutes until completely dissolved.

[0058] Mixing and Preparation: Weigh 5.5 g of magnesium-containing tricalcium phosphate powder, add all the magnesium-containing tricalcium phosphate powder to the collagen solution, and stir with a tetrafluoro stirring paddle at 400 rpm for 10 minutes. Add 1 mL of 1 wt.% glutaraldehyde solution to the mixed solution. Stir with a tetrafluoro stirring paddle at 400 rpm for 1 minute.

[0059] Perfusion Molding: Completely immerse the 3D printed scaffold in the mixed slurry, put it into a dryer, vacuum filter until the vacuum degree reaches 0.090 MPa, then release the air, and repeat the vacuum pumping 5 times to make the internal pores completely filled with the mixed slurry. Then crosslink the material at -4 °C for 1 hour, and then put the mold into a -40 °C low-temperature storage cabinet to freeze for 4 hours. After freeze-drying, obtain a preliminarily formed composite artificial bone material.

[0060] Crosslinking and Cleaning: Immerse the preliminarily formed composite artificial bone material in 1 wt.% glutaraldehyde solution for 12 hours, and put it into pure water and stir and clean at 400 rpm for 12 hours.

[0061] Freeze-Drying: Put the cleaned sample into a -40 °C refrigerator to freeze for 4 hours, and then freeze-dry for 36 hours to finally obtain a 3D printed composite artificial bone material. Figure 1 This is a photo of the obtained composite artificial bone.

[0062] Detection: The 3D printed composite artificial bone material has good liquid wettability, see Figure 2 .

[0063] The porosity is 52%, the compressive strength is 11.4 MPa, and the compressive strength shows no obvious decrease after being immersed in tris buffer solution with pH = 7.3 for 1 month in the in vitro degradation test. See Figure 3 .

[0064] In the composite artificial bone material, the collagen sponge part can induce blood vessels and new bone to grow into the material internally 1 month after being implanted into the femoral condyle of a rabbit, and has excellent osteogenic effect 3 months after implantation. See Figure 4 , which can effectively solve the problem of nonunion of large segmental bone defects.

[0065] Example 2

[0066] Mixing and kneading: Weigh 400 g of polycaprolactone and 300 g of magnesium-containing tricalcium phosphate using an analytical balance, add them to a kneader and mix at 120 °C for 2 hours. Take out the product, put it into a granulator to crush it, and then add it back into the kneader and mix at 120 °C for 0.5 hour.

[0067] Pelletizing and wire making: Put the mixed material into a granulator to crush it, and sieve to obtain mixed granular materials with a size of 0.2 - 5 mm. Then put them into a screw extruder to make wires. The wire-making temperature is set at 80 °C, the wire-making speed is 1.2 m / min, and the average wire diameter is 2.85 mm.

[0068] Printing and forming: Connect the prepared wire material to a 3D printer and import the digital model. The model filling rate is set at 50%, the printing temperature is set at 200 °C, the layer height is 0.2 mm, the printing speed is 10 mm / s, and the wire width is 0.4 mm. Print to obtain a 3D printed porous scaffold.

[0069] Weighing and dissolving: Weigh 50 g of 1 wt.% acetic acid solution using an analytical balance, add 0.5 g of bovine type I collagen sponge, and stir with a tetrafluoro stirring paddle at 400 rpm for 30 minutes until completely dissolved.

[0070] Mixing and preparing: Weigh 3 g of magnesium-containing tricalcium phosphate powder, add all the magnesium-containing tricalcium phosphate powder into the collagen solution, and stir with a tetrafluoro stirring paddle at 400 rpm for 10 minutes. Add 0.5 mL of 1 wt.% glutaraldehyde solution to the mixed solution. Stir with a tetrafluoro stirring paddle at 400 rpm for 1 minute.

[0071] Perfusion and forming: Completely immerse the 3D printed scaffold in the mixed slurry, put it into a dryer and vacuum filter until the vacuum degree reaches 0.090 MPa and then release the air. Repeat the vacuum pumping 5 times to make the internal pores completely filled with the mixed slurry. Then crosslink the material at -4 °C for 1 hour, and then put the mold into a -40 °C low-temperature storage cabinet and freeze it for 4 hours. After freeze-drying, obtain the preliminarily formed composite artificial bone material.

[0072] Crosslinking cleaning: Immerse the preliminarily formed composite artificial bone material in a 1 wt.% glutaraldehyde solution for 12 hours, and then place it in pure water and stir-clean at 400 rpm for 12 hours.

[0073] Freeze-drying: Place the cleaned sample in a -40°C refrigerator and freeze it for 4 hours, and then freeze-dry it for 36 hours to finally obtain the 3D printed composite artificial bone material.

[0074] Detection: The mechanical strength of the 3D printed composite artificial bone material is 14.5 MPa, and the porosity is 44%.

[0075] Comparative example

[0076] Mixing and kneading: Weigh 300 g of polycaprolactone and 150 g of magnesium-containing tricalcium phosphate using an analytical balance, add them to a kneader and mix at 100°C for 1 hour. Take out the product, crush it in a pelletizer, and then add it back to the kneader and mix at 100°C for 0.5 hour.

[0077] Pelletizing and wire-making: Place the mixed material in a pelletizer to crush it, and sieve it to obtain a mixed granular material with a size of 0.2 - 5 mm. Then put it into a screw extruder to make wires. The wire-making temperature is set at 90°C, the wire-making speed is 1.5 m / min, and the average wire diameter is 2.85 mm.

[0078] Printing and forming: Connect the prepared wire material to a 3D printer and import the digital model. The model filling rate is set at 40%, the printing temperature is set at 200°C, the layer height is 0.2 mm, the printing speed is 10 mm / s, and the wire width is 0.4 mm. Print to obtain a 3D printed porous scaffold.

[0079] Detection: The 3D printed porous scaffold without composite collagen sponge has poor liquid wettability, it is difficult to induce the growth of cells in the blood into it, and the new bone growth is slow. See Figure 5 .

[0080] Although the 3D printed porous scaffold of the comparative example maintains a through-porous structure, it has no guiding effect on liquids, and it is difficult for blood and cells to enter autonomously. In the example, by compounding collagen sponge / magnesium-containing tricalcium phosphate in the 3D printed scaffold, the blood wettability of the material is significantly improved. The excellent wettability helps the cells in the blood to quickly penetrate the inside of the scaffold after the scaffold is implanted, promoting new bone growth.

[0081] In the example, a 3D printed polycaprolactone / magnesium-containing tricalcium phosphate porous scaffold is constructed, and the crosslinked collagen sponge / magnesium-containing tricalcium phosphate is perfused to form a composite artificial bone material, which is a fully degradable system. At the same time, active magnesium elements are doped in the tricalcium phosphate, which can significantly enhance the bioactivity of the composite material and promote the osteogenic differentiation of cells.

[0082] In the example, a 3D printed polycaprolactone / magnesium-containing tricalcium phosphate porous scaffold was constructed and infused with a cross-linked collagen sponge to form a composite artificial bone material. The expected degradation time of the collagen sponge part in the composite artificial bone is 3 - 6 months, which can effectively promote early blood vessel growth during the treatment of large segment bone defects and nonunion. At the same time, the 3D printed polycaprolactone / magnesium-containing tricalcium phosphate degrades after 1 - 2 years of implantation, and can provide mechanical support for the defect site for 6 - 12 months, ultimately achieving bone reconstruction at the large segment bone defect and nonunion sites.

[0083] As shown in this specification and the claims, the terms "comprising" and "including" only indicate the inclusion of the explicitly identified steps and elements, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements. The term "and / or" used herein includes any combination of one or more of the related listed items.

[0084] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. If there are contradictions or inconsistencies between the definitions used herein and those contained in other published documents, the definitions used herein shall prevail.

[0086] The above examples are only for illustrating the technical concept and features of the present invention. They are a preferred example, and their purpose is to enable those familiar with this technology to understand the content of the present invention and implement it accordingly. However, the protection scope of the present invention cannot be limited thereby. Any equivalent transformation or modification made according to the principle of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for preparing a composite artificial bone, characterized in that: The steps include: (1) mixing polycaprolactone and calcium phosphate and preparing a filament; (2) According to a preset model, the filament is used to print and shape to obtain a 3D printed porous scaffold, wherein the porous scaffold has a pore structure; (3) injecting a filling slurry into the pore structure of the porous scaffold, wherein the filling slurry contains collagen and calcium phosphate, and drying to obtain a formed composite artificial bone.

2. The method for preparing the composite artificial bone according to claim 1, characterized in that: The preparation method further comprises the following steps: (4) chemically cross-linking the composite artificial bone and the cross-linking agent, washing and drying after cross-linking to obtain a composite artificial bone product.

3. The method for preparing the composite artificial bone according to claim 2, characterized in that: In step (4), the composite artificial bone is immersed in a crosslinking agent solution, wherein the crosslinking agent comprises a combination of one or more selected from genipin, glutaraldehyde, and carbodiimide, and the concentration of the crosslinking agent is 0.05-5wt.%.

4. The method for preparing a composite artificial bone according to claim 1 or 2, characterized in that: In steps (1) and (3), the calcium phosphate comprises a combination of one or more selected from tricalcium phosphate, magnesium-containing tricalcium phosphate, and hydroxyapatite, and the calcium phosphate is in the form of powder with a d50 particle size of 500nm-50μm.

5. The method for preparing the composite artificial bone according to claim 4, characterized in that: In step (1), the content of calcium phosphate in the wire is 10-50wt.%, the intrinsic viscosity of polycaprolactone is 0.5-1.2dL / g, polycaprolactone and calcium phosphate powders are added to an internal mixer and heated and mixed to obtain a uniformly mixed polycaprolactone / calcium phosphate composite material, which is extruded through a screw extruder to obtain a wire with a diameter of 1-3mm, and the wire outlet temperature is set to 70-120°C.

6. The method for preparing a composite artificial bone according to claim 1 or 2, characterized in that: In step (2), the printing temperature is set to 120-250° C., the printing layer height is 0.1-0.3 mm, the printing speed is 5-50 mm / s, the filling rate is 20-100%, and the filament diameter is 0.1-0.8 mm.

7. The method for preparing a composite artificial bone according to claim 1 or 2, characterized in that: In step (3), collagen is added to an acid solution and stirred until completely dissolved, calcium phosphate powder and a cross-linking agent are added in sequence, and stirred evenly to obtain the mixed slurry, the porous scaffold is immersed in the mixed slurry, and vacuum filtration is performed so that the internal pores of the porous scaffold are filled with the mixed slurry; wherein the mass ratio of collagen to calcium phosphate powder in the mixed slurry is 0.1-1, the concentration of the acid solution is 0.2-2wt.%, the concentration of collagen is 5-30mg / mL, the concentration of the cross-linking agent is 10-500ppm, the vacuum degree of the vacuum filtration is 0.060-0.095MPa, the acid solution includes hydrochloric acid or acetic acid, and the cross-linking agent includes genipin and / or glutaraldehyde.

8. The method for preparing composite artificial bone according to claim 1, characterized in that: The preparation method is specifically implemented as follows: The polycaprolactone and calcium phosphate powders are added to an internal mixer and heated and mixed, the heating temperature is 60-150° C., and the mixing time is 0.5-12 hours; the obtained mixed material is put into a pelletizer and crushed to form a granular material with a diameter of 0.2-5 mm; then the mixed material is added to the internal mixer and mixed again, the heating temperature is 60-150° C., and the mixing time is 0.5-1 hour to obtain a uniformly mixed polycaprolactone / calcium phosphate composite material, wherein the calcium phosphate content is 10-50wt%; the polycaprolactone / calcium phosphate powder composite material is added to a screw extruder, the wire outlet temperature is set to 70-120° C., and the wire diameter is 1-3 mm; According to the preset model, the filament is used for printing to obtain a 3D printed porous scaffold, wherein the printing temperature is 120-250° C., the printing layer height is 0.1-0.3 mm, the printing speed is 5-50 mm / s, the filling rate is 20-100%, and the filament diameter is 0.1-0.8 mm; The collagen is added to the acid solution, and stirred at 200-600 rpm with a PTFE stirring paddle for 1-10 minutes until it is completely dissolved; the calcium phosphate powder is added to the collagen solution, and the stirring is continued for 5-30 minutes; the cross-linking agent is added to the mixed slurry, and the mixture is stirred for 1-5 minutes; the porous scaffold is completely immersed in the mixed slurry, and the scaffold is placed in a dryer and vacuumed to completely fill the internal pores with the mixed slurry, and a preliminarily formed composite artificial bone is obtained after freeze drying; the mass ratio of collagen to calcium phosphate powder in the mixed slurry is 0.1-1; The composite artificial bone is immersed in a crosslinking agent solution for 6-48 hours, and after crosslinking, is placed in pure water for stirring and washing at 200-600 rpm for 6-24 hours, and is freeze-dried to obtain a composite artificial bone product.

9. A composite artificial bone, characterized in that: It comprises a porous support with a pore structure and a filling material injected into the pore structure, wherein the porous support comprises polycaprolactone and calcium phosphate, and the filling material comprises collagen and calcium phosphate.

10. The composite artificial bone according to claim 1, characterized in that: The porous scaffold is a 3D printed porous scaffold, the porosity of the porous scaffold is 20-70%, the compressive strength is 10-15Mpa, the silk thread constituting the porous scaffold is composed of polycaprolactone and calcium phosphate, wherein the content of calcium phosphate is 10-50wt.%, and the diameter of the silk thread is 0.1-0.8mm; the mass ratio of collagen to calcium phosphate powder in the filling material is 0.1-1.

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