3D printing artificial bone with hypoxia-induced vascularization function as well as preparation method and application of 3D printing artificial bone

Abstract

The invention belongs to the technical field of bone repair materials, and discloses a 3D printing artificial bone with the hypoxia-induced vascularization function and a preparation method and application of the 3D printing artificial bone with the hypoxia-induced vascularization function. The secondary pore structures are communicated with pores of the artificial bone and are uniformly distributed on the surface of the artificial bone, and the secondary pore structures are semi-closed structures. According to the artificial bone, a semi-closed pore structure with a small opening and a large inner cavity is constructed, under the limitation of a structural space, an anoxic microenvironment is gradually formed in pores of the artificial bone, an anoxic induction factor HIF-1alpha is generated by gathered cells, and blood vessel growth is induced and promoted through an anoxic induction signal channel. According to the 3D printing artificial bone, medicine does not need to be added, the processing cost is reduced, the side effect of the medicine is avoided, the hypoxia induction mechanism is achieved through the internal semi-closed structure design, vascularization is accelerated, bone tissue regeneration is promoted, and the vascularization bone forming function of the 3D printing artificial bone is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and specifically relates to a 3D printed artificial bone with hypoxia-induced vascularization function, and a preparation method and application thereof. Background Art

[0002] Bone tissue damage affects millions of people around the world. In recent decades, the development of artificial bone has been at the forefront of this field. As researchers gain a deeper understanding of physiology and healing mechanisms, various bionic materials have been reported. However, artificial bones currently have the problem of poor vascularization and low repair ability. Achieving vascularized bone regeneration is one of the effective solutions to bone defect repair. Since the formation of new blood vessels in the process of bone defect repair is earlier than the osteogenic repair effect, the new capillaries will form new microcirculation in the cartilage and callus, and bring in osteoblasts to promote the formation of new bone. Traditional calcium phosphate artificial bone cannot provide sufficient blood supply to the defect area, so problems such as bone central necrosis and repair failure often occur. The key to repairing bone defects is to achieve vascularization and ensure adequate blood supply. Therefore, the development of effective artificial bones that can promote vascular regeneration is an urgent problem to be solved.

[0003] At present, methods for promoting angiogenesis include loading vascular endothelial growth factor (VEGF) (CN202311823004.5, a calcium phosphate bone repair scaffold and its preparation method and application). The main calcium phosphate component of the 3D printed calcium phosphate artificial bone loaded with VEGF in this invention has good osteoinductivity, and the loaded VEGF can effectively promote angiogenesis and the formation of vascular network in the defect area. In addition, there are small molecule drug-loaded methods to promote blood vessel growth (CN202310851700.0, a method for preparing a bone repair scaffold that can sustainably release small molecule drugs). However, these methods have problems such as drug non-specificity and continuous release of growth factors. New methods are needed to promote angiogenesis, thereby further promoting the growth of bone tissue.

[0004] In order to solve this problem, researchers have found that cells can show different physiological responses under specific environmental conditions. In a hypoxic environment, cell gene transcription can be induced, which can promote blood vessel regeneration. A variety of transcription factors can respond to hypoxia and induce or inhibit genes, leading to the initiation of adaptive transcriptional responses. The most important of these is hypoxia-inducible factor (HIF), which is called the "master regulator" of the animal's adaptive response to hypoxia. It can promote vascular endothelial growth factor (VEGF) and assist angiogenesis. At present, most of them use drug-loaded treatment methods to achieve the regulation of hypoxic environments. For example, ion release is used to regulate the expression of angiogenic active factors in tissues (CN202210290471.5, a polyetheretherketone / lithium-containing bioceramic bone repair material and its preparation method); it can also promote angiogenesis by loading drugs, such as deferoxamine (DFO), so as to better promote bone repair (CN202110603045.8, a vascularized bone biomimetic multifunctional tissue engineering scaffold with anti-inflammatory effect and its preparation method). This method has problems such as drug release, drug non-specificity, and drug toxicity, and its clinical use is limited. Therefore, new methods are needed to regulate the hypoxic microenvironment to promote vascular ingrowth and tissue regeneration. The specific structure in natural biological tissues plays an important role in their physiological functions. The base structure design of porous implants can be optimized to regulate cell behavior and guide tissue regeneration repair. Literature (Adv Sci (Weinh), 2023, 10 (15): e2207224) found in animal experiments that HIF-1α is highly expressed in the center of artificial bone, but its expression location cannot be applied to the whole.

[0005] In summary, hypoxia induction is a new way to promote the vascularization of artificial bone. Existing hypoxia-inducible functional scaffolds usually use drugs to promote the release of hypoxia-inducible factors, but there are still problems such as drug controlled release and drug side effects. Structural induction can also achieve hypoxia regulation. It is urgent to develop a new structural design of artificial bone with hypoxia-induced blood vessel growth to improve the vascularization of artificial bone in bone defect regeneration and repair. Summary of the invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a 3D printed artificial bone with hypoxia-induced vascularization function.

[0007] Another object of the present invention is to provide a method for preparing a 3D printed artificial bone with hypoxia-induced vascularization function, which mainly adopts a 3D printing molding process. By designing the three-dimensional pore structure of the 3D printed model, cells can aggregate in the space to form an oxygen-deficient cellular microenvironment, activate the hypoxia-induced signal pathway, and further promote the growth of blood vessels and tissues.

[0008] Another object of the present invention is to provide the application of the above-mentioned 3D printed artificial bone with hypoxia-induced vascularization function. The present invention provides a new method to activate the hypoxia-induced signaling pathway, which can be applied to tissue repair.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A 3D printed artificial bone with hypoxia-induced vascularization function, wherein a secondary pore structure is arranged on the surface of the artificial bone, the secondary pore structure is connected with the pores of the artificial bone and is evenly distributed on the surface of the artificial bone, and the secondary pore structure is a semi-closed pore structure with a small opening and a large inner cavity.

[0011] Preferably, a secondary pore structure is provided on the cubic crossbeam support;

[0012] Preferably, T-shaped grooves are respectively provided on both sides of the secondary pore structure, and the T-shaped grooves have an inner cavity 2 and an opening 1 that are interconnected, wherein the width of the inner cavity is greater than the width of the opening;

[0013] The two T-shaped grooves in the secondary pore structure are axially symmetrically distributed along the T-shaped transverse straight line, and the distances from the two T-shaped grooves to the symmetry axis are equal.

[0014] Preferably, the inner cavity width B of the secondary pore structure is 500-1000 μm, the opening width A is 150-500 μm, the T-slot pore length C is 500-1000 μm, and the pore depth D is 200-800 μm.

[0015] Preferably, the overall porosity of the 3D printed artificial bone with hypoxia-induced vascularization function is 60%-70%.

[0016] A method for preparing a 3D printed artificial bone with hypoxia-induced vascularization function, comprising the following steps:

[0017] Step 1: Design the model using 3D drawing software;

[0018] Step 2: Print the model through a 3D molding process, and then obtain the 3D printed artificial bone with the hypoxia-induced vascularization function through post-processing.

[0019] Preferably, the 3D forming process in step 1 is laser melting sintering, stereolithography or digital light processing;

[0020] Step 1: The first layer printing time of the 3D molding process is 60-75s, the printing time of each layer is 4-6s, and the optical power is 20-25mW / cm 2 .

[0021] Preferably, the slurry for 3D printing in step 2 comprises: ceramic powder or metal powder or photocurable polymer monomer, photosensitive resin and photoinitiator.

[0022] Preferably, the ceramic powder is at least one of hydroxyapatite, tricalcium phosphate, wollastonite, and magnesium leucapatite;

[0023] The photosensitive resin is at least one of 1,6-hexanediol diacrylate, hydroxyethyl acrylate or 3-hydroxymethylpropane-3-methacrylate, and the photosensitive resin is more preferably a mixture of 1,6-hexanediol diacrylate, hydroxyethyl acrylate and 3-hydroxymethylpropane-3-methacrylate, with a volume ratio of 6:3:1;

[0024] The photosensitive resin and the ceramic powder are added in a mass ratio of 1-3:1-3.

[0025] The photoinitiator is 2,4,6-trimethylbenzoyl-diphenylphosphine oxide or bisacylphosphine oxide or 1-hydroxycyclohexyl phenyl ketone.

[0026] Preferably, the amount of the photoinitiator added is 1-5% of the total volume of the photosensitive resin.

[0027] Preferably, the post-processing in step 2 includes cleaning, drying, high-temperature sintering and sterilization;

[0028] Preferably, the high temperature sintering temperature is 800-1150°C.

[0029] The sterilization is high temperature and high pressure sterilization, ultraviolet irradiation sterilization or radiation sterilization.

[0030] The application of the above-mentioned 3D printed artificial bone with hypoxia-induced vascularization function in bone tissue regeneration and repair.

[0031] The present invention has the following advantages and beneficial effects:

[0032] (1) The method of artificial bone pore structure design is adopted to design a semi-closed pore structure with a small opening and a large inner cavity. Under the limitation of structural space, a hypoxic microenvironment is gradually formed inside the pores of this artificial bone, creating a hypoxic microenvironment for tissue regeneration. This 3D printed artificial bone does not require the addition of drugs, which reduces processing costs and avoids the side effects of drugs. The internal semi-closed structure design realizes the mechanism of hypoxia induction to accelerate vascularization and promote bone tissue regeneration;

[0033] (2) By precisely regulating the size of the semi-closed pore structure, the HIF-1α / VEGF signaling pathway is effectively activated, promoting the directional migration of endothelial cells and the expression of genes related to angiogenesis;

[0034] (3) The first artificial bone with coordinated regulation of "structure-oxygen microenvironment-vascularization" combined with biochemical signals to achieve efficient reconstruction of functional vascular network after implantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the structural diagram of the 3D printed artificial bone model with hypoxia-induced vascularization function. In order to be applied to different models, the red frame part is the edge area after stacking in the right picture and cutting with a cylinder, 1-opening, 2-inner cavity, A-opening width, B-inner cavity width, C-pore length, D-pore depth.

[0036] Figure 2 This is a solid picture of a 3D-printed artificial bone with hypoxia-induced vascularization function. The upper row shows that the semi-closed pore structure is evenly arranged on a flat plate, and the opening sizes are 0μm (control group), 175μm, 250μm, and 375μm; the lower row shows that the semi-closed pore structure is applied to a three-dimensional artificial bone structure, and the opening sizes are 0μm (control group), 175μm, 250μm, and 375μm, respectively.

[0037] Figure 3 Process diagram of 3D printed artificial bone with hypoxia-induced vascularization function.

[0038] Figure 4 This is a test for the release of hypoxia-inducible factor by RAW246.7 cells on artificial bone.

[0039] Figure 5 Image of vascular ingrowth in 3D-printed artificial bone with hypoxia-induced vascularization. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with specific embodiments, but the embodiments of the present invention are not limited thereto. For process parameters not particularly noted, conventional techniques may be used.

[0041] Example 1

[0042] Preparation of a 3D printed tricalcium phosphate ceramic artificial bone for regulating cellular hypoxic microenvironment

[0043] Step 1: Use 3D software to design the model. Figure 1 As shown in the figure, a semi-enclosed pore is designed on the cubic crossbeam support (taking a rectangular pore as an example), the inner cavity width B is 500μm, the opening width A is designed between 175μm and 375μm, the external pore is connected to the overall pore, the pore depth D is 300μm, and the pore length C is 500μm. This pore structure is evenly distributed on the surface of the model. The model is converted to STL format and sliced ​​with a thickness of 25μm.

[0044] Step 2: Use digital light processing (DLP) 3D printing to prepare the tricalcium phosphate β-TCP sample designed in step 1: tricalcium phosphate, photosensitive resin (by volume percentage, 60% 1,6-hexanediol diacrylate; 30% hydroxyethyl acrylate; 10% 3-hydroxymethylpropane-3-methylacrylate), photosensitive resin and ceramic powder are added in a mass ratio of 1:1, and the photoinitiator 2,4,6-trimethylbenzoyl-diphenylphosphine oxide is 3% of the total volume of the photosensitive resin. The ball milling frequency is 40Hz and the ball milling time is 6h. Using DLP 3D printing technology, the first layer printing time is 69-75s, the printing time of each layer is 4-6s, and the light power is 20-25mW / cm 2 .

[0045] The blanks were ultrasonically cleaned in ultrapure water and then ultrasonically cleaned again. After drying at 50°C for 12 minutes, the samples were subjected to high temperature treatment in a muffle furnace at 800-1150°C.

[0046] Step 3: Sterilize the sample in step 2 under high temperature and high pressure.

[0047] Step 4: Apply the samples from step 3 to the cell experiment. Place the samples in a 48-well plate and fully moisten it with culture medium. Then, inoculate macrophages RAW246.7 and co-culture for three days, then perform immunofluorescence staining to observe the expression of HIF-1α. Figure 4 As shown, it can be observed that the fluorescence intensity of HIF-1α in the artificial bone with an opening size of 375 μm is the strongest, and the expression level of cellular HIF-1α in the sample with semi-closed pores is increased.

[0048] Step five: Apply the sample from step three to the chorioallantoic membrane of the chicken embryo. Incubate the fertilized chicken embryo in an incubator at a temperature of 38.8°C and a humidity of 60%. After 3 days of incubation, extract 1 mL of egg liquid to expand the air chamber to facilitate the subsequent implantation of the sample. After 6 days of incubation, open the chicken embryo to expose the air chamber, observe the growth of the chicken embryo, and evaluate the formation of blood vessels; use a sealing film to seal the open window, paying attention to sterility and sealing. After 8 days of incubation, implant the sample on the chorioallantoic membrane above the chicken embryo, continue to incubate for 4 days, remove the sample, rinse it twice with saline, and observe and record it under a stereo microscope. Figure 5 As shown, compared with the control group without semi-closed pores, the pores with openings of 275μm and 375μm formed a rich vascular network and promoted blood vessel growth, while the control group without semi-closed pores only induced a small amount of blood vessel proliferation.

[0049] Example 2

[0050] Preparation of a 3D printed bone repair magnesium leucasite ceramic artificial bone for regulating cellular hypoxic microenvironment

[0051] Step 1: Use 3D software to design semi-enclosed pores on the cubic crossbeam pillars. The inner cavity pore width B is 500μm, the opening width A is 175μm and 250μm, the external pores are connected to the overall pores, the semi-enclosed pore depth D is 200μm, and the pore length C is 500μm. This pore structure is evenly distributed on the model surface. Convert the model to STL format and slice it with a thickness of 25μm.

[0052] Step 2: Use stereolithography (SLA) 3D printing to form. The sample blank is ultrasonically cleaned in ultrapure water and then ultrasonically cleaned again. After drying at 50℃ for 30 minutes, the chip blank is heated in a muffle furnace at 800-1150℃.

[0053] Step 3: Sterilize the sample in step 3 under high temperature and high pressure.

[0054] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A 3D printed artificial bone with hypoxia-induced vascularization function, characterized in that: A secondary pore structure is arranged on the surface of the artificial bone, the secondary pore structure is connected with the pores of the artificial bone and is evenly distributed on the surface of the artificial bone, and the secondary pore structure is a semi-closed pore structure.

2. The 3D printed artificial bone with hypoxia-induced vascularization function according to claim 1, characterized in that: T-shaped grooves are respectively arranged on both sides of the secondary pore structure, and the T-shaped grooves have an opening 1 and an inner cavity 2 which are connected to each other, wherein the width of the inner cavity is greater than the width of the opening; Two of the secondary pore structures are axially symmetrically distributed along the T-shaped transverse straight line.

3. The 3D printed artificial bone with hypoxia-induced vascularization function according to claim 2, characterized in that: The inner cavity width of the secondary pore structure is 500-1000 μm, the opening width is 150-500 μm, the T-slot pore length is 500-1000 μm, and the pore depth is 200-800 μm.

4. The 3D printed artificial bone with hypoxia-induced vascularization function according to claim 1, characterized in that: The overall porosity of the 3D printed artificial bone with hypoxia-induced vascularization function is 60%-70%.

5. A method for preparing a 3D printed artificial bone with hypoxia-induced vascularization function according to any one of claims 1 to 4, characterized in that: The steps include: Step 1: Design the model using 3D drawing software; Step 2: Print the model through a 3D molding process, and then obtain the 3D printed artificial bone with the hypoxia-induced vascularization function through post-processing.

6. The method for preparing 3D printed artificial bone with hypoxia-induced vascularization function according to claim 5, characterized in that: The 3D forming process in step 1 is laser melting sintering, stereolithography or digital light processing; Step 1: The first layer printing time of the 3D molding process is 60-75s, the printing time of each layer is 4-6s, and the optical power is 20-25mW / cm 2 .

7. The method for preparing 3D printed artificial bone with hypoxia-induced vascularization function according to claim 5, characterized in that: The 3D printing slurry in step 2 includes: ceramic powder or metal powder or photocurable polymer monomer, photosensitive resin and photoinitiator.

8. The method for preparing 3D printed artificial bone with hypoxia-induced vascularization function according to claim 7, characterized in that: The ceramic powder is at least one of hydroxyapatite, tricalcium phosphate, wollastonite and magnesium leucapatite; The photosensitive resin is at least one of 1,6-hexanediol diacrylate, hydroxyethyl acrylate or 3-hydroxymethylpropane-3-methacrylate; The photoinitiator is 2,4,6-trimethylbenzoyl-diphenylphosphine oxide or bisacylphosphine oxide or 1-hydroxycyclohexyl phenyl ketone.

9. The method for preparing 3D printed artificial bone with hypoxia-induced vascularization function according to claim 5, characterized in that: The post-processing in step 2 includes cleaning, drying, high temperature sintering and sterilization; The sterilization is high temperature and high pressure sterilization, ultraviolet irradiation sterilization or radiation sterilization.

10. Use of the 3D printed artificial bone with hypoxia-induced vascularization function according to any one of claims 1 to 4 in bone tissue regeneration and repair.

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