Artificial bone for promoting osteoporotic bone defect repair and preparation method thereof

Abstract

The invention discloses an artificial bone for promoting osteoporotic bone defect repair and a preparation method thereof, and belongs to the technical field of biomedical materials. The artificial bone disclosed by the invention is prepared by compounding the following components in percentage by weight: 89.65 to 89.85 weight percent of polylactic acid-glycolic acid copolymer, 9.85 to 10.15 weight percent of nano hydroxyapatite and 0.1 to 0.5 weight percent of zoledronic acid. In the artificial bone, the nano-hydroxyapatite and zoledronic acid are uniformly dispersed in the polylactic acid-glycolic acid copolymer, and the porous structure on the surface of the artificial bone is combined, so that osteoblasts can be attached to the surface of the artificial bone, and new bone tissues can grow in the artificial bone; phosphonate radicals of zoledronic acid can be chelated with nano-hydroxyapatite to generate stable chemical bonds, the stable chemical bonds are introduced into the polylactic acid-glycolic acid copolymer, effective loading of zoledronic acid can be achieved, and the effective loading of zoledronic acid can inhibit bone resorption increase caused by overactivation of osteoclasts and repair osteoporotic bone defects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to an artificial bone for promoting the repair of osteoporotic bone defects and a preparation method thereof. Background Art

[0002] Osteoporosis is a metabolic bone disease with a decline in bone regeneration ability, which easily causes delayed healing, nonunion of fractures, and even the occurrence of bone defects. The osteogenic ability of clinical bone grafts at the bone defect site is significantly lower than the bone destruction caused by osteoclast activation, and ideal bone defect healing cannot be achieved. Bone tissue engineering has great application potential in the field of bone defect repair. Tissue engineering scaffolds are one of the three major elements of bone tissue engineering, providing an anchoring spatial structure and mechanical support for cells and inducing the formation of new bone in the scaffolds. However, the accumulation of senescent cells in the osteoporosis state not only affects the quantity and function of bone marrow mesenchymal stem cells (BMSCs), but also can release senescence-related factors to promote senescence in a self-regulatory manner, or trigger the senescence of neighboring cells in a paracrine manner, exacerbating bone loss and disease progression. Therefore, it is very important to develop ideal bone tissue engineering scaffold materials.

[0003] Poly(lactic-co-glycolic acid) (PLGA), as a synthetic copolymer, has become one of the most attractive biopolymers for preparing bone tissue engineering scaffolds due to its excellent biocompatibility and controllable biodegradability. However, the mechanical properties of 3D-printed PLGA scaffolds are poor and cannot be used alone for the preparation of bone tissue engineering scaffolds. Therefore, PLGA is usually combined with other materials, such as ceramics, bioactive glass, or appropriately modified to improve mechanical properties and osteoconductivity, thereby enhancing bone regeneration. Nano-hydroxyapatite (nHA) is an inorganic filler that is widely used in bone repair due to its non-toxicity, bioactivity, osteoconductivity, and similarity to natural bone minerals. Adding nHA as a reinforcing phase to the PLGA matrix can improve the mechanical and biological properties of the scaffold material. Existing research results show that compared with PLGA, PLGA / nHA porous scaffolds can promote cell proliferation, differentiation, and bone mineral formation. However, the problem of the imbalance of the osteoblast-osteoclast microenvironment caused by excessive osteoclast activation still cannot be solved. In addition, the existing PLGA / nHA composites are synthesized by simply physically mixing nHA with PLGA, and the uniform dispersion of nHA in the PLGA matrix cannot be achieved.

[0004] As a first-line clinical anti-osteoporosis drug, bisphosphonates can exert their effects through multiple pathways. It has the ability to inhibit the osteoclastic differentiation of pre-osteoclasts, inhibit the proliferation of osteoclasts, promote the apoptosis of osteoclasts, and can regulate the osteogenic differentiation ability of osteoblasts and bone marrow mesenchymal stem cells. Therefore, it has the function of reversing the imbalance of the osteoclast-osteoblast microenvironment. However, the bioavailability of bisphosphonates is relatively low under systemic administration methods such as oral and intravenous injection, resulting in insufficient drug concentration reaching the prosthesis-host bone integration interface and making it difficult to effectively play a regulatory role. This situation makes it a challenge to precisely regulate the osteoblast-osteoclast balance at the prosthesis interface. Summary of the Invention

[0005] The purpose of the present invention is to provide an artificial bone for promoting the repair of osteoporotic bone defects and its preparation method. This artificial bone not only has high strength but also can inhibit the over-activation of osteoclasts at the local bone defect of osteoporosis patients to inhibit excessive bone resorption, thereby improving new bone formation and achieving the purpose of repairing bone defects.

[0006] To achieve the above purpose, the technical solutions adopted by the present invention are as follows.

[0007] In the first aspect, the present invention provides an artificial bone for promoting the repair of osteoporotic bone defects, which is composed of 89.65wt%-89.85wt% poly(lactic-co-glycolic acid) (PLGA), 9.85wt%-10.15wt% nano-hydroxyapatite (nHA), and 0.1wt%-0.5wt% zoledronic acid (ZOL).

[0008] Further, the artificial bone for promoting the repair of osteoporotic bone defects is composed of 89.775wt% poly(lactic-co-glycolic acid), 9.975wt% nano-hydroxyapatite, and 0.25wt% zoledronic acid.

[0009] Further, the molecular weight of the poly(lactic-co-glycolic acid) is 35000-40000.

[0010] Further, the molar ratio of lactic acid to glycolic acid in the poly(lactic-co-glycolic acid) is 75:25.

[0011] Further, the artificial bone for promoting the repair of osteoporotic bone defects has a microporous structure with a pore size of 400-450μm and a porosity of 40%-45%.

[0012] In the second aspect, the present invention also provides a preparation method for the above artificial bone for promoting the repair of osteoporotic bone defects, including the following steps:

[0013] (1)Dissolve poly (lactic-co-glycolic acid), nano-hydroxyapatite, and zoledronic acid in chloroform according to the ratio, and uniformly blend them to obtain a mixed solution. After removing chloroform, filtering, and drying, a bio-ink is obtained.

[0014] (2)Design an artificial bone model using software.

[0015] (3)Use the bio-ink prepared in step (1) to print the artificial bone model in step (2) through three-dimensional (3D) printing technology to obtain an artificial bone for promoting the repair of osteoporotic bone defects.

[0016] Further, step (1) includes:

[0017] (1-1)Dissolve poly (lactic-co-glycolic acid) in chloroform, repeat stirring and ultrasonic treatment multiple times until uniformly dispersed, add nano-hydroxyapatite and zoledronic acid, and heat and stir until completely dissolved and uniformly mixed to obtain a mixed solution.

[0018] (1-2)Under stirring, add the mixed solution to 5-10 times the volume of absolute ethanol, transfer the obtained solid to absolute ethanol for soaking for 2-4 h, filter, air-dry, and then dry in an oven to obtain the bio-ink.

[0019] Furthermore, in step (1-1), each stirring time is 5-10 min, each ultrasonic treatment time is 5-10 min, and it is repeated three times.

[0020] Furthermore, in step (1-1), the heating and stirring temperature is 45-50 °C, and the heating and stirring time is 24-48 h.

[0021] Furthermore, in step (1-1), the concentration of the mixed solution is 0.01-0.1 g / mL.

[0022] Furthermore, in step (1-2), the drying time is 2-3 days.

[0023] Further, in step (2), the software is SolidWorks, the laying pattern is from -45° to 45°, and the line spacing is 400-450 μm.

[0024] Further, step (3) includes:

[0025] (3-1)Put the bio-ink prepared in step (1) into the heating cylinder of a 3D printer, set the bottom plate temperature to 35-40 °C, and set the print head temperature to 160-165 °C.

[0026] (3-2) Import the artificial bone model in step (2) into Simplify3D software to generate a G-code file, and input it into a 3D printer. The 3D printer prints according to a preset path to obtain an artificial bone for promoting the repair of osteoporotic bone defects.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] The artificial bone for promoting the repair of osteoporotic bone defects of the present invention uses nano-hydroxyapatite, poly(lactic-co-glycolic acid), and zoledronic acid as raw materials. The three are uniformly blended by chloroform dissolution method, so as to realize the uniform dispersion of nano-hydroxyapatite and zoledronic acid in the poly(lactic-co-glycolic acid) matrix. Nano-hydroxyapatite and zoledronic acid are uniformly distributed on the surface and inside of the artificial bone. Combining with the porous structure on the surface of the artificial bone is beneficial to the attachment of osteoblasts on the surface. After the artificial bone is implanted into the treatment site, it can adjust the imbalance state of the local osteogenic-osteoclastic microenvironment of osteoporosis patients, which is beneficial to the ingrowth of new bone tissue and achieves the purpose of repairing osteoporotic bone defects.

[0029] In the artificial bone for promoting the repair of osteoporotic bone defects of the present invention, the phosphonate group of zoledronic acid can chelate with nano-hydroxyapatite to form a stable chemical bond. Introducing it into poly(lactic-co-glycolic acid) can achieve the effective loading of zoledronic acid. The effective loading of zoledronic acid can inhibit the increased bone resorption caused by the over-activation of osteoclasts, thereby adjusting the imbalance state of the local osteogenic-osteoclastic microenvironment of osteoporosis patients and achieving the purpose of repairing osteoporotic bone defects.

[0030] The artificial bone for promoting the repair of osteoporotic bone defects of the present invention can 3D print the artificial bone according to the shape of the bone defect site, realizing personalized customization and fitting well with the defect site. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0032] Figure 1 It is the scanning electron microscope image of PLGA / nHA / 0.25%ZOL prepared in Example 1 of the present invention.

[0033] Figure 2 It is the infrared spectrum of PLGA / nHA / 0.25%ZOL prepared in Example 1 of the present invention and PLGA / nHA prepared in Comparative Example 1.

[0034] Figure 3The surface and cross-sectional microtopographies of PLGA / nHA / 0.25%ZOL prepared in Example 1 of the present invention; wherein, a is the scanning electron microscopy (SEM) image of the surface of PLGA / nHA / 0.25%ZOL, b is the partial enlarged view of the g region in a, c is the partial enlarged view of the h region in b, d is the SEM image of the cross-section of PLGA / nHA / 0.25%ZOL, e is the partial enlarged view of the i region in d, and f is the partial enlarged view of the j region in e.

[0035] Figure 4 The surface element distribution map of PLGA / nHA / 0.25%ZOL prepared in Example 1 of the present invention; wherein, a is the distribution map of all elements, b is the C element distribution, c is the N element distribution, d is the O element distribution, e is the P element distribution, and f is the Ca element distribution.

[0036] Figure 5 The cross-sectional element distribution map of PLGA / nHA / 0.25%ZOL prepared in Example 1 of the present invention; wherein, a is the distribution map of all elements, b is the C element distribution, c is the N element distribution, d is the O element distribution, e is the P element distribution, and f is the Ca element distribution.

[0037] Figure 6 The alizarin red staining bright-field images of artificial bone containing different mass percentages of zoledronic acid co-cultured with BMSCs on the 14th day; wherein, a is PLGA / nHA, b is PLGA / nHA / 0.01%ZOL, c is PLGA / nHA / 0.05%ZOL, and d is PLGA / nHA / 0.25%ZOL.

[0038] Figure 7 The alizarin red staining bright-field images of artificial bone containing different mass percentages of zoledronic acid co-cultured with BMSCs under 20× magnification on the 14th day; wherein, a is PLGA / nHA, b is PLGA / nHA / 0.01%ZOL, c is PLGA / nHA / 0.05%ZOL, and d is PLGA / nHA / 0.25%ZOL.

[0039] Figure 8 The ALP staining bright-field images of artificial bone containing different mass percentages of zoledronic acid co-cultured with BMSCs on the 7th day, a is PLGA / nHA, b is PLGA / nHA / 0.01%ZOL, c is PLGA / nHA / 0.05%ZOL, and d is PLGA / nHA / 0.25%ZOL.

[0040] Figure 9Bright-field images of ALP staining on day 14 of co-culture of artificial bone containing zoledronic acid with different mass percentages and BMSCs. a is PLGA / nHA, b is PLGA / nHA / 0.01%ZOL, c is PLGA / nHA / 0.05%ZOL, and d is PLGA / nHA / 0.25%ZOL.

[0041] Figure 10 Bright-field images of TRAP staining on day 7 of co-culture of artificial bone containing zoledronic acid with different mass percentages and RAW264.7 (mouse mononuclear macrophage leukemia cells). a is PLGA / nHA, b is PLGA / nHA / 0.01%ZOL, c is PLGA / nHA / 0.05%ZOL, and d is PLGA / nHA / 0.25%ZOL. Arrows in the figure indicate TRAP-stained positive cells (i.e., osteoclasts).

[0042] Figure 11 Fluorescence images of F-actin staining on day 7 of co-culture of artificial bone containing zoledronic acid with different mass percentages and RAW264.7. a is PLGA / nHA, b is PLGA / nHA / 0.01%ZOL, c is PLGA / nHA / 0.05%ZOL, and d is PLGA / nHA / 0.25%ZOL. Detailed implementation manners

[0043] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with embodiments.

[0044] In the following embodiments, various processes and methods not described in detail are conventional methods well known in the art. Materials, reagents, devices, instruments, equipment, etc. used in the following embodiments can be obtained from commercial channels without special instructions.

[0045] Embodiment 1

[0046] An artificial bone for promoting the repair of osteoporotic bone defects, which is composed of a copolymer of polylactic acid and glycolic acid, nano-hydroxyapatite, and zoledronic acid. The mass percentages of the copolymer of polylactic acid and glycolic acid, nano-hydroxyapatite, and bisphosphonate are 89.775%, 9.975%, and 0.25% respectively.

[0047] The preparation method of the above-mentioned artificial bone for promoting the repair of osteoporotic bone defects includes the following steps:

[0048] (1) Weigh 8.9775 g of PLGA (molecular weight 35000 - 40000, molar ratio of polylactic acid to glycolic acid is 75:25), add it to 100 mL of chloroform, stir for 5 min and then sonicate for 5 min, repeat three times to ensure sufficient dispersion of PLGA, and obtain a mixed solution;

[0049] Add 0.9975 g of nHA powder and 0.025 g of ZOL to the mixed solution, heat to 50 °C and stir for 24 h. Under stirring, slowly add it to 5 - 10 times the volume of absolute ethanol. Transfer the obtained solid to absolute ethanol and soak for 2 h, filter, air-dry and then dry in an oven for 2 - 3 days to obtain the bioink.

[0050] (2) Use SolidWorks software to design an artificial bone support model (dimensions: length 10 mm, width 10 mm, height 2 mm), the laying pattern is from -45° to 45°, and the line spacing is 400 - 450 μm.

[0051] (3) Put the bioink into the heating cylinder of a 3D printer (FDM), set the bottom plate temperature to 35 - 40 °C and the print head temperature to 160 - 165 °C; import the artificial bone model into simplify3D software to generate a G-code file, input it into the 3D printer, and the 3D printer prints according to the preset path to obtain an artificial bone for promoting the repair of osteoporotic bone defects, denoted as PLGA / nHA / 0.25%ZOL. The scanning electron micrograph is as shown in Figure 1 shown. It can be seen from Figure 1 that the pore size of the artificial bone for promoting the repair of osteoporotic bone defects in Example 1 is 400 - 450 μm.

[0052] Example 2

[0053] Replace the mass percentage of zoledronic acid in the artificial bone for promoting the repair of osteoporotic bone defects with 0.01% respectively, and the others are the same as in Example 1, to obtain an artificial bone for promoting the repair of osteoporotic bone defects, denoted as PLGA / nHA / 0.01%ZOL.

[0054] Example 3

[0055] Replace the mass percentage of zoledronic acid in the artificial bone for promoting the repair of osteoporotic bone defects with 0.05% respectively, and the others are the same as in Example 1, to obtain an artificial bone for promoting the repair of osteoporotic bone defects, denoted as PLGA / nHA / 0.05%ZOL.

[0056] Comparative Example 1

[0057] The mass percentage of zoledronic acid in the artificial bone for promoting the repair of osteoporotic bone defects was replaced with 0% respectively, and the others were the same as in Example 1, to obtain an artificial bone for promoting the repair of osteoporotic bone defects, denoted as PLGA / nHA.

[0058] Comparative Example 2

[0059] The mass percentage of zoledronic acid in the artificial bone for promoting the repair of osteoporotic bone defects was replaced with 1.25% respectively, and the others were the same as in Example 1. The obtained bioink was prone to clogging the nozzle of the 3D printer, and the artificial bone could not be obtained.

[0060] Infrared spectroscopy tests were carried out on the PLGA / nHA / 0.25%ZOL prepared in Example 1 and the PLGA / nHA prepared in Comparative Example 1. The results are as Figure 2 shown. Characteristic peaks of PLGA, nHA, and ZOL were detected on the surface of PLGA / nHA / 0.25%ZOL. The characteristic peaks of nHA in the range of 900 - 1200 cm -1 showed the P–O stretching vibration of -PO4 3- The characteristic peaks of PLGA in the range of 1730 - 1780 cm -1 showed the C=O stretching vibration of the ester group. The characteristic peaks of ZOL in the range of 2900 - 3180 cm -1 showed the C-H stretching vibration peak, and the characteristic peaks in the range of 2900 - 3180 cm -1 showed the stretching vibration peak of N-H. The microscopic morphologies of the surface and cross-section of PLGA / nHA / 0.25%ZOL are as Figure 3 shown. A small amount of nHA particles can be observed on the surface ( Figure 3 a, b, c in) and cross-section ( Figure 3 d, e, f in) of the artificial bone, indicating that HA nanoparticles were loaded into the PLGA material. At the same time, the distribution maps of all elements, C element, N element, O element, P element, and Ca element on the surface of PLGA / nHA / 0.25%ZOL obtained based on energy-dispersive X-ray spectroscopy analysis are respectively as Figure 4 shown in a, b, c, d, e, f in, and the distribution maps of all elements, C element, N element, O element, P element, and Ca element of the cross-section of PLGA / nHA / 0.25%ZOL are respectively as Figure 5 shown in a, b, c, d, e, f in, indicating that nHA and ZOL are uniformly distributed in the artificial bone. The above results show that the artificial bone for promoting the repair of osteoporotic bone defects of the present invention successfully loaded ZOL and is evenly distributed in the artificial bone.

[0061] Verify the ability of the artificial bone for promoting the repair of osteoporotic bone defects in Examples 1-3 and Comparative Example 1 to regulate the osteoblast-osteoclast balance.

[0062] The experiment for regulating the osteogenic process is as follows: In a 24-well plate, 2×10 4 BMSCs were co-cultured with the artificial bone in each well, and osteogenic induction medium (500 ml of complete DMEM medium, 10% fetal bovine serum, 1% penicillin-streptomycin double antibody, 12.8 mg / L vitamin C, 2.16 g / L β-glycerophosphate, 5 mmol / L dexamethasone) was used. After culturing in a 37°C, 5% CO 2 cell incubator for 7 days, alkaline phosphatase (ALP) staining kit was used for ALP staining, and after 14 days, ALP and alizarin red staining kits were used for ALP and alizarin red staining. The results are as shown in Figure 6 a-d in Figure 7 a-d in Figure 8 a-d in and Figure 9 a-d in.

[0063] The experiment for regulating the osteoclastic process is as follows: In a 6-well plate, 2×10 4 RAW264.7 (as osteoclast precursor cells) were co-cultured with the artificial bone in each well, and MEM-α medium containing 100 ng / ml osteoclast induction factor (RANKL) was used to induce the directional differentiation of RANW264.7 cells into osteoclasts. After culturing in a 37°C, 5% CO 2 cell incubator for 7 days, tartrate-resistant acid phosphatase (TRAP) staining kit was used to count the number of formed osteoclasts. The F-Actin ring of the tight sealing zone formed by the bone resorption of mature OCs was stained with FITC-labeled phalloidin, and its bone resorption ability was reflected by the fluorescence area of its tight sealing zone. The results are as shown in Figure 10 a-d in and Figure 11 a-d in.

[0064] It can be seen from Figures 6 - 11 that the artificial bone of the present invention can inhibit the formation of osteoclasts (TRAP staining indicates that the number of osteoclasts is extremely small) and the tight sealing zone formed by bone resorption (the fluorescence area of the F-Actin ring is extremely small), and at the same time can promote the expression of alkaline phosphatase (ALP staining) and the deposition of calcium nodules (alizarin red staining) of bone marrow mesenchymal stem cells, and play the biological function of regulating the osteoblast-osteoclast balance in the microenvironment. And the artificial bone containing 0.25% by mass of zoledronic acid has the strongest ability to regulate osteoblast-osteoclast.

[0065] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the embodiments. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. An artificial bone for promoting the repair of osteoporotic bone defects, characterized in that: The composite is composed of 89.65wt%-89.85wt% of polylactic acid-glycolic acid copolymer, 9.85wt%-10.15wt% of nano-hydroxyapatite and 0.1wt%-0.5wt% of zoledronic acid.

2. The artificial bone for promoting the repair of osteoporotic bone defects according to claim 1, characterized in that: It is composed of 89.775wt% polylactic acid-glycolic acid copolymer, 9.975wt% nano-hydroxyapatite and 0.25wt% zoledronic acid.

3. The artificial bone for promoting the repair of osteoporotic bone defects according to claim 1, characterized in that: The molecular weight of the polylactic acid-glycolic acid copolymer is 35000-40000.

4. The artificial bone for promoting the repair of osteoporotic bone defects according to claim 1, characterized in that: The molar ratio of polylactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 75:

25.

5. The artificial bone for promoting the repair of osteoporotic bone defects according to claim 1, characterized in that: The artificial bone for promoting the repair of osteoporotic bone defects has a pore size of 400-450 μm and a porosity of 40%-45%.

6. The method for preparing an artificial bone for promoting the repair of osteoporotic bone defects according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) uniformly blending polylactic acid-glycolic acid copolymer, nano-hydroxyapatite and zoledronic acid according to the ratio by chloroform dissolution method to obtain a mixed solution, and obtaining a bio-ink after removing chloroform, filtering and drying; (2) Designing artificial bone models using software; (3) Using the bio-ink prepared in step (1), the artificial bone model of step (2) is printed by three-dimensional printing technology to obtain an artificial bone that promotes the repair of osteoporotic bone defects.

7. The method for preparing an artificial bone for promoting the repair of osteoporotic bone defects according to claim 6, characterized in that: The step (1) comprises: (1-1) dissolving poly(lactic acid-co-glycolic acid) copolymer in chloroform, repeatedly stirring and ultrasonicating for multiple times until the copolymer is uniformly dispersed, adding hydroxyapatite and zoledronic acid, heating and stirring until the copolymer is completely dissolved and uniformly mixed, to obtain a mixed solution; (1-2) The mixed solution is added to 5-10 times the volume of anhydrous ethanol under stirring, and the obtained solid is transferred to anhydrous ethanol and soaked for 2-4 hours, filtered, dried and then baked to obtain bio-ink.

8. The method for preparing an artificial bone for promoting the repair of osteoporotic bone defects according to claim 7, characterized in that: In step (1-1), each stirring time is 5-10 min, each ultrasonic treatment time is 5-10 min, and it is repeated three times; In step (1-1), the heating and stirring temperature is 45-50°C, and the heating and stirring time is 24-48h; In step (1-2), the drying time is 2-3 days.

9. The method for preparing an artificial bone for promoting the repair of osteoporotic bone defects according to claim 6, characterized in that: In step (2), the software is SolidWorks, the laying mode is -45° to 45°, and the line spacing is 400-450 μm.

10. The method for preparing an artificial bone for promoting the repair of osteoporotic bone defects according to claim 6, characterized in that: The step (3) comprises: (3-1) placing the bio-ink prepared in step (1) into a heating cylinder of a 3D printer, setting the base plate temperature to 35-40°C and the print head temperature to 160-165°C; (3-2) The artificial bone model of step (2) is imported into simplify3D software to generate a G code file, which is then input into a 3D printer. The 3D printer prints according to a preset path to obtain an artificial bone that promotes the repair of osteoporotic bone defects.

Images