3D printed titanium alloy biomimetic porous artificial bone for promoting bone differentiation and preparation method and application thereof

Abstract

The application discloses a 3D-printed porous biomimetic artificial bone of titanium alloy for promoting bone differentiation, a preparation method and application, and belongs to the field of biomedical materials. The artificial bone is composed of a titanium metal framework and has a porous reticular structure inside; the porous reticular structure is designed by imitating the three-dimensional microstructure of human fourth lumbar cancellous bone, the average pore diameter is 60.3-73.9 mu m, and the porosity is 67.9%-80.5%. The preparation method is as follows: fourth lumbar tomography images with faster bone density recovery are obtained, the cancellous bone structure is extracted for three-dimensional reconstruction, and the selected laser melting technology is used for printing forming. The specific physical topological structure of the application can effectively activate the Notch2 signal pathway of bone marrow mesenchymal stem cells and induce osteogenic differentiation. When applied to critical bone defect repair, the amount of new bone formation at the defect site can be increased, the elastic modulus and maximum bending stress of the healing area can be improved, and good bone integration can be realized.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and medical device technology, specifically to a 3D-printed titanium alloy biomimetic porous artificial bone that promotes bone differentiation, its preparation method, and its application. Background Technology

[0002] In most cases, small bone defects can heal spontaneously after receiving standard treatment. However, when the defect is too large to heal on its own, surgical bone grafting or other methods are often required to achieve healing. This type of bone defect is called a critical bone defect. Critical bone defects remain a common challenge in orthopedics, typically caused by bone trauma, bone infection, or tumor resection. Approximately 20% to 40% of open fractures of the diaphysis caused by high-energy injuries are accompanied by bone defects. These defects often lead to poor prognoses, such as chronic pain, compartment syndrome, delayed union, and nonunion. Therefore, bone defects severely impact patients' health and quality of life, and impose a significant medical burden on society. Immediate bone grafting surgery is usually necessary to promote bone regeneration and healing in patients with critical bone defects. For this surgical intervention, choosing the appropriate bone graft material is crucial. Commonly used bone graft materials include autologous bone, allogeneic bone, and xenograft bone. However, these bone graft materials have many limitations, including donor site complications, limited bone volume available for transplantation, immune rejection, the risk of disease transmission, and high costs. To address these issues, 3D-printed biomimetic porous artificial bone may be a promising approach to effectively promote bone repair in patients with borderline bone defects. Titanium alloys are considered ideal metal bone graft materials due to their excellent biocompatibility, low weight, elastic modulus close to that of bone tissue, long-term inertness, and superior mechanical properties. Furthermore, the 3D structure and interconnected porous structure of bone grafts have excellent osteoinductive properties, inducing bone marrow mesenchymal stem cells (BMSCs) to differentiate into osteoblasts, playing a crucial role in promoting bone healing. In addition, 3D printing technology can utilize metal powder materials to construct porous structures through layer-by-layer deposition. Advances in 3D printing technology have made it possible to manufacture 3D-printed titanium alloy biomimetic porous artificial bone with specific shapes and multidimensional porous structures. Because different three-dimensional porous structures can effectively promote cell growth, proliferation, and differentiation, as well as nutrient transport, thereby promoting new bone formation, some scientists are working to find the optimal structure for 3D-printed porous scaffolds, including ideal porosity, pore size, and pore shape. Based on these theories, biomimetic artificial bone structures that match the structure of natural bone exhibit excellent performance and can bring better treatment outcomes for patients with severe bone defects.

[0003] Natural bone typically consists of an inner cancellous bone and an outer cortical bone. Cancellous bone, also known as trabecular bone or spongy bone, has a porosity of 50% to 90% and a rich 3D network structure, while cortical bone has a porosity as low as 5% to 10%. Currently, most biomimetic porous implants are designed based on the structure of cancellous bone. Since cancellous bone has been shown to possess a variety of 3D network structures with different porosities, pore shapes, and pore sizes, biomimetic porous artificial bone that mimics different types of cancellous bone has remarkable osteoinductive potential, further improving the repair rate of critical bone defects. A possible mechanism is that the diverse 3D network structure of the implant affects the activation of the Notch signaling pathway, a classic signaling pathway regulating bone repair and modulating osteogenic differentiation of bone marrow mesenchymal stem cells, further accelerating bone repair, bone regeneration, and new bone formation. However, few studies have focused on evaluating the potential of different biomimetic porous artificial bones to promote osteogenic differentiation of BMSCs. Further research is needed to identify the optimal structure of natural bone and to design 3D-printed biomimetic porous artificial bone that mimics this structure to facilitate critical bone defects.

[0004] Osteoporosis is a common bone resorption disease characterized by increased bone resorption, decreased bone formation, reduced bone mass, and deterioration of bone tissue microstructure. The severity of osteoporosis varies in different locations, and the recovery rate of different bones also differs after proper medical intervention. This phenomenon may be due to the different 3D network structures of different cancellous bones, which have varying abilities to promote osteogenic differentiation of bone mesenchymal stem cells (BMSCs) and new bone formation. Therefore, after treatment, the bone tissue in a particular location recovers faster from osteoporosis, and the corresponding cancellous bone with a superior 3D network structure is likely to be superior to that in other locations. Therefore, it is possible to identify better cancellous bone 3D network structures from the rehabilitation process of osteoporosis patients to guide the design of 3D-printed biomimetic porous artificial bone. In summary, the main objective of this study is to evaluate the bone-promoting effects of different 3D-printed biomimetic porous artificial bones in order to identify a better structure of natural cancellous bone, design a more ideal 3D-printed biomimetic porous artificial bone by mimicking this structure, explore the regulatory mechanism of 3D structure on new bone formation, and ultimately provide experimental evidence for the use of appropriate bone grafts to treat severe bone defects. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes a 3D-printed titanium alloy artificial bone that promotes bone differentiation, its preparation method, and its application.

[0006] The technical solution of this invention is as follows: A biomimetic porous artificial bone includes a metal skeleton, the metal skeleton being a porous mesh structure with interconnected structures; the porous mesh structure has the following topological parameters: average pore size of 60.3-73.9 μm, porosity of 67.9-80.5%, tortuosity of 1.79-2.51, and permeability of 1642.0-2105.2 mD / C.

[0007] Furthermore, the physical density of the metal skeleton is 3.25-4.55 g / mL.

[0008] Furthermore, the material of the metal skeleton is Ti6Al4V titanium alloy.

[0009] Furthermore, the physical environment formed by the interconnected porous mesh structure can activate the Notch2 signaling pathway of bone marrow mesenchymal stem cells.

[0010] A method for preparing the aforementioned biomimetic porous artificial bone includes the following steps: S1. Obtain continuous cross-sectional anatomical images of the fourth lumbar vertebra of the human body, perform image segmentation on the continuous cross-sectional anatomical images, divide the cancellous bone region, and extract the cancellous bone image; S2. Perform three-dimensional reconstruction on the cancellous bone image to obtain a porous digital model that reflects the natural morphology and spatial organization of the fourth lumbar vertebra. S3. Using the aforementioned porous structure digital model as a processing model, titanium alloy powder is used to construct the biomimetic porous artificial bone through selective laser melting 3D printing.

[0011] Furthermore, in step S1, the thickness and slice interval of the continuous tomographic anatomical images are both 0.1 mm.

[0012] Further, in step S1, the specific process of extracting the cancellous bone image is as follows: convert the continuous tomographic anatomical image into DICOM format and import it into the three-dimensional visualization software, adjust the grayscale threshold of image segmentation to 25, and manually identify and divide the cancellous bone region.

[0013] Furthermore, in step S3, the titanium alloy powder material is Ti6Al4V.

[0014] The application of the biomimetic porous artificial bone in the preparation of implantable medical devices for bone defect repair.

[0015] Furthermore, the bone defect repair implantable medical device is used to repair critical bone defects.

[0016] Compared with the prior art, the present invention has at least the following advantages: 1. This invention relates to a 3D-printed titanium alloy biomimetic porous artificial bone that promotes bone differentiation and its preparation method. Based on dynamic bone density recovery data from clinical osteoporosis patients, this invention selects the fourth lumbar vertebra (L4) cancellous bone with optimal bone regeneration potential as a biomimetic template. The actual microstructure is extracted using tomographic scanning and three-dimensional reconstruction, and then fabricated using selective laser melting 3D printing technology. The artificial bone prepared by this method possesses specific physical topological parameters (average pore size 67.12±6.79 μm, porosity 74.24±6.34%). In vitro cell experiments show that, compared with conventional biomimetic structures, this specific topological structure can act as a physical signal stimulus, specifically activating the Notch2 signaling pathway in bone marrow mesenchymal stem cells (BMSCs), significantly upregulating the expression levels of N2ICD and its downstream molecule Hes1, promoting the differentiation of stem cells into osteoblasts, generating more calcium nodules after 14 days of culture, and significantly increasing the expression levels of osteogenic-related genes and proteins such as Col1A1 and Runx2.

[0017] 2. This invention relates to the application of a 3D-printed titanium alloy biomimetic porous artificial bone that promotes bone differentiation. When used as an orthopedic implant to repair critical bone defects, the interconnected porous network within the artificial bone provides effective channels for cell migration, proliferation, and nutrient delivery. In vivo experiments using a rat femoral critical bone defect model showed that, 8 weeks after implantation, micro-CT and histological sections revealed co-growth of bone tissue and the implant, with abundant collagen fiber deposition within the pores. Quantitative analysis showed a significant increase in relative bone volume (BV / TV), trabecular bone number, and thickness. Biomechanical testing showed that the maximum bending strain of the repaired bone reached 0.036±0.001 mm, and the elastic modulus reached 13367.42±1289.00 MPa. All mechanical properties were significantly higher than those of the conventional structure group and the sham surgery group, effectively improving the load-bearing capacity of the newly formed bone and the osseointegration effect of the implant. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0019] Figure 1This is a graph showing the changes in bone mineral density (BMD) and the selection results for optimal cancellous bone structure in different osteoporosis patients. A is a scatter plot showing the distribution of BMD values ​​of different vertebrae among the included osteoporosis patients by gender, age, and vertebral body; B is a line graph showing the trend of BMD of different lumbar vertebrae with age for all patients; C is a line graph showing the trend of BMD of different lumbar vertebrae with age for the same patient; D is a heatmap of the rate of change of lumbar vertebral BMD for all screened patients; E is a bar chart showing the proportion of vertebrae with the largest rate of change of BMD (Gender: gender; male: male; female: female; Number of lumbar vertebra: lumbar vertebral body number; Patient ID: patient number; BMD: bone mineral density value; Age: age; Patient: patient; ΔBMD%: rate of change of bone mineral density; L1-L4: first to fourth lumbar vertebrae).

[0020] Figure 2 This document presents a comparison of the design concepts, 3D printing manufacturing process, and parameters of biomimetic porous artificial bone. A shows a schematic diagram of the design and manufacturing process for L2 and L4 porous artificial bones, where a is a schematic diagram of volunteer sectional anatomy, b is a continuous sectional section image, c is the extracted microstructure of trabeculae, d is the constructed 3D digital model, and e is the manufactured 3D printed product. B shows a comparison of the digital models of the L2 and L4 structures and their corresponding 3D printed entities. C is a bar chart comparing the parameters of the digital model and the 3D printed object in bone volume (BV), relative bone volume (BV / TV), connectivity density (Conn.D), number of trabeculae (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). (Volunteer; Serial sheet sections; Microstructure of vertebras; Digital model; 3Dprinted) products: 3D printed products; BV: bone volume; BV / TV: relative bone volume; Conn.D: connection density; Tb.N: number of trabeculae; Tb.Th: trabecular thickness; Tb.Sp: trabecular separation; ns: no statistical difference.

[0021] Figure 3This is a diagram illustrating the microscopic characterization and mechanical properties of 3D-printed porous artificial bone. A shows scanning electron microscope images of L2 and L4 artificial bones; B shows the stress-strain curves of L2 and L4 artificial bones; C shows a quantitative comparison bar chart of maximum bending strain, elastic modulus, and maximum bending stress for L2 and L4; D shows the pore distribution of L2 and L4 as detected by mercury porosimetry; E shows a quantitative comparison bar chart of L2 and L4 in terms of average pore diameter, permeability, porosity, bone density, and tortuosity (Loading; Displacement; Maximum bending strain; Elasticity modulus; Maximum bending stress; Average pore diameter; Permeability; Porosity; Skeletal density; Tortuosity; millidarcy).

[0022] Figure 4 This is a diagram illustrating the identification of bone marrow mesenchymal stem cells and the biocompatibility evaluation of artificial bone. A shows the phenotypic identification of primary bone marrow mesenchymal stem cells by flow cytometry; B shows the morphology and multi-directional differentiation results of bone marrow mesenchymal stem cells (including undifferentiated morphology, osteogenic differentiation, adipogenic differentiation, and chondrogenic differentiation); C shows the observation of live / dead cell fluorescence staining after 72 hours of culture on L2 and L4 artificial bone with bone marrow mesenchymal stem cells (Count: cell count; SSC-H: side scatter height; FSC-H: forward scatter height; FITC-H / APC-H / PerCP-H / PE-H: flow cytometry fluorescence detection channel abbreviation; CD90 / CD73 / CD105 / CD45 / CD34 / CD11b / CD19 / HLA-DR: cell surface differentiation cluster / recipient name; BMSCs: bone marrow mesenchymal stem cells; Osteogenic differentiation: osteogenic differentiation; Adipogenic differentiation: adipogenic differentiation; Chondrogenic differentiation: chondrogenic differentiation).

[0023] Figure 5This is a diagram comparing the in vitro osteogenic induction capacity of L2 and L4 artificial bones and verifying their mechanisms. A shows the Alizarin Red staining of L2 and L4 artificial bone 14 days after osteogenic differentiation induction; B is a bar chart of OD value quantitative analysis of Alizarin Red staining in Figure A; C is a graph of relative gene expression levels of Col1A1 and Runx2 in L2 and L4 detected by RT-PCR; D is a bar chart of Western blot (WB) detection of osteogenic-related proteins (Col1A1, Runx2); E is a graph of quantitative analysis of the relative expression levels of proteins corresponding to the WB bands in Figure D; F is a heatmap of differentially expressed genes between L2 and L4; G is a volcano diagram of differentially expressed genes between L2 and L4; H is a bubble chart of KEGG pathway enrichment of differentially expressed genes between L2 and L4; I is a graph of relative gene expression levels of Notch2 and Hes1 detected by RT-PCR; J is a bar chart of WB detection of key proteins (N2ICD, HES1) in the Notch2 pathway; K is a graph of quantitative analysis of the relative expression levels of proteins corresponding to the WB bands in Figure J (ARS: Alizarin Red staining; OD Value: absorbance value; relative to...). GAPDH: Expression level relative to the internal reference GAPDH; COL1A1: Type I collagen α1 chain; Runx2: Runt-related transcription factor 2, a marker of osteogenic differentiation; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; kD: kilodaltons; Down regulation: Downregulated; Not Significant: No significant change; Up regulation: Upregulated; Fold-change: Fold change; Top 30 of Pathway Enrichment: Top 30 enriched pathways; Rich Factor: Enrichment factor; Gene_number: Number of genes; q_value: p-value after multiple test correction; N2ICD: Notch2 receptor intracellular domain; HES1: Trichoderma enhancer 1, a marker of Notch signaling pathway activation.

[0024] Figure 6 This is a reverse verification diagram showing the elimination of the difference in bone induction capacity between L2 and L4 by blocking the Notch2 pathway (adding the Notch pathway inhibitor DAPT). A is an Alizarin Red staining image of L2 and L4 artificial bone after adding DAPT to the culture medium; B is a bar chart of quantitative analysis of Alizarin Red staining after adding DAPT; C is an RT-PCR analysis of the relative expression levels of Col1A1, Runx2, Notch2, and Hes1 in L2 and L4 after adding DAPT; D is a Western blot (WB) band image of related proteins after adding DAPT; E is a quantitative analysis of the relative expression levels of each protein detected by WB after adding DAPT (+DAPT: adding DAPT solution / Notch signaling pathway inhibitor).

[0025] Figure 7This is an in vivo experimental evaluation diagram of 3D-printed porous artificial bone repairing critical bone defects in rats. A shows a real-world diagram of the experimental procedure and tissue harvesting process in rats (in sequence: 3D printing of bone grafts, creation of bone defects, implantation of grafts, femoral tissue harvesting, and micro-CT scanning); B shows three-dimensional images of bone healing after implantation of L2 and L4 artificial bones using micro-CT reconstruction; C shows a bar chart comparing parameters BV, BV / TV, Conn.D, Tb.N, Tb.Th, and Tb.Sp obtained from quantitative analysis using micro-CT; D shows transmission electron microscopy (TEM) images of ultrathin sections of samples from the implantation site, with black arrows indicating collagen fibers; E shows the stress-strain curves of the femur in the sham-operated group and the groups implanted with L2 and L4 artificial bones; F shows a quantitative comparison bar chart of maximum bending strain, elastic modulus, and maximum bending stress in the sham-operated group, L2 group, and L4 group (3D printed bone grafts; Critical bone defect; Transplantation of bone grafts; Femur was harvested; Micro-CT). scanning: micro-computed tomography; Sham: sham surgery group). Detailed Implementation

[0026] The present invention will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0027] This invention provides a general and / or specific description of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods; all reagents or instruments used, unless otherwise specified, are commercially available conventional products prepared or used using conventional methods.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0029] Example 1: Clinical screening and digital model construction of optimal biomimetic porous structures This embodiment provides a method for screening and extracting the optimal biomimetic porous structure based on clinical bone mineral density (BMD) data.

[0030] To determine the ideal three-dimensional porous structure, bone mineral density was measured in patients with osteoporosis. A total of 538 patients were screened and included in this study; 441 patients were excluded due to not meeting the inclusion criteria (exclusion criteria included: 67 with poor medical records, 281 who refused follow-up, 12 over 90 years of age, 27 with pathological fractures, and 54 with spinal tumors or infections). Finally, 97 patients were included, comprising 84 women and 13 men, with a mean age of 73.5 ± 9.3 years. Patient basic information distribution is as follows: Figure 1 As shown in Figure A.

[0031] like Figure 1 As shown in Figure B, this invention compared the age-related trends of bone mineral density (BMD) in different lumbar vertebrae across all patients; it also examined the age-related trends of BMD in different lumbar vertebrae within the same patient. The results showed that the age-related trends of BMD in each lumbar vertebra differed; that is, with increasing age, the BMD of some lumbar vertebrae may increase, while the BMD of other lumbar vertebrae may decrease in the same patient (e.g., ...). Figure 1 (As shown in C). This phenomenon indicates that the three-dimensional porous structure of different cancellous bones affects bone formation, and differences in the porous structure within different cancellous bones lead to different bone density recovery rates. Therefore, by selecting vertebrae with faster recovery based on bone density, a good three-dimensional porous structure can be obtained.

[0032] Further screening of the aforementioned 97 patients excluded 47 patients who received standardized anti-osteoporosis treatment and had their bone mineral density measured regularly. The rate of change in bone mineral density (ΔBMD%) was calculated using the formula: ΔBMD% = ΔBMD / ΔAge = (BMD2 - BMD1) / (Age2 - Age1) (where BMD1 is the initial value of bone mineral density of a specific lumbar vertebra measured at the initial time point, Age1 is the patient's age at the corresponding initial time point; BMD2 is the follow-up value of bone mineral density of the same lumbar vertebra measured at subsequent follow-up time points, Age2 is the patient's age at the corresponding follow-up time point; ΔBMD is the absolute change in bone mineral density between the two measurements; ΔAge is the time interval between the two measurements, with age in years). Figure 1 (As shown in D). Simultaneously, the percentage of bone mineral density (BMD) of each vertebra was compared, and the vertebra with the greatest rate of change was identified. Results showed that among the 50 patients, 17 had the highest BMD in the fourth lumbar vertebra (L4), 10 in the third lumbar vertebra (L3), 9 in the second lumbar vertebra (L2), and 14 in the first lumbar vertebra (L1). Overall, the fourth lumbar vertebra (L4) exhibited the largest positive rate of change, while the second lumbar vertebra (L2) showed a relatively slower recovery rate. The difference in change between the second lumbar vertebra (L2) and the fourth lumbar vertebra (L4) was the greatest (e.g., ...). Figure 1 (As shown in E). Therefore, L2 and L4 were selected for subsequent three-dimensional structure extraction and comparative studies.

[0033] Example 2: Design and fabrication of 3D-printed porous artificial bone Design concept and model acquisition of bionic artificial bone: For L2 and L4 determined in Example 1, high-resolution tomographic anatomical images of the lumbar spine of volunteers were acquired from the Department of Digital Medicine, College of Biomedical Engineering and Medical Imaging, Army Medical University. Figure 2 Aa、 Figure 2 The slice thickness of the image (Ab) is 0.1 mm. The high-resolution original image was converted to DICOM format and imported into the 3D visualization analysis software Amira (version 5.0). In the software parameter settings, both the slice thickness and slice interval were set to 0.1 mm, and the grayscale threshold for image segmentation was adjusted to 25. Subsequently, based on the anatomical characteristics of the human sectional anatomical image, the cancellous bone region was manually identified and segmented, and cortical bone and soft tissue interference were removed to obtain a high-resolution image of the cancellous bone microstructure. Figure 2 Finally, the extracted cancellous bone images were reconstructed in three dimensions to generate a porous digital model that reflects the true natural lumbar spine morphology and spatial tissue structure. Figure 2 The data was then exported as an STL file for subsequent selective laser melting (SLM) 3D printing. Following standard SLM printing procedures, using a Renishaw AM-400 machine, Ti6Al4V titanium alloy, a 400W laser, a scanning speed of 1.5m / s, and a powder layer thickness of 30μm, realistic porous metal artificial bone was produced (Figures 2A-e).

[0034] The final obtained L2 and L4 corresponding digital models are as follows: Figure 2 As shown in Figure B, L2 measures 50 mm × 42 mm × 20 mm, and L4 measures 57 mm × 48 mm × 22 mm. Subsequently, CT scans were performed on the 3D-printed artificial bones to obtain relevant parameters, which were then compared with the digital model. The results showed no significant differences between the digital model and the 3D-printed artificial bone in bone volume (BV), relative bone volume (BV / TV), connectivity density (Conn.D), number of trabeculae (Tb.N), and trabecular separation (Tb.Sp). For the 3D-printed artificial bone, the bone volume, bone volume to total volume ratio, and trabecular thickness (Tb.Th) of group L4 were all greater than those of group L2. However, there were no significant differences between the two groups in connectivity density (Conn.D), number of trabeculae (Tb.N), and trabecular separation (Tb.Sp). Figure 2 C).

[0035] Example 3: Characteristic Analysis of 3D Printed Porous Artificial Bone Microscopic morphology and property analysis of porous artificial bone: The structure of the artificial bone was evaluated using scanning electron microscopy (SEM). Figure 3 As shown in Figure A, the artificial bone has a rough surface covered with metal particles and no microcracks or defects. The SEM image also shows that it has anisotropic and interconnected porous structure.

[0036] Mechanical tests were performed on 3D-printed porous artificial bone, and the corresponding stress-strain curves are shown below. Figure 3 As shown in Figure B. The results show that there are no statistically significant differences in maximum bending strain, elastic modulus, and maximum bending stress between L2 and L4 (e.g., Figure 3 (as shown in C).

[0037] The internal structure of 3D-printed artificial bone was evaluated using mercury porosimetry, and the corresponding pore size-cumulative intrusion curves are shown below. Figure 3 As shown in Figure D, the average pore size of L4 was higher than that of L2 (L4 = 67.12 ± 6.79 μm, L2 = 51.58 ± 5.77 μm). The permeability (L2 = 4169.89 ± 478.67 mdarcy, L4 = 1873.63 ± 231.60 mdarcy) and tortuosity (L2 = 4.10 ± 0.55, L4 = 2.15 ± 0.36) of L4 were both higher than those of L2. No significant differences were found in porosity (L2 = 75.82 ± 5.77% vs. L4 = 74.24 ± 6.34%) and physical density (L2 = 3.57 ± 0.47 g / mL vs. L4 = 3.90 ± 0.65 g / mL). The quantitative comparison results of the above structural parameters are shown below. Figure 3 The bar chart for E is shown below.

[0038] Example 4: Identification of bone marrow mesenchymal stem cells and evaluation of in vitro biocompatibility of artificial bone Extraction and Phenotypic Identification of Primary Bone Marrow Mesenchymal Stem Cells (BMSCs): Primary BMSCs were isolated from human bone marrow and their phenotypic characteristics were identified. Flow cytometry results showed that the extracted BMSCs expressed CD90, CD73, and CD105, but did not express CD45, CD34, CD11b, CD19, or HLA-DR (e.g., CD90, CD73, and CD105). Figure 4 As shown in Aa). Figure 4 As shown in Figure Ab, adherent BMSCs are spindle-shaped and grow in a whorl pattern; after differentiation induction, they can differentiate into osteoblasts, adipocytes, and chondrocytes. The isolated BMSCs meet the criteria for stem cells defined by the International Society for Cell Therapy (ISCAP). Figure 4 B has a scale bar of 200 μm.

[0039] In vitro cell compatibility of artificial bone: The above-mentioned BMSCs were seeded onto 3D-printed artificial bone and cultured to assess their biocompatibility. After 72 hours of culture, the samples were stained for live / dead cells. The results showed that the BMSCs not only adhered to the artificial bone but also maintained their inherent spindle-shaped morphology. Cell viability assays showed that all cells were stained with green fluorescence, and no signs of cell death were observed. This demonstrates that both L4 and L2 artificial bone types exhibit extremely strong cell compatibility (e.g., ...). Figure 4 As shown in B, Figure 4 In diagram B, the scale bar is 200 μm in the main image and 100 μm in the high-power image.

[0040] Example 5: Verification of osteogenic induction capacity and its molecular mechanism In vitro osteogenic induction capacity test: To compare the osteogenic induction capacity of L2 and L4 printed materials, BMSCs were seeded on 3D printed porous artificial bone and induced to undergo osteogenic differentiation for 14 days using VivaCell's finished human bone marrow mesenchymal stem cell osteogenic differentiation induction medium.

[0041] Alizarin Red S staining (ARS) analysis showed that L4 artificial bone contained more calcium nodules than L2 artificial bone (e.g., Figure 5 As shown in A); quantitative analysis of ARS staining also showed that more alizarin red was formed in L4 artificial bone than in L2 artificial bone (as shown in A). Figure 5 As shown in B).

[0042] Osteogenic differentiation markers were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting (WB). The results showed that the expression levels of osteogenic-related genes and proteins Col1A1 and Runx2 were significantly higher in L4 artificial bone than in L2 artificial bone (e.g., ...). Figure 5 C- Figure 5 (As shown in E). The above results indicate that, compared with the L2 structure, the L4 structure artificial bone has a stronger potential to induce osteogenic differentiation of BMSCs.

[0043] Transcriptome sequencing and Notch2 signaling pathway validation: To explore the underlying mechanisms, cells were collected from L4 and L2 artificial bone 14 days after osteogenic differentiation induction, and RNA was extracted for transcriptome sequencing. Heatmaps of differentially expressed genes were created (e.g., […]). Figure 5 (as shown in F) and volcano diagram (as shown in F) Figure 5 As shown in G), this revealed a significant difference in gene expression between the two groups. KEGG enrichment analysis was performed on the differentially expressed genes, and the results were visualized using bubble charts (e.g., G). Figure 5(As shown in H), the analysis revealed significant enrichment of multiple bone-related signaling pathways, including the Notch signaling pathway and the MAPK signaling pathway.

[0044] Further detection of the expression of key factors in the Notch2 pathway, RT-PCR results (e.g.) Figure 5 (as shown in Figure I) and WB detection results (as shown in Figure I) Figure 5 J、 Figure 5 As shown in K), compared with L2 artificial bone, L4 artificial bone promoted the expression of more N2ICD (Notch2 intracellular domain) and its downstream molecule Hes1.

[0045] DAPT pathway blockade reverse verification experiment: 10 μM DAPT (Notch signaling pathway inhibitor) was added to the culture medium to block the activation of the Notch2 signaling pathway. With the addition of DAPT, ARS assay confirmed that the amount of calcium nodules produced by L2 and L4 artificial bone was approximately the same, eliminating previous differences in osteogenicity (e.g., Figure 6 A, Figure 6 As shown in B).

[0046] Meanwhile, RT-PCR showed that after the addition of DAPT, there were no longer significant differences in gene expression of Col1A1, Runx2, N2ICD, or Hes1 between L2 and L4 artificial bones (e.g., Figure 6 (As shown in C). Western blotting further confirmed that, in the absence of DAPT, the protein expression levels of the above genes in group L4 were higher than those in group L2; however, no significant difference was observed with the addition of 10 μM DAPT (as shown in C). Figure 6 D、 Figure 6 (As shown in E). These results definitively demonstrate that L4-structured artificial bone promotes osteogenic differentiation of BMSCs by activating the Notch2 signaling pathway.

[0047] The primer sequences involved in this example are: GAPDH-F GGAGCGAGATCCCTCCAAAAT (SEQ ID NO. 1), GAPDH-R GGCTGTTGTCATACTTCTCATGG (SEQ ID NO. 2); Col1A1-FGAGGGCCAAGACGAAGACATC (SEQ ID NO. 3), Col1A1-R CAGATCACGTCATCGCACAAC (SEQ ID NO. 4); Runx2-F AGGCAGTTCCCAAGCATTTCATCC(SEQ ID NO.5), Runx2-RGGCAGGTAGGTGTGGTAGTGAG(SEQ ID NO.6); Notch2-F CCTTCCACTGTGAGTGTCTGA(SEQ ID NO.7), Notch2-R AGGTAGCATCATTCTGGCAG(SEQ ID NO.8); Hes1-FACGTGCGAGGGCGTTAATAC(SEQ ID NO.9), Hes1-R GGGGTAGGTCATGGCATTGA (SEQ ID NO. 10).

[0048] Example 6: In vivo experimental evaluation of porous artificial bone in repairing critical bone defects This embodiment uses an in vivo implantation experiment in rats to verify the bone repair effect of 3D-printed porous artificial bone in vivo. Since a fully printed lumbar vertebra is too large, a sampling method was used to randomly select nine small cylinders (2 mm base diameter × 6 mm height) from the digital model and 3D print them for animal experiments.

[0049] A critical bone defect measuring 1 mm × 6 mm was created in the femur of SD rats, and then repaired by bone grafting using 3D-printed porous artificial bone designed with L2 and L4 cancellous bone structures, respectively. Eight weeks later, the femur was removed to assess bone formation within the artificial bone (sampling illustration shown). Figure 7 (As shown in A).

[0050] Micro-CT scans were performed on all samples, and the results showed that bone tissue grew together with the implant, and the internal pores of the implant were well filled with bone tissue (e.g., Figure 7 (As shown in B). Quantitative CT analysis showed that rats implanted with the L4 structure had significantly higher bone volume (BV), relative bone volume (BV / TV), trabecular number (Tb.N), connectivity density (Conn.D), and trabecular thickness (Tb.Th) than the L2 group, while the trabecular separation (Tb.Sp) of the L4 group was lower than that of the L2 group (as shown in B). Figure 7(as shown in C).

[0051] Ultrathin sections of the sample were examined using transmission electron microscopy (TEM). The results showed that the area surrounding the metallic material was filled with newly formed bone tissue, especially exhibiting a large amount of collagen fibers (such as...). Figure 7 As shown in D, the scale bar in the figure is 5μm.

[0052] Finally, mechanical tests were performed on the femur (sham surgery group) and the femur filled with printed artificial bone (L2 or L4 group). The corresponding stress-strain curves are shown below. Figure 7 As shown in E. Mechanical data show that: the maximum bending strain of group L4 was greater than that of the sham-operated group and group L2 (sham-operated group 0.028±0.005 mm, group L2 0.033±0.003 mm, group L4 0.036±0.001 mm, P<0.05); in terms of elastic modulus, group L4 was greater than group L2 or the sham-operated group (sham-operated group 6735.00±766.2 MPa, group L2 10153.45±1693.20 MPa, group L4 13367.42±1289.00 MPa, P<0.05); in terms of maximum bending stress, L4 was also greater than group L2 or the sham-operated group (sham-operated group 121.78±14.22 MPa, group L2 229.24±10.12 MPa, group L4 254.00±9.53 MPa, P<0.05) (as shown in E). Figure 7 As shown in F).

[0053] In vivo experiments have fully demonstrated that, under real physiological conditions, the comprehensive mechanical and biological capabilities of the L4-specific microstructure in inducing new bone formation and repairing critical bone defects are significantly greater than those of the L2 structure.

[0054] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A biomimetic porous artificial bone, characterized in that, The biomimetic porous artificial bone includes a metal skeleton, which is a porous mesh structure with interconnected structures. The porous mesh structure has the following topological parameters: average pore size of 60.3-73.9 μm, porosity of 67.9-80.5%, tortuosity of 1.79-2.51, and permeability of 1642.0-2105.2 mD / Cy.

2. The biomimetic porous artificial bone according to claim 1, characterized in that, The physical density of the metal skeleton is 3.25-4.55 g / mL.

3. The biomimetic porous artificial bone according to claim 1, characterized in that, The metal skeleton is made of Ti6Al4V titanium alloy.

4. The biomimetic porous artificial bone according to any one of claims 1-3, characterized in that, The physical environment formed by the interconnected porous mesh structure can activate the Notch2 signaling pathway in bone marrow mesenchymal stem cells.

5. A method for preparing a biomimetic porous artificial bone according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Obtain continuous cross-sectional anatomical images of the fourth lumbar vertebra of the human body, perform image segmentation on the continuous cross-sectional anatomical images, divide the cancellous bone region, and extract the cancellous bone image. S2. Perform three-dimensional reconstruction on the cancellous bone image to obtain a porous digital model that reflects the natural morphology and spatial organization of the fourth lumbar vertebra. S3. Using the aforementioned porous structure digital model as a processing model, titanium alloy powder is used to construct the biomimetic porous artificial bone through selective laser melting 3D printing.

6. The preparation method according to claim 5, characterized in that, In step S1, the thickness and slice interval of the continuous tomographic anatomical images are both 0.1 mm.

7. The preparation method according to claim 5, characterized in that, In step S1, the specific process of extracting the cancellous bone image is as follows: convert the continuous tomographic anatomical image into DICOM format and import it into the three-dimensional visualization software, adjust the grayscale threshold of image segmentation to 25, and manually identify and divide the cancellous bone region.

8. The preparation method according to claim 5, characterized in that, In step S3, the titanium alloy powder material is Ti6Al4V.

9. The use of a biomimetic porous artificial bone as described in any one of claims 1 to 4 in the preparation of implantable medical devices for bone defect repair.

10. The application according to claim 9, characterized in that, The bone defect repair implantable medical device is used to repair critical bone defects.

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