Phospholipid membrane-loaded orthopedic implants, methods of making and using the same

Abstract

The application provides a phospholipid membrane loaded orthopedic implant and a preparation method and application thereof. The orthopedic implant comprises a base material and a phospholipid membrane attached to the surface of the base material. The phospholipid membrane loaded orthopedic implant can significantly improve cell proliferation activity, cell compatibility and promote osteogenesis.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to an orthopedic implant loaded with a phospholipid membrane, its preparation method, and its application. Background Technology

[0002] Tissue engineering is playing an increasingly important role in the field of orthopedic repair, and the biocompatibility and functionality of the surface of orthopedic implants are key factors that determine the repair effect of bone tissue defects.

[0003] Traditional orthopedic implant surface treatment techniques mainly focus on physical morphology modification or inorganic coating modification. Although they have achieved certain results, they still have limitations in promoting the targeted recruitment and differentiation of stem cells. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide an orthopedic implant loaded with a phospholipid membrane, its preparation method and application.

[0005] Based on the bioactive properties of phospholipids, this invention optimizes the implant-tissue interface reaction by simulating the natural cell membrane microenvironment, providing a more effective solution for bone defect repair.

[0006] As one of the most abundant cell types in the human body, red blood cells exhibit unique application value in the field of tissue engineering due to their cell membrane structure. Recent studies have found that phospholipids, the main component of the red blood cell membrane, not only possess excellent biocompatibility but also specifically interact with hydroxyapatite, the main inorganic component of bone tissue, playing a crucial role in bone regeneration.

[0007] As a natural biomembrane system, the erythrocyte membrane possesses unique advantages and application potential in the field of tissue engineering. Structurally, the erythrocyte membrane is mainly composed of a phospholipid bilayer (approximately 50%), membrane skeletal proteins (approximately 30%), and carbohydrates (approximately 20%). This unique composition endows the erythrocyte membrane with excellent mechanical stability, biocompatibility, and multifunctionality. In biomedical applications, the erythrocyte membrane has been widely used in drug delivery systems, immune modulation, and the construction of biomimetic materials.

[0008] The unique value of erythrocyte membranes in orthopedic applications is mainly reflected in the following aspects: First, their phospholipid components (such as phosphatidylcholine and phosphatidylethanolamine) contain abundant phosphate groups, which can specifically interact with calcium ions and hydroxyapatite crystals. Studies have shown that this interaction may be involved in regulating the early mineralization core formation process. Second, specific proteins on the surface of erythrocyte membranes (such as band 3 proteins and blood group glycoproteins) can mediate cell-material signal transduction, affecting stem cell adhesion and differentiation behavior. Furthermore, erythrocyte membranes have excellent antifouling properties, reducing non-specific protein adsorption and thus creating favorable conditions for subsequent selective cell adhesion.

[0009] From a clinical application perspective, red blood cells, as a byproduct of routine blood tests, are widely available and easily obtained. High-purity red blood cell membrane components can be obtained through standardized separation and purification processes, providing a feasible basis for their large-scale biomedical applications. It is noteworthy that patient-derived red blood cell membranes exhibit lower immunogenicity, primarily due to the specific expression pattern of surface antigens during circulation. This characteristic is particularly important for reducing implant-related immune rejection.

[0010] The technical solution of this invention is as follows: In a first aspect, the present invention provides an orthopedic implant loaded with a phospholipid membrane, the orthopedic implant comprising a base material and a phospholipid membrane attached to the surface of the base material.

[0011] More preferably, the phospholipid membrane is a membrane structure formed by the self-assembly of phospholipid molecules in the erythrocyte membrane after the erythrocyte membrane has been broken down.

[0012] More preferably, the substrate material is a porous material, and the porous material is at least one of titanium alloy, hydroxyapatite, and tricalcium phosphate.

[0013] More preferably, the orthopedic implant includes a porous inorganic substrate material and a phospholipid membrane attached to the surface of the porous inorganic substrate material; the phospholipid membrane is a membrane structure formed by the self-assembly of phospholipid molecules after the red blood cell membrane is broken.

[0014] In a second aspect, the present invention provides a method for preparing the above-mentioned orthopedic implant loaded with a phospholipid membrane, characterized in that the preparation method includes the following steps: S1. Extracting red blood cell membranes; S2. The red blood cell membrane obtained in step S1 is broken down to obtain phospholipid molecules, and then the phospholipid molecules are prepared into a phospholipid molecule solution. S3. Immerse the substrate material in the solution of phospholipid molecules obtained in step S2. The phospholipid molecules self-assemble into a membrane structure and attach to the surface of the substrate material to obtain an orthopedic implant loaded with a phospholipid membrane.

[0015] More preferably, in step S1, red blood cell membranes are extracted by a combination of hypotonic lysis and gradient centrifugation.

[0016] More preferably, the hypotonic lysis combined with gradient centrifugation method is as follows: the sample and double-distilled water are lysed and broken down at a volume ratio of 1:3-10 for 15-20 minutes. The lysed sample solution is centrifuged using a high-speed centrifuge, the hemoglobin is discarded, the precipitate is washed, and the red blood cell membrane is obtained.

[0017] More preferably, the concentration of the phospholipid molecule solution is 2.81-90 mg / mL.

[0018] More preferably, the concentration of the phospholipid molecule solution is 5.63-90 mg / mL.

[0019] More preferably, the concentration of the phospholipid molecule solution is 45 mg / mL.

[0020] More preferably, the high-speed centrifuge is set with parameters of 5,000-10,000g and 10-30min.

[0021] More preferably, in step S2, the red blood cell membrane obtained in step S1 is broken up by ultrasound.

[0022] More preferably, the ultrasound settings are: power 200-400W, time 0.5-2min, intermittent mode.

[0023] More preferably, in step S3, the conditions for immersing the substrate material in the phospholipid molecule solution are: soaking at room temperature for 10-14 hours.

[0024] In a third aspect, the present invention provides the application of the above-described orthopedic implant loaded with a phospholipid membrane or the orthopedic implant loaded with a phospholipid membrane prepared by the above-described preparation method in the preparation of bone implant scaffold products.

[0025] Compared with the prior art, the present invention has the following beneficial effects: 1. The orthopedic implants loaded with phospholipid membranes provided by the present invention have shown in experimental results that: the orthopedic implants treated with phospholipid molecules not only promote the formation of osteogenic cores and improve the proliferation and differentiation behavior of subsequent cells; the orthopedic implants modified with phospholipid molecules did not cause obvious inflammatory reactions or ectopic ossification and other adverse reactions, indicating that the orthopedic implants loaded with phospholipid membranes provided by the present invention have good biocompatibility.

[0026] 2. The method for preparing orthopedic implants loaded with phospholipid membranes provided by the present invention has a simple preparation process. It only requires coating the surface of the orthopedic implant substrate material with phospholipid molecules extracted from red blood cell membranes, thereby preparing an orthopedic implant that can significantly improve the cell proliferation capacity and cell compatibility of the orthopedic implant surface.

[0027] 3. The orthopedic implants loaded with phospholipid membranes provided by this invention have been confirmed by lipid metabolomics analysis to be able to effectively enrich various bioactive phospholipid components in erythrocyte membranes, such as hydroxyapatite, tricalcium phosphate, and other inorganic porous substrate materials, through hydroxyapatite and tricalcium phosphate. Their enrichment effect is superior to that of organic polymer materials such as polycaprolactone, and they can construct a bioactive interface on the implant surface that simulates the microenvironment of natural cell membranes. Attached Figure Description

[0028] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a schematic diagram illustrating the in vitro proliferation results of osteogenic precursor cells (BMSCs) promoted by the porous bone implant scaffold provided in Experimental Example 1 of the present invention; wherein, Figure 1 Figure A shows a bar chart of the in vitro proliferation activity of osteoblast precursor cells (BMSCs). Figure 1 Figure B in the middle is a bar chart showing the expression results of the osteogenic progenitor cell BMSC proliferation gene CCNA2; Figure 2 This is a schematic diagram showing the results of the porous bone implant scaffold provided in Experimental Example 1 of the present invention promoting the expression of osteogenic genes RUNX2, OPN, OCN, ALP, and OSX in bone mesenchymal stem cells (BMSCs). Figure 3 This is a schematic diagram showing the results of the porous bone implant scaffold provided in Experimental Example 1 of the present invention promoting bone tissue formation. Figure 3 Figure A in the middle is a schematic diagram of the Micro-CT analysis results 4 weeks after implantation; Figure 3 Figure B in the middle is a schematic diagram of the histological section observation results of a rat femoral defect model. Figure 4 This is a schematic diagram of the CCK8 assay results showing that the porous hydroxyapatite bone implant scaffold treated with erythrocyte membrane phospholipids promoted the proliferation of human MG63 cells, as provided in Experimental Example 2 of this invention. Figure 5 This is a schematic diagram of the PCR detection results showing that the porous hydroxyapatite bone implant scaffold treated with erythrocyte membrane phospholipids promoted the expression of osteogenic genes ALP and RUNX2 in human MG63 cells, as provided in Experimental Example 2 of the present invention. Figure 6This is a schematic diagram of the RNA-seq transcriptome sequencing results of human MG63 cells in a porous hydroxyapatite bone implant scaffold treated with erythrocyte membrane phospholipids provided in Experimental Example 2 of the present invention. Figure 7 This is a schematic diagram of the lipid metabolomics analysis results of the hydroxyapatite and tricalcium phosphate implants after erythrocyte membrane phospholipid treatment provided in Experimental Example 3 of the present invention. Figure 8 This is a schematic diagram showing the comparison of the phospholipid enrichment capacity of hydroxyapatite, tricalcium phosphate, and polycaprolactone materials after phospholipid treatment of erythrocyte membranes, as provided in Experimental Example 3 of this invention. Detailed Implementation

[0029] The present invention will now be described in detail with reference to specific embodiments.

[0030] The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

[0031] In the following embodiments, the methods for extracting red blood cell membranes and preparing phospholipid molecule solutions are the same, as detailed below: Extraction of red blood cell membranes: Take 100 mL of blood sample from the patient during the operation. Mix the blood sample with sterile double-distilled water at a volume ratio of 1:3 for lysis and lysis for 20 min. Centrifuge the lysed sample solution at 10,000 g for 10 min using a high-speed centrifuge. Discard the hemoglobin supernatant and wash the precipitate three times with PBS to obtain the red blood cell membrane.

[0032] Preparation of phospholipid molecule solution: The obtained red blood cell membranes were placed in Tris-HCl buffer (pH 7.4) and disrupted by sonication. The sonication parameters were: power 200W, time 2min, intermittent mode, to obtain phospholipid molecules. Phospholipid molecules were then used as a solvent to prepare a phospholipid molecule solution of the required concentration.

[0033] First Embodiment This embodiment provides an orthopedic implant loaded with a phospholipid membrane. In this embodiment, the substrate material is a porous titanium alloy, and the concentration of the phospholipid molecule solution is 2.81 mg / mL.

[0034] The method for preparing the orthopedic implant loaded with a phospholipid membrane in this embodiment is as follows: Porous titanium alloys were soaked in PBS, and their surfaces were activated by plasma cleaning. The pretreated porous titanium alloys were then immersed in a phospholipid molecule solution with a concentration of 2.81 mg / mL at room temperature for 12 hours. During the immersion process, the phospholipid molecules in the solution self-assembled to form a membrane structure (phospholipid membrane) that adhered to the surface of the titanium alloy, resulting in orthopedic implants loaded with phospholipid membranes.

[0035] Second Embodiment This embodiment provides an orthopedic implant loaded with a phospholipid membrane. In this embodiment, the substrate material is a porous titanium alloy, and the concentration of the phospholipid molecule solution is 5.63 mg / mL.

[0036] The method for preparing the orthopedic implant loaded with a phospholipid membrane in this embodiment is as follows: Porous titanium alloys were soaked in PBS, and their surfaces were then activated by plasma cleaning. The pretreated porous titanium alloys were then immersed in a phospholipid molecule solution with a concentration of 5.63 mg / mL at room temperature for 12 hours. During the immersion process, the phospholipid molecules in the solution self-assembled to form a membrane structure (phospholipid membrane) that adhered to the surface of the titanium alloy, resulting in orthopedic implants loaded with a phospholipid membrane.

[0037] Third Embodiment This embodiment provides an orthopedic implant loaded with a phospholipid membrane. In this embodiment, the substrate material is a porous titanium alloy, and the concentration of the phospholipid molecule solution is 11.25 mg / mL.

[0038] The method for preparing the orthopedic implant loaded with a phospholipid membrane in this embodiment is as follows: Porous titanium alloys were soaked in PBS, and their surfaces were then activated by plasma cleaning. The pretreated porous titanium alloys were then immersed in a phospholipid molecule solution with a concentration of 11.25 mg / mL and soaked at room temperature for 12 h. During the immersion process, the phospholipid molecules in the solution self-assembled to form a membrane structure (phospholipid membrane) that adhered to the surface of the titanium alloy, resulting in orthopedic implants loaded with phospholipid membranes.

[0039] Fourth embodiment This embodiment provides an orthopedic implant loaded with a phospholipid membrane. In this embodiment, the substrate material is a porous titanium alloy, and the concentration of the phospholipid molecule solution is 22.5 mg / mL.

[0040] The method for preparing the orthopedic implant loaded with a phospholipid membrane in this embodiment is as follows: Porous titanium alloys were soaked in PBS, and their surfaces were then activated by plasma cleaning. The pretreated porous titanium alloys were then immersed in a phospholipid molecule solution with a concentration of 22.5 mg / mL at room temperature for 12 hours. During the immersion process, the phospholipid molecules in the solution self-assembled to form a membrane structure (phospholipid membrane) that adhered to the surface of the titanium alloy, resulting in orthopedic implants loaded with a phospholipid membrane.

[0041] Fifth embodiment This embodiment provides an orthopedic implant loaded with a phospholipid membrane. In this embodiment, the substrate material is a porous titanium alloy, and the concentration of the phospholipid molecule solution is 45 mg / mL.

[0042] The method for preparing the orthopedic implant loaded with a phospholipid membrane in this embodiment is as follows: Porous titanium alloys were soaked in PBS, and their surfaces were then activated by plasma cleaning. The pretreated porous titanium alloys were then immersed in a phospholipid molecule solution with a concentration of 45 mg / mL for 12 hours at room temperature. During the immersion process, the phospholipid molecules in the solution self-assembled to form a membrane structure (phospholipid membrane) that adhered to the surface of the titanium alloy, resulting in orthopedic implants loaded with a phospholipid membrane.

[0043] Sixth Embodiment This embodiment provides an orthopedic implant loaded with a phospholipid membrane. In this embodiment, the base material is a porous titanium alloy, and the concentration of the phospholipid molecule solution is 90 mg / mL.

[0044] The method for preparing the orthopedic implant loaded with a phospholipid membrane in this embodiment is as follows: Porous titanium alloys were soaked in PBS, and their surfaces were then activated by plasma cleaning. The pretreated porous titanium alloys were then immersed in a phospholipid molecule solution with a concentration of 90 mg / mL for 12 hours at room temperature. During the immersion process, the phospholipid molecules in the solution self-assembled to form a membrane structure (phospholipid membrane) that adhered to the surface of the titanium alloy, resulting in orthopedic implants loaded with a phospholipid membrane.

[0045] Seventh Embodiment This embodiment provides an orthopedic implant loaded with a phospholipid membrane. In this embodiment, the base material is porous hydroxyapatite, and the concentration of the phospholipid molecule solution is 45 mg / mL.

[0046] The method for preparing the orthopedic implant loaded with a phospholipid membrane in this embodiment is as follows: Porous hydroxyapatite was soaked in PBS, and its surface was activated by plasma cleaning. The pretreated porous hydroxyapatite was then immersed in a phospholipid molecule solution with a concentration of 45 mg / mL at room temperature for 12 h. During the immersion process, the phospholipid molecules in the solution self-assembled to form a membrane structure (phospholipid membrane) that adhered to the surface of the hydroxyapatite, resulting in an orthopedic implant loaded with a phospholipid membrane.

[0047] Eighth embodiment This embodiment provides an orthopedic implant loaded with a phospholipid membrane. In this embodiment, the base material is porous tricalcium phosphate, and the concentration of the phospholipid molecule solution is 45 mg / mL.

[0048] The method for preparing the orthopedic implant loaded with a phospholipid membrane in this embodiment is as follows: Porous tricalcium phosphate was soaked in PBS, and its surface was activated by plasma cleaning. The pretreated porous tricalcium phosphate was then immersed in a phospholipid molecule solution with a concentration of 45 mg / mL and soaked at room temperature for 12 h. During the soaking process, the phospholipid molecules in the solution self-assembled to form a membrane structure (phospholipid membrane) that adhered to the surface of the tricalcium phosphate, resulting in an orthopedic implant loaded with a phospholipid membrane.

[0049] Application examples This application example selects the orthopedic implants loaded with phospholipid membranes provided in the above embodiments and prepares them into porous bone implant scaffolds. The preparation method is as follows: Orthopedic implants loaded with phospholipid membranes were mixed with sterile saline at a ratio of 1:10 and incubated with shaking in a 37°C, 5% CO2 cell culture incubator for 30 minutes to obtain a porous bone implant scaffold.

[0050] Experimental Example 1 This experimental example selects the orthopedic implants with phospholipid membranes provided in the first to sixth embodiments, and prepares porous bone implant scaffolds using the method provided in the application examples. The following experiments comprehensively evaluate the in vitro biological effects and in vivo osteogenic effects of the porous bone implant scaffolds described in this invention. A control group (Control) is also set up: porous titanium alloys without phospholipid molecule solution treatment are used, and porous bone implant scaffolds prepared using the method provided in the application examples are employed.

[0051] The experimental groups are set up as follows: Control group: The porous bone implant scaffold was prepared from a porous titanium alloy that had not been treated with phospholipid molecule solution; Group PL0.5: The porous bone implant scaffold was prepared from the orthopedic implant loaded with a phospholipid membrane provided in the first embodiment (phospholipid molecule solution concentration 2.81 mg / mL); Group PL1: The porous bone implant scaffold was prepared from the orthopedic implant loaded with a phospholipid membrane provided in the second embodiment (phospholipid molecule solution concentration 5.63 mg / mL); Group PL2: The porous bone implant scaffold was prepared from the orthopedic implant loaded with a phospholipid membrane provided in the third embodiment (phospholipid molecule solution concentration 11.25 mg / mL); Group PL4: The porous bone implant scaffold was prepared from the orthopedic implant loaded with a phospholipid membrane provided in the fourth embodiment (phospholipid molecule solution concentration 22.5 mg / mL); Group PL8: The porous bone implant scaffold was prepared from the orthopedic implant loaded with a phospholipid membrane provided in the fifth embodiment (phospholipid molecule solution concentration 45 mg / mL); Group PL16: The porous bone implant scaffold was prepared from the orthopedic implant loaded with a phospholipid membrane provided in the sixth embodiment (phospholipid molecule solution concentration 90 mg / mL).

[0052] 1. In vitro experiments (1) CCK8 detection Osteoblastic precursor cells (BMSCs) were co-incubated with the porous bone implantation scaffolds of the above groups at 37°C in a 5% CO2 cell culture incubator. After 8 days of in vitro co-incubation, cell viability was detected using a CCK8 staining kit (Beyotime, Shanghai).

[0053] Experimental results are as follows Figure 1 As shown in Figure A, after 3 days of in vitro co-incubation, the cell activity of porous bone implant scaffolds treated with phospholipid molecule solution in groups PL4 (22.5 mg / mL), PL2 (11.25 mg / mL), PL1 (5.63 mg / mL), and PL0.5 (2.81 mg / mL) was significantly higher than that in the control group. After 8 days of in vitro co-incubation, the cell proliferation activity of porous bone implant scaffolds treated with phospholipid molecule solution in group PL8 (45 mg / mL) was significantly increased and significantly higher than that in the control group. This indicates that the porous bone implant scaffold provided by this invention can significantly enhance the proliferation activity of cells on the surface of orthopedic implants.

[0054] After 8 days of in vitro co-incubation with the porous bone implant scaffold, the expression levels of a cell proliferation-related gene (CCNA2) were analyzed using quantitative real-time PCR. The results are as follows: Figure 1 As shown in Figure B, in groups PL1, PL2, and PL16, the expression level of the CCNA2 gene in cells co-incubated with the porous bone implant scaffold was significantly higher than that in the control group, indicating that the porous bone implant scaffold treated with phospholipid molecule solution can improve cell proliferation performance.

[0055] In summary, the porous bone implant scaffold provided by this invention can enhance the proliferative activity of cells on the surface of orthopedic implants. The porous bone implant scaffold in this experiment was prepared from the orthopedic implant loaded with a phospholipid membrane provided by this invention. This also demonstrates that the orthopedic implant loaded with a phospholipid membrane provided by this invention does not have a toxic effect on cells, has excellent cell compatibility, and can improve cell proliferation performance.

[0056] (2) Osteogenic differentiation potential test Osteogenic precursor cells (BMSCs) were co-incubated with the aforementioned porous bone implantation scaffolds at 37°C in a 5% CO2 cell culture incubator. After 10 days of osteogenic induction culture using osteogenic induction cell culture medium (Cyagen Biosciences, Shanghai), the red calcium nodule regions were stained with Alizarin Red to verify osteogenic performance. The expression levels of key osteogenic-related genes (RUNX2, OPN, OCN, ALP, OSX) in BMSCs were analyzed using quantitative real-time PCR. The results are as follows: Figure 2 As shown, for the RUNX2 gene, in group PL16 (90 mg / mL), the expression level of key osteogenic genes in porous bone implant scaffolds prepared with orthopedic implants treated with phospholipid molecule solution was increased compared with the control group; for the OPN gene, the expression level of key osteogenic genes in porous bone implant scaffolds prepared with orthopedic implants treated with phospholipid molecule solution was increased compared with the control group; for the OCN gene, the expression level of key osteogenic genes in porous bone implant scaffolds prepared with orthopedic implants treated with phospholipid molecule solution was increased compared with the control group; for the ALP gene... Therefore, in the PL1 group (5.63 mg / mL), PL8 group (45 mg / mL), and PL16 group (90 mg / mL), the expression levels of key osteogenic genes in the porous bone implant scaffolds prepared with orthopedic implants treated with phospholipid molecule solution were all increased compared with the control group. For the OSX gene, the expression levels of key osteogenic genes in the porous bone implant scaffolds prepared with orthopedic implants treated with phospholipid molecule solution were all increased compared with the control group in the PL1 group (5.63 mg / mL), PL8 group (45 mg / mL), and PL16 group (90 mg / mL). These results indicate that the phospholipid-loaded orthopedic implants provided by this invention can significantly promote the osteogenic phenotype of bone marrow mesenchymal stem cells (BMSCs), which is beneficial for promoting osteogenic differentiation.

[0057] 2. In vivo experiments This experiment used porous bone implant scaffolds prepared in group PL8 (phospholipid molecule solution concentration of 45 mg / mL). These scaffolds were implanted into an SD rat femoral defect model. Micro-CT analysis was performed on the rat femoral defect model at different time points. A control group was also included: the porous bone implant scaffold was prepared from porous titanium alloy that had not been treated with phospholipid molecule solution.

[0058] The results are as follows Figure 3 As shown in Figure A, at 4 weeks post-implantation, the new bone volume fraction (BV / TV) in the treatment group reached 35.7±3.2%, significantly higher than that in the control group (22.1±2.8%). Histological section observation results are as follows... Figure 3As shown in Figure B, continuous bone tissue was formed at the bone interface of the porous bone implant scaffold in the treatment group, while obvious fibrous tissue septa were present in the control group. This indicates that the orthopedic implant with phospholipid membrane provided by the present invention can promote the formation of osteogenic core and is beneficial to promoting the healing of bone injury sites.

[0059] A long-term observation (12 months) was conducted on a rat femoral defect animal model implanted with a porous bone implant scaffold. The results showed that the porous bone implant scaffold provided by this invention did not cause significant inflammatory reactions or ectopic ossification and other adverse reactions. This indicates that the orthopedic implant with a phospholipid membrane provided by this invention has good biocompatibility.

[0060] Treatment group: The porous bone implant scaffold was prepared from the orthopedic implant loaded with a phospholipid membrane provided in the fifth embodiment (phospholipid molecule solution concentration 45 mg / mL); Control group: The porous bone implant scaffold was prepared from a porous titanium alloy that had not been treated with phospholipid molecule solution.

[0061] Experimental Example 2 To further verify the biological effects of the orthopedic implants loaded with phospholipid membranes provided by this invention when hydroxyapatite is used as the base material, and to compare them with other types of cell membrane treatment methods, this experimental example is based on the seventh embodiment, and human MG63 osteoblast-like cells are used for in vitro functional evaluation.

[0062] The porous bone implant scaffold used in this experimental example was prepared by selecting the orthopedic implant with a phospholipid membrane loaded as provided in the seventh example (porous hydroxyapatite substrate material, phospholipid molecule solution concentration 45mg / mL) and preparing it into a porous bone implant scaffold according to the method provided in the application example.

[0063] Meanwhile, a control group was set up: osteoblast membrane phospholipid molecule solution of the same concentration (45 mg / mL) was used instead of erythrocyte membrane phospholipid molecule solution, and porous hydroxyapatite was treated in the same way to prepare control porous bone implant scaffolds.

[0064] The method for extracting osteoblast membranes is as follows: collect cultured osteoblasts, extract osteoblast membranes using a combination of hypotonic lysis and gradient centrifugation, and then follow the same steps as for erythrocyte membrane processing.

[0065] The experimental groups are set up as follows: Erythrocyte membrane phase: Human MG63 cells were co-incubated with the porous hydroxyapatite bone implant scaffold treated with erythrocyte membrane phospholipids provided in the seventh embodiment; Control group (osteoblast membrane group): Human MG63 cells were co-incubated with porous hydroxyapatite bone implant scaffolds treated with osteoblast membrane phospholipids.

[0066] (1) CCK8 detection Human MG63 osteoblast-like cells were seeded into porous hydroxyapatite bone implantation scaffolds in the erythrocyte membrane group and the control group, respectively, and co-incubated in a 37°C, 5% CO2 cell culture incubator. Cell proliferation activity was detected by CCK8 assay.

[0067] Experimental results are as follows Figure 4 As shown, the erythrocyte membrane group promoted the proliferation of human MG63 cells inside the hydroxyapatite implant more effectively than the control group (osteoblast membrane group). This result indicates that phospholipid molecules derived from erythrocyte membranes have a significant advantage in promoting cell proliferation inside orthopedic implants.

[0068] (2) Osteogenic gene expression detection Human MG63 osteoblast-like cells were seeded into porous hydroxyapatite bone implant scaffolds in the erythrocyte membrane group and the control group, respectively, and then incubated. The expression of osteoblast genes alkaline phosphatase (ALP) and Runt-related transcription factor 2 (RUNX2) was detected by real-time quantitative PCR.

[0069] Experimental results are as follows Figure 5 As shown, the erythrocyte membrane group significantly enhanced the osteogenic phenotype of human MG63 cells within the hydroxyapatite implant compared to the control group (osteoblast membrane group), with significantly higher expression levels of ALP and RUNX2 genes. This result further indicates that phospholipid molecules derived from erythrocyte membranes can effectively promote osteogenic differentiation of cells within orthopedic implants.

[0070] (3) RNA-seq transcriptome sequencing analysis To further explore the molecular mechanism by which erythrocyte membrane phospholipid-modified orthopedic implants promote cell function, RNA-seq transcriptome sequencing analysis was performed on human MG63 cells cultured in porous hydroxyapatite bone implant scaffolds in both the erythrocyte membrane group and the control group.

[0071] Experimental results are as follows Figure 6 As shown, the erythrocyte membrane group, compared with the control group (osteoblast membrane group), significantly enhanced the adhesion, proliferation, and expression of genes related to rapid hypoxia response of human MG63 cells within the hydroxyapatite implant. These results reveal, at the transcriptomic level, that erythrocyte membrane phospholipid-modified orthopedic implants improve the biological effects of the implant-cell interface by promoting cell adhesion, proliferation, and enhancing hypoxia adaptation.

[0072] Experiment Example 3 To clarify the specific binding components of erythrocyte membrane phospholipid molecules on the surface of orthopedic implants and the differences in phospholipid enrichment among different substrate materials, this study used lipid metabolomics to analyze different orthopedic implants treated with erythrocyte membrane phospholipids.

[0073] (1) Phospholipid-binding component analysis of hydroxyapatite and tricalcium phosphate implants Orthopedic implants loaded with phospholipid membranes provided in the seventh example (porous hydroxyapatite, phospholipid molecule solution concentration 45 mg / mL) and the eighth example (porous tricalcium phosphate, phospholipid molecule solution concentration 45 mg / mL) were selected and lipidomics sequencing analysis was performed using lipid metabolomics.

[0074] Experimental results are as follows Figure 7 As shown, both hydroxyapatite and tricalcium phosphate orthopedic implants can effectively bind various bioactive components in erythrocyte membrane phospholipids, including phosphatidylcholine (PC), sphingomyelin (SM), acetal phosphatidylcholine (PCp), triglycerides (TG), and sphingolipids (Sph). This result indicates that the porous inorganic substrate material described in this invention can effectively enrich various bioactive phospholipid components in the erythrocyte membrane. The enrichment of these phospholipid components helps to construct a bioactive interface on the implant surface that mimics the natural cell membrane microenvironment.

[0075] (2) Comparison of the ability of different substrate materials to enrich phospholipid components To verify the advantages of the porous inorganic substrate material described in this invention in phospholipid enrichment compared to other common bone implant materials, polycaprolactone was selected as a comparative material in addition to the aforementioned hydroxyapatite and tricalcium phosphate. Porous hydroxyapatite, porous tricalcium phosphate, and polycaprolactone materials were treated with a erythrocyte membrane phospholipid molecule solution of the same concentration (45 mg / mL), and lipid metabolomics was used to compare the phospholipid enrichment capabilities of the three materials.

[0076] Experimental results are as follows Figure 8 As shown, hydroxyapatite and tricalcium phosphate, compared with polycaprolactone, can better enrich various bioactive components such as phosphatidylcholine (PC), sphingomyelin (SM), triglycerides (TG), and sphingolipids (Sph). This result indicates that inorganic porous materials, represented by hydroxyapatite and tricalcium phosphate, have a stronger affinity and binding capacity with phospholipid molecules in erythrocyte membranes. This may be related to the specific interaction between calcium ions and phosphate groups on the surface of inorganic materials and the phosphate head groups in phospholipid molecules, further confirming the rationality and superiority of selecting inorganic porous materials such as hydroxyapatite and tricalcium phosphate as substrate materials in this invention.

[0077] In summary, the results of Experiments 1 to 3 demonstrate that the orthopedic implants loaded with phospholipid membranes provided by this invention not only promote the formation of osteogenic cores but also significantly improve cell proliferation and differentiation behavior. Phospholipid molecules derived from erythrocyte membranes exhibit superior proliferative and osteodifferentiation-promoting effects compared to phospholipid molecules derived from other cell membranes. The inorganic porous substrate materials such as hydroxyapatite and tricalcium phosphate used in this invention can effectively enrich various bioactive phospholipid components from erythrocyte membranes, meeting clinical requirements for bone tissue defect repair, exhibiting good biocompatibility, and playing a crucial role in bone regeneration.

[0078] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in this application can be arbitrarily combined with each other.

Claims

1. An orthopedic implant loaded with a phospholipid membrane, characterized in that, The orthopedic implant includes a base material and a phospholipid membrane attached to the surface of the base material.

2. The orthopedic implant loaded with a phospholipid membrane according to claim 1, characterized in that, The phospholipid membrane is a membrane structure formed by the self-assembly of phospholipid molecules within the erythrocyte membrane after the membrane has been broken down.

3. The orthopedic implant loaded with a phospholipid membrane according to claim 1, characterized in that, The substrate material is a porous material, and the porous material is at least one of titanium alloy, hydroxyapatite, and tricalcium phosphate.

4. A method for preparing an orthopedic implant loaded with a phospholipid membrane as described in any one of claims 1-3, characterized in that, The preparation method includes the following steps: S1. Extracting red blood cell membranes; S2. The red blood cell membrane obtained in step S1 is broken down to obtain phospholipid molecules, and then the phospholipid molecules are prepared into a phospholipid molecule solution. S3. Immerse the substrate material in the phospholipid molecule solution obtained in step S2. The phospholipid molecules self-assemble into a membrane structure and attach to the surface of the substrate material to obtain an orthopedic implant loaded with a phospholipid membrane.

5. The method for preparing the orthopedic implant loaded with a phospholipid membrane according to claim 4, characterized in that, In step S1, red blood cell membranes are extracted by a combination of hypotonic lysis and gradient centrifugation.

6. The method for preparing the orthopedic implant loaded with a phospholipid membrane according to claim 5, characterized in that, The hypotonic lysis combined with gradient centrifugation method is as follows: the sample and double-distilled water are lysed at a volume ratio of 1:3-10 for 15-20 minutes. The lysed sample solution is centrifuged using a high-speed centrifuge, the hemoglobin is discarded, the precipitate is washed, and the red blood cell membrane is obtained.

7. The method for preparing the orthopedic implant loaded with a phospholipid membrane according to claim 6, characterized in that, The high-speed centrifuge is set to 5,000-10,000g for 10-30 minutes.

8. The method for preparing the orthopedic implant loaded with a phospholipid membrane according to claim 4, characterized in that, In step S2, the red blood cell membrane obtained in step S1 is broken up by ultrasound.

9. The method for preparing the orthopedic implant loaded with a phospholipid membrane according to claim 8, characterized in that, The ultrasound settings are: power 200-400W, time 0.5-2min, intermittent mode.

10. The use of the orthopedic implant loaded with a phospholipid membrane according to any one of claims 1-3 or the orthopedic implant loaded with a phospholipid membrane prepared by the preparation method according to any one of claims 4-9 in the preparation of bone implant scaffold products.

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