A mixed cell membrane functionalized bone repair matrix for regulating inflammatory microenvironment and preparation method thereof

Abstract

The present invention relates to a mixed cell membrane functionalized bone repair matrix for regulating an inflammatory microenvironment and a preparation method thereof, and belongs to the technical field of bone repair matrix preparation. The present invention discloses a preparation method of a mixed cell membrane functionalized bone repair matrix for regulating an inflammatory microenvironment, wherein the amino groups in polycaprolactone nanofibers and mixed cell membranes (anti-inflammatory macrophage membranes and osteogenic differentiated bone marrow mesenchymal stem cell membranes) are reacted with the carboxyl groups on biotin to perform biotin modification to obtain biotin-modified polycaprolactone nanofibers and biotin-modified mixed cell membranes, and then streptavidin is added for coupling reaction to obtain a mixed cell membrane functionalized bone repair matrix for regulating an inflammatory microenvironment.

Description

Technical Field

[0001] The invention belongs to the technical field of bone repair preparation, and relates to a mixed cell membrane functionalized bone repair matrix for regulating inflammatory microenvironment and a preparation method thereof. Background Art

[0002] Bone defect is a common clinical disease in orthopedics. Autologous bone transplantation is the most advantageous treatment for bone defect. However, the amount of autologous bone is limited, and symptoms such as infection and pain after surgery affect the application of autologous bone transplantation. With the development of bone tissue engineering, bone repair matrix has a significant effect in the treatment of bone defects. Common bone tissue engineering matrix can not only simulate the three-dimensional structure of natural extracellular matrix, have good mechanical properties and biocompatibility, but also has the ability to promote the adhesion, proliferation and osteogenic differentiation of osteoblasts; at the same time, the process of bone tissue regeneration and bone defect repair is complex and dynamic, accompanied by the infiltration of inflammatory cells, the proliferation, migration and differentiation of osteoprogenitor cells, and the formation of extracellular matrix around bones, ultimately achieving bone tissue reconstruction. Bionic bone extracellular matrix (bECM) needs to have the ability to regulate inflammation and regenerate the microenvironment.

[0003] As a common bECM material, electrospun micro / nanofiber scaffolds have good porosity and three-dimensional connectivity and have been widely used in biomedical fields such as wound healing, tissue engineering and drug delivery. However, after transplantation, the material will non-specifically adsorb a large number of immune cells (such as monocytes, macrophages, neutrophils, dendritic cells, etc.). Inflammatory cells will degrade the surface of the material and secrete proinflammatory factors, leading to an imbalance in the local tissue inflammatory microenvironment; macrophages are important cells that regulate the body's immune and inflammatory responses in bone tissue repair; proinflammatory macrophages (M1 phenotype) and pro-repair macrophages (M2 phenotype) have unique synergistic effects in maintaining the stability of the tissue microenvironment, and regulating macrophage phenotypic transformation has become an attractive treatment method in bone tissue engineering. Therefore, targeting the cellular behavior and function of immune cells may potentially regulate bone regeneration.

[0004] There are many traditional methods to regulate the immune response and improve the regenerative microenvironment after the implantation of electrospun materials, including the addition of bone promoters and immunomodulators, such as protein growth factors, cytokines, chemokines, etc. However, these substances have some disadvantages, including unstable biological activity in vivo, easy degradation, explosive release causing systemic toxicity, etc. Micro-nanocarriers disguised with natural cell membranes have the physicochemical properties of natural cells and have similar biological functions to natural cells. In recent decades, biological cell membranes extracted from red blood cells, platelets, macrophages and stem cells have been widely used in drug delivery, detoxification, immunomodulation, detection, imaging and photoactivated therapy. At the same time, the interaction of biological molecules such as cell membrane surface proteins on the cell membrane surface involves life activities, including cell growth and metabolism, endocytosis and exocytosis, signal transduction, migration, apoptosis, etc. Differences in surface proteins contained in cell membranes from different cell sources may affect cell function.

[0005] Therefore, finding a suitable cell membrane is expected to explore new avenues for bone tissue engineering. At present, there are no hybrid membrane-functionalized electrospun nanofibers based on immune cells and mesenchymal stem cells, and there are no reports on hybrid membrane-functionalized matrices synergistically promoting bone defect regeneration. It is necessary to study and prepare hybrid membrane-functionalized matrices so that they can play the functions of immune cell membranes and mesenchymal stem cell membranes, synergistically promote inflammation regulation and bone defect repair, and provide a promising option for the preparation of bone regeneration materials. Summary of the invention

[0006] In view of this, one of the objects of the present invention is to provide a mixed cell membrane functionalized bone repair matrix for regulating the inflammatory microenvironment; a second object of the present invention is to provide a method for preparing a mixed cell membrane functionalized bone repair matrix for regulating the inflammatory microenvironment; a third object of the present invention is to provide an application of a mixed cell membrane functionalized bone repair matrix for regulating the inflammatory microenvironment.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] 1. A method for preparing a mixed cell membrane functionalized bone repair matrix for regulating inflammatory microenvironment, the preparation method comprising the following steps:

[0009] (1) Preparation of biotin-modified polycaprolactone nanofibers: a 0.1-1.0 mM biotin solution, a 0.1-1.0 M 1-(3-dimethylaminopropyl)-3-ethylcarbamide ester solution, and a 0.1-1.0 M N-hydroxysuccinimide solution were mixed, and then polycaprolactone nanofibers were added to react at 37° C. for 2-6 h, so that the carboxyl group of biotin reacts with the amino group of the polycaprolactone nanofiber to obtain biotin-modified polycaprolactone nanofibers;

[0010] (2) preparing a mixed cell membrane: mixing equal amounts of anti-inflammatory macrophage membrane and osteogenic differentiated bone marrow mesenchymal stem cell membrane, ultrasonically treating for 15 to 30 minutes, and extruding through a 100 to 400 nm polycarbonate porous membrane for 10 to 20 times to obtain a mixed cell membrane;

[0011] (3) preparing a biotin-modified mixed cell membrane: mixing a 0.1-1.0 mM biotin solution, a 0.1-1.0 M 1-(3-dimethylaminopropyl)-3-ethylcarbamide ester solution, and a 0.1-1.0 M N-hydroxysuccinimide solution, adding the mixed cell membrane and treating overnight at 4° C. to allow the carboxyl group of biotin to react with the amino group of the mixed cell membrane to obtain a biotin-modified mixed cell membrane;

[0012] (4) The biotin-modified polycaprolactone nanofibers prepared in step (1), the biotin-modified mixed cell membrane prepared in step (3) and streptavidin are mixed to form a mixture, and the mixture is coupled for 2 to 6 hours to obtain a mixed cell membrane functionalized bone repair matrix that regulates the inflammatory microenvironment.

[0013] Preferably, in step (1), the polycaprolactone nanofibers are prepared according to the following method: pure polycaprolactone (poly-ε-caprolactone, PCL) particles are dissolved in hexafluoroisopropanol, transported at a speed of 1 mL / h through a jet pump, a 16-22G passivated stainless steel needle and a 15kv high-voltage power supply, and collected on aluminum foil;

[0014] The aluminum foil is wrapped on an open cage drum collector with a rotation speed of no more than 1000 rpm.

[0015] Preferably, in step (1), the ratio of biotin, 1-(3-dimethylaminopropyl)-3-ethylcarbamide salt ester, N-hydroxysuccinimide and the surface area of ​​polycaprolactone nanofibers in the biotin solution is 1.22:0.96:0.58:1-4.82:0.40:0.11:1, mg:mg:mg:cm 2 .

[0016] Preferably, in step (2), the anti-inflammatory macrophage membrane is prepared according to the following method:

[0017] (1) RAW264.7 macrophages were cultured for 7 to 14 days in conditioned medium containing 10 to 100 ng / mL interleukin 4 (IL-4) to obtain anti-inflammatory macrophages;

[0018] (2) The anti-inflammatory macrophage suspension was obtained by cell scraping, added to a low osmotic lysis buffer containing a protease inhibitor, cooled in an ice bath for 15 to 30 minutes, placed in liquid nitrogen and a 37°C water bath in turn and repeatedly frozen and thawed to lyse and break the cells, and the anti-inflammatory macrophage membrane precipitate was obtained by gradient centrifugation. The anti-inflammatory macrophage membrane precipitate was resuspended in triple distilled water, and extruded through a 100 to 400 nm polycarbonate porous membrane for 10 to 20 times to obtain an anti-inflammatory macrophage membrane.

[0019] Preferably, in step (2), the osteogenic differentiated bone marrow mesenchymal stem cell membrane is prepared according to the following method:

[0020] (1) A solution containing 1 mM sodium pyruvate and 10 -7 Bone marrow mesenchymal stem cells are cultured in an osteogenic induction medium containing 100 μM dexamethasone, 50 μg / mL vitamin C, and 10 mM β-glycerophosphate for 7 to 14 days to obtain osteogenic differentiated bone marrow mesenchymal stem cells;

[0021] (2) Obtain osteogenic differentiated bone marrow mesenchymal stem cell suspension by cell scraping, add it to a low osmotic lysis buffer containing a protease inhibitor, cool it in an ice bath for 15 to 30 minutes, place it in liquid nitrogen and a 37°C water bath in turn to lyse and break the cells, obtain osteogenic differentiated bone marrow mesenchymal stem cell membrane precipitate by gradient centrifugation, resuspend the osteogenic differentiated bone marrow mesenchymal stem cell precipitate in triple distilled water, and extrude it through a 100 to 400 nm polycarbonate porous membrane for 10 to 20 times to obtain osteogenic differentiated bone marrow mesenchymal stem cell membrane.

[0022] Further preferably, the repeated freezing and thawing is specifically: placing the cell suspension in liquid nitrogen for rapid freezing for 10 minutes, and then rapidly thawing in a 37° C. water bath for 10 minutes;

[0023] The gradient centrifugation is specifically as follows: the broken cells are centrifuged at 800-1000 rpm for 5-10 min at 4°C to obtain a supernatant; the supernatant is centrifuged at 300-4000 rpm for 10-20 min at 4°C; and the supernatant is centrifuged again at 13000-15000 rpm for 20-30 min at 4°C.

[0024] Preferably, in step (3), the mass ratio of biotin, 1-(3-dimethylaminopropyl)-3-ethylcarbamide ester, N-hydroxysuccinimide and mixed cell membrane in the biotin solution is 1.22:0.96:0.58:100 to 4.82:0.40:0.11:100.

[0025] Preferably, in step (4), the surface area of ​​the biotin-modified polycaprolactone nanofibers and the mass ratio of the biotin-modified mixed cell membranes is 1:100, cm 2 :mg;

[0026] The concentration of streptavidin in the mixture is 0.1-1.0 mM.

[0027] 2. A mixed cell membrane functionalized bone repair matrix for regulating the inflammatory microenvironment prepared according to the above preparation method.

[0028] 3. Application of the above-mentioned mixed cell membrane functionalized bone repair matrix for regulating the inflammatory microenvironment in regulating the inflammatory microenvironment.

[0029] The beneficial effects of the present invention are as follows: the present invention discloses a method for preparing a mixed cell membrane functionalized bone repair matrix for regulating an inflammatory microenvironment, wherein the amino groups in polycaprolactone nanofibers and a mixed cell membrane (anti-inflammatory macrophage membrane and osteogenic differentiated bone marrow mesenchymal stem cell membrane) are reacted with the carboxyl groups on biotin to perform biotin modification to obtain biotin-modified polycaprolactone nanofibers and biotin-modified mixed cell membranes, and then streptavidin is added for a coupling reaction to obtain a mixed cell membrane functionalized bone repair matrix for regulating an inflammatory microenvironment. The mixed cell membrane functionalized bone repair matrix for regulating the inflammatory microenvironment prepared by the preparation method of the present invention has specific proteins on the surface of anti-inflammatory macrophage membranes and osteogenic differentiated bone marrow mesenchymal stem cell membranes, which can regulate the phenotypic transformation of macrophages and the osteogenic differentiation of bone marrow mesenchymal stem cells, enriching the function of a single cell membrane; on the other hand, it is an attempt to combine the two micro / nano structures of the mixed cell membrane (anti-inflammatory macrophage membrane and osteogenic differentiated bone marrow mesenchymal stem cell membrane) with electrospinning, breaking through the application scope of cell membrane materials, and can realize the repair of rat skull defects, with potential medical prospects.

[0030] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0032] Figure 1 is a flow chart for preparing different materials in Example 1;

[0033] Figure 2is a transmission electron microscopy (TEM) image of the hybrid cell membrane (HM) prepared in Example 1;

[0034] Figure 3 Zeta potential diagrams of the anti-inflammatory macrophage membrane (M2), bone marrow mesenchymal stem cell membrane (oiM) and mixed cell membrane (HM) prepared in Example 1;

[0035] Figure 4 This is a field emission scanning electron microscopy (SEM) image of the PCL nanofibers and hybrid cell membrane functionalized bone repair matrix (HM / PCL) prepared in Example 1;

[0036] Figure 5 Figure 2 shows the gene expression of macrophages regulated by different materials (PCL, M2 / PCL, oiM / PCL and HM / PCL), where A is the gene expression of interleukin-6 (IL-6); B is the gene expression of tumor necrosis factor-α (TNF-α); C is the gene expression of interleukin-1β (IL-1β); D is the gene expression of chemokine receptor 7 (CCR7); E is the gene expression of interleukin-10 (IL-10); F is the gene expression of CD206; G is the transforming growth factor-β1 (TGF-β1); H is the gene expression of interleukin-1ra (IL-1ra);

[0037] Figure 6 These are micro-CT images of rat skull defects repaired with different materials (PCL, M2 / PCL, oiM / PCL and HM / PCL), where A is the micro-CT reconstruction image; B is the bone volume fraction (bone volume / total volume, BV / TV); C is the bone mineral density (bone mineral density, BMD);

[0038] Among them, 1 is anti-inflammatory macrophages, 2 is osteogenic differentiated bone marrow mesenchymal stem cells, 3 is cell membrane extraction process, 4 is anti-inflammatory macrophage membrane (M2), 5 is bone-differentiated bone marrow mesenchymal stem cell membrane (oiM), 6 is polycarbonate porous membrane, 7 is mixed cell membrane (HM), 8 is biotin, 9 is biotin-modified mixed cell membrane, 10 is hexafluoroisopropanol solution of pure polycaprolactone particles, 11 is electrospinning process, 12 is polycaprolactone nanofibers (PCL nanofibers), 13 is biotin-modified polycaprolactone nanofibers, 14 is streptavidin, 15 is mixed cell membrane functionalized bone repair matrix (HM / PCL), 16 is anti-inflammatory macrophage membrane functionalized bone repair matrix (M2 / PCL), and 17 is bone-differentiated bone marrow mesenchymal stem cell membrane functionalized bone repair matrix (oiM / PCL). DETAILED DESCRIPTION

[0039] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0040] Example 1

[0041] The mixed cell membrane functionalized bone repair matrix is ​​prepared according to the following steps:

[0042] (1) First, RAW264.7 (provided by the Cell Resource Center of Shanghai Institute of Life Sciences, China) was used as the macrophage cell line and cultured with conditioned medium containing 100 ng / mL IL-4 for 7 days to obtain anti-inflammatory macrophages. Then, the anti-inflammatory macrophage suspension was obtained by cell scraping and added to a low osmotic lysis buffer containing protease inhibitors (1 mM MgCl, 1 mM EDTA, 10 mM The cells were then placed in a 37°C water bath (Tris-HCl, 1 mM protease inhibitor, pH 7.4), cooled in an ice bath for 15 minutes, and repeatedly frozen and thawed (the anti-inflammatory macrophage suspension was quickly frozen in liquid nitrogen for 10 minutes, and then quickly thawed in a 37°C water bath for 10 minutes) three times to fully lyse and break the cells. The broken cells were subjected to gradient centrifugation (the broken cells were centrifuged at 1000 rpm at 4°C for 5 minutes to obtain the supernatant; the supernatant was centrifuged at 4000 rpm at 4°C for 10 minutes; and the supernatant was centrifuged again at 15000 rpm at 4°C for 30 minutes) to obtain the anti-inflammatory macrophage membrane precipitate; finally, the anti-inflammatory macrophage membrane precipitate was resuspended in triple distilled water, and the membrane was squeezed 10 times through 400 nm and 100 nm polycarbonate porous membranes using a micro extruder to obtain the anti-inflammatory macrophage membrane (M2).

[0043] (2) First, primary mesenchymal stem cells were isolated from the tibia and femur of 4-week-old female SD rats (all SD rats were used for cell isolation experiments under specific pathogen-free conditions). Osteogenic induction medium (containing 1 mM sodium pyruvate, 10 -7 M dexamethasone, 50 μg / mL vitamin C, 10 mM β-glycerophosphate) were cultured for 7 days to obtain osteogenic differentiated bone marrow mesenchymal stem cells; then the anti-inflammatory macrophage suspension was obtained by cell scraping and added to a low osmotic lysis buffer containing protease inhibitors (1 mM MgCl, 1 mM EDTA, 10 mM The cells were then placed in a 37°C water bath (Tris-HCl, 1 mM protease inhibitor, pH 7.4), cooled in an ice bath for 15 minutes, and repeatedly frozen and thawed three times (first, the osteogenic differentiated bone marrow mesenchymal stem cell suspension was quickly frozen in liquid nitrogen for 10 minutes, and then quickly thawed in a 37°C water bath for 10 minutes) to fully lyse and break the cells. The broken cells were subjected to gradient centrifugation (the broken cells were centrifuged at 1000 rpm for 5 minutes at 4°C to obtain the supernatant; the supernatant was centrifuged at 4000 rpm for 10 minutes at 4°C; and the supernatant was centrifuged again at 15000 rpm for 30 minutes at 4°C) to obtain osteogenic differentiated bone marrow mesenchymal stem cell membrane precipitation; finally, the osteogenic differentiated bone marrow mesenchymal stem cell precipitation was resuspended in triple distilled water, and the membrane was squeezed 10 times through 400nm and 100nm polycarbonate porous membranes using a micro extruder to obtain osteogenic differentiated bone marrow mesenchymal stem cell membrane (oiM).

[0044] (3) M2 obtained in step (1) and oiM obtained in step (2) were mixed in a mass ratio of 1:1, and then ultrasonically treated for 15 minutes. The membrane was extruded 10 times through 400 nm and 100 nm polycarbonate porous membranes using a micro extruder to obtain a hybrid cell membrane (HM).

[0045] (4) Pure polycaprolactone (pure PCL) particles were dissolved in hexafluoroisopropanol solution and transported through a jet pump, a 22G passivated stainless steel needle and a 15 kV high-voltage power supply (the delivery rate was 1 mL / h). The PCL nanofibers were collected on aluminum foil (the aluminum foil was wrapped on an open cage drum collector at a rotation speed of 300 rpm) to obtain polycaprolactone nanofibers (PCL nanofibers).

[0046] (5) A 1 mM biotin solution was used to treat (react at 37°C for 2 hours) PCL nanofibers by adding 1-(3-dimethylaminopropyl)-3-ethylcarbonamide (the 1-(3-dimethylaminopropyl)-3-ethylcarbonamide was derived from a 0.1 M 1-(3-dimethylaminopropyl)-3-ethylcarbonamide solution) and N-hydroxysuccinimide (the N-hydroxysuccinimide was derived from a 0.1 M N-hydroxysuccinimide solution) to allow the carboxyl group of biotin to bind to the amino group of the PCL nanofiber (the ratio of biotin, 1-(3-dimethylaminopropyl)-3-ethylcarbonamide, N-hydroxysuccinimide to the surface area of ​​the polycaprolactone nanofiber in the biotin solution was 1.22:0.96:0.58:1, mg:mg:mg:cm 2 ) to obtain biotin-modified polycaprolactone nanofibers.

[0047] (6) A biotin solution at a concentration of 1 mM was used to prepare a biotin-modified mixed cell membrane by adding 1-(3-dimethylaminopropyl)-3-ethylcarbonamide (the 1-(3-dimethylaminopropyl)-3-ethylcarbonamide was derived from a 1-(3-dimethylaminopropyl)-3-ethylcarbonamide solution at a concentration of 0.1 M) and N-hydroxysuccinimide (the N-hydroxysuccinimide was derived from a 0.1 M N-hydroxysuccinimide solution) to treat the mixed cell membrane (reacting overnight at 4°C) so that the carboxyl group of biotin was combined with the amino group of the mixed cell membrane (the mass ratio of biotin in the biotin solution, 1-(3-dimethylaminopropyl)-3-ethylcarbonamide, N-hydroxysuccinimide and the mixed cell membrane was 1.22:0.96:0.58:100).

[0048] (7) mixing the biotin-modified polycaprolactone nanofibers obtained in step (5) and the biotin-modified mixed cell membranes obtained in step (6) with streptavidin to form a mixture (wherein the mass ratio of the surface area of ​​the biotin-modified polycaprolactone nanofibers to the biotin-modified mixed cell membranes is 1:100, cm 2 :mg, and the concentration of streptavidin in the mixture was 0.1 mm) for 2 hours to prepare the HM / PCL bone repair matrix.

[0049] Example 2

[0050] The mixed cell membrane functionalized bone repair matrix is ​​prepared according to the following steps:

[0051] (1) First, RAW264.7 (provided by the Cell Resource Center of Shanghai Institute of Life Sciences, China) was used as the macrophage cell line and cultured with conditioned medium containing 100 ng / mL IL-4 for 14 days to obtain anti-inflammatory macrophages. Then, the anti-inflammatory macrophage suspension was obtained by cell scraping and added to a low osmotic lysis buffer containing protease inhibitors (1 mM MgCl, 1 mM EDTA, 10 mM The cells were then placed in a 37°C water bath (Tris-HCl, 1 mM protease inhibitor, pH 7.4), cooled in an ice bath for 30 minutes, and repeatedly frozen and thawed (the anti-inflammatory macrophage suspension was quickly frozen in liquid nitrogen for 10 minutes, and then quickly thawed in a 37°C water bath for 10 minutes) three times to fully lyse and break the cells. The broken cells were subjected to gradient centrifugation (the broken cells were centrifuged at 800 rpm for 10 minutes at 4°C to obtain the supernatant; the supernatant was centrifuged at 300 rpm at 4°C for 20 minutes; and the supernatant was centrifuged again at 13000 rpm at 4°C for 20 minutes) to obtain the anti-inflammatory macrophage membrane precipitate; finally, the anti-inflammatory macrophage membrane precipitate was resuspended in triple distilled water, and the membrane was squeezed 20 times through 200 nm and 100 nm polycarbonate porous membranes using a micro extruder to obtain an anti-inflammatory macrophage membrane (M2).

[0052] (2) First, primary mesenchymal stem cells were isolated from the tibia and femur of 4-week-old female SD rats (all SD rats were used for cell isolation experiments under specific pathogen-free conditions). Osteogenic induction medium (containing 1 mM sodium pyruvate, 10 -7M dexamethasone, 50 μg / mL vitamin C, 10 mM β-glycerophosphate) were cultured for 14 days to obtain osteogenic differentiated bone marrow mesenchymal stem cells; then, anti-inflammatory macrophage suspension was obtained by cell scraping and added to a low osmotic lysis buffer containing protease inhibitors (1 mM MgCl, 1 mM EDTA, 10 mM The cells were then placed in a 37°C water bath (Tris-HCl, 1 mM protease inhibitor, pH 7.4), cooled in an ice bath for 30 minutes, and repeatedly frozen and thawed three times (first, the osteogenic differentiated bone marrow mesenchymal stem cell suspension was quickly frozen in liquid nitrogen for 10 minutes, and then quickly thawed in a 37°C water bath for 10 minutes) to fully lyse and break the cells. The broken cells were subjected to gradient centrifugation (the broken cells were centrifuged at 800 rpm for 10 minutes at 4°C to obtain the supernatant; the supernatant was centrifuged at 300 rpm at 4°C for 20 minutes; and the supernatant was centrifuged again at 13000 rpm at 4°C for 20 minutes) to obtain osteogenic differentiated bone marrow mesenchymal stem cell membrane precipitation; finally, the osteogenic differentiated bone marrow mesenchymal stem cell precipitation was resuspended in triple distilled water, and the membrane was squeezed 20 times through 200 nm and 100 nm polycarbonate porous membranes using a micro extruder to obtain osteogenic differentiated bone marrow mesenchymal stem cell membrane (oiM).

[0053] (3) M2 obtained in step (1) and oiM obtained in step (2) were mixed in a mass ratio of 1:1 and ultrasonically treated for 30 minutes. The membrane was extruded 20 times through 200 nm and 100 nm polycarbonate porous membranes using a micro extruder to obtain a hybrid cell membrane (HM).

[0054] (4) Pure polycaprolactone (pure PCL) particles were dissolved in hexafluoroisopropanol solution and transported through a jet pump, a 16G passivated stainless steel needle and a 15 kV high-voltage power supply (the delivery rate was 1 mL / h). The PCL nanofibers were collected on aluminum foil (the aluminum foil was wrapped on an open cage drum collector at a rotation speed of 1000 rpm) to obtain polycaprolactone nanofibers (PCL nanofibers).

[0055] (5) A 0.1 mM biotin solution was used to treat PCL nanofibers (reacted at 37°C for 6 hours) by adding 1-(3-dimethylaminopropyl)-3-ethylcarbonamide (the 1-(3-dimethylaminopropyl)-3-ethylcarbonamide was derived from a 1.0 M 1-(3-dimethylaminopropyl)-3-ethylcarbonamide solution) and N-hydroxysuccinimide (the N-hydroxysuccinimide was derived from a 1.0 M N-hydroxysuccinimide solution) to allow the carboxyl group of biotin to bind to the amino group of the PCL nanofiber (the ratio of biotin, 1-(3-dimethylaminopropyl)-3-ethylcarbonamide, N-hydroxysuccinimide to the surface area of ​​the polycaprolactone nanofiber in the biotin solution was 4.82:0.40:0.11:1, mg:mg:mg:cm 2 ) to obtain biotin-modified polycaprolactone nanofibers.

[0056] (6) A biotin solution at a concentration of 0.1 mM was used to treat (react overnight at 4°C) a mixed cell membrane by adding 1-(3-dimethylaminopropyl)-3-ethylcarbonamide (the 1-(3-dimethylaminopropyl)-3-ethylcarbonamide was derived from a 1.0 M 1-(3-dimethylaminopropyl)-3-ethylcarbonamide solution) and N-hydroxysuccinimide (the N-hydroxysuccinimide was derived from a 1.0 M N-hydroxysuccinimide solution) to allow the carboxyl group of biotin to bind to the amino group of the mixed cell membrane (the mass ratio of biotin in the biotin solution, 1-(3-dimethylaminopropyl)-3-ethylcarbonamide, N-hydroxysuccinimide and the mixed cell membrane was 4.82:0.40:0.11:100) to prepare a biotin-modified mixed cell membrane.

[0057] (7) mixing the biotin-modified polycaprolactone nanofibers obtained in step (5) and the biotin-modified mixed cell membranes obtained in step (6) with streptavidin to form a mixture (wherein the mass ratio of the surface area of ​​the biotin-modified polycaprolactone nanofibers to the biotin-modified mixed cell membranes is 1:100, cm 2 :mg, and the concentration of streptavidin in the mixture was 1.0mM) for 6 hours to prepare the HM / PCL bone repair matrix.

[0058] Example 3

[0059] The mixed cell membrane functionalized bone repair matrix is ​​prepared according to the following steps:

[0060] (1) First, RAW264.7 (provided by the Cell Resource Center of Shanghai Institute of Life Sciences, China) was used as the macrophage cell line and cultured with conditioned medium containing 100 ng / mL IL-4 for 10 days to obtain anti-inflammatory macrophages. Then, the anti-inflammatory macrophage suspension was obtained by cell scraping and added to a low osmotic lysis buffer containing protease inhibitors (1 mM MgCl, 1 mM EDTA, 10 mM The cells were then placed in a 37°C water bath (Tris-HCl, 1 mM protease inhibitor, pH 7.4), cooled in an ice bath for 20 minutes, and repeatedly frozen and thawed (the anti-inflammatory macrophage suspension was quickly frozen in liquid nitrogen for 10 minutes, and then quickly thawed in a 37°C water bath for 10 minutes) three times to fully lyse and break the cells. The broken cells were subjected to gradient centrifugation (the broken cells were centrifuged at 900 rpm for 8 minutes at 4°C to obtain the supernatant; the supernatant was centrifuged at 2000 rpm at 4°C for 15 minutes; and the supernatant was centrifuged again at 14000 rpm at 4°C for 25 minutes) to obtain the anti-inflammatory macrophage membrane precipitate; finally, the anti-inflammatory macrophage membrane precipitate was resuspended in triple distilled water, and the membrane was squeezed 15 times through 100 nm and 400 nm polycarbonate porous membranes using a micro extruder to obtain the anti-inflammatory macrophage membrane (M2).

[0061] (2) First, primary mesenchymal stem cells were isolated from the tibia and femur of 4-week-old female SD rats (all SD rats were used for cell isolation experiments under specific pathogen-free conditions). Osteogenic induction medium (containing 1 mM sodium pyruvate, 10 -7 M dexamethasone, 50 μg / mL vitamin C, 10 mM β-glycerophosphate) were cultured for 10 days to obtain osteogenic differentiated bone marrow mesenchymal stem cells; then, anti-inflammatory macrophage suspension was obtained by cell scraping and added to a low osmotic lysis buffer containing protease inhibitors (1 mM MgCl, 1 mM EDTA, 10 mM The cells were then placed in a 37°C water bath (Tris-HCl, 1 mM protease inhibitor, pH 7.4), cooled in an ice bath for 20 minutes, and repeatedly frozen and thawed three times (first, the osteogenic differentiated bone marrow mesenchymal stem cell suspension was quickly frozen in liquid nitrogen for 10 minutes, and then quickly thawed in a 37°C water bath for 10 minutes) to fully lyse and break the cells. The broken cells were subjected to gradient centrifugation (the broken cells were centrifuged at 800 rpm for 8 minutes at 4°C to obtain the supernatant; the supernatant was centrifuged at 2000 rpm at 4°C for 15 minutes; and the supernatant was centrifuged again at 14000 rpm at 4°C for 20 minutes) to obtain osteogenic differentiated bone marrow mesenchymal stem cell membrane precipitation; finally, the osteogenic differentiated bone marrow mesenchymal stem cell precipitation was resuspended in triple distilled water, and the membrane was squeezed 10 times through 100 nm and 400 nm polycarbonate porous membranes using a micro extruder to obtain osteogenic differentiated bone marrow mesenchymal stem cell membrane (oiM).

[0062] (3) M2 obtained in step (1) and oiM obtained in step (2) were mixed in a mass ratio of 1:1 and ultrasonically treated for 20 minutes. The membrane was extruded 15 times through 100 nm and 400 nm polycarbonate porous membranes using a micro extruder to obtain a hybrid cell membrane (HM).

[0063] (4) Pure polycaprolactone (pure PCL) particles were dissolved in hexafluoroisopropanol solution and transported through a jet pump, a 20G passivated stainless steel needle and a 15 kV high-voltage power supply (the delivery rate was 1 mL / h). The PCL nanofibers were collected on aluminum foil (the aluminum foil was wrapped on an open cage drum collector at a rotation speed of 800 rpm) to obtain polycaprolactone nanofibers (PCL nanofibers).

[0064] (5) A 0.5 mM biotin solution was used to treat PCL nanofibers (reacted at 37°C for 4 hours) by adding 1-(3-dimethylaminopropyl)-3-ethylcarbonamide (the 1-(3-dimethylaminopropyl)-3-ethylcarbonamide was derived from a 0.5 M 1-(3-dimethylaminopropyl)-3-ethylcarbonamide solution) and N-hydroxysuccinimide (the N-hydroxysuccinimide was derived from a 0.5 M N-hydroxysuccinimide solution) to allow the carboxyl group of biotin to bind to the amino group of the PCL nanofiber (the ratio of biotin, 1-(3-dimethylaminopropyl)-3-ethylcarbonamide, N-hydroxysuccinimide to the surface area of ​​the polycaprolactone nanofiber in the biotin solution was 1.22:0.96:0.58:1, mg:mg:mg:cm 2 ) to obtain biotin-modified polycaprolactone nanofibers.

[0065] (6) Using a 0.5 mM biotin solution, a mixed cell membrane was treated (reacted overnight at 4°C) with 1-(3-dimethylaminopropyl)-3-ethylcarbonamide (the 1-(3-dimethylaminopropyl)-3-ethylcarbonamide is derived from a 0.5 M 1-(3-dimethylaminopropyl)-3-ethylcarbonamide solution) and N-hydroxysuccinimide (the N-hydroxysuccinimide is derived from a 0.5 M N-hydroxysuccinimide solution) to allow the carboxyl group of biotin to bind to the amino group of the mixed cell membrane (the mass ratio of biotin in the biotin solution, 1-(3-dimethylaminopropyl)-3-ethylcarbonamide, N-hydroxysuccinimide and mixed cell membrane is 4.82:0.40:0.11:100) to prepare a biotin-modified mixed cell membrane.

[0066] (7) mixing the biotin-modified polycaprolactone nanofibers obtained in step (5) and the biotin-modified mixed cell membranes obtained in step (6) with streptavidin to form a mixture (wherein the mass ratio of the surface area of ​​the biotin-modified polycaprolactone nanofibers to the biotin-modified mixed cell membranes is 1:100, cm 2 :mg, and the concentration of streptavidin in the mixture was 0.5mm) for 4 hours to prepare the HM / PCL bone repair matrix.

[0067] Comparative Example 1

[0068] The anti-inflammatory macrophage membrane (M2) was prepared as M2 / PCL of the PCL nanofiber coating, and the specific method is as follows:

[0069] The anti-inflammatory macrophage membrane (M2) prepared in step (1) of Example 1 and the PCL nanofibers prepared in step (4) of Example 1 were combined by electrostatic adsorption to prepare M2 / PCL at 4° C. overnight.

[0070] Comparative Example 2

[0071] The osteogenic differentiated bone marrow mesenchymal stem cell membrane (oiM) was prepared as M2 / PCL of the PCL nanofiber coating, and the specific method is as follows:

[0072] The osteogenic differentiated bone marrow mesenchymal stem cell membrane (oiM) prepared in step (2) of Example 1 was combined with the PCL nanofibers prepared in step (4) of Example 1 by electrostatic adsorption, and oiM / PCL was prepared at 4°C overnight.

[0073] The preparation process of different materials in the above embodiment 1 is as follows: Figure 1 As shown, 1 is anti-inflammatory macrophages, 2 is osteogenic differentiated bone marrow mesenchymal stem cells, 3 is cell membrane extraction process, 4 is anti-inflammatory macrophage membrane (M2), 5 is osteogenic differentiated bone marrow mesenchymal stem cell membrane (oiM), 6 is polycarbonate porous membrane, 7 is mixed cell membrane (HM), 8 is biotin, 9 is biotin-modified mixed cell membrane, 10 is hexafluoroisopropanol solution of pure polycaprolactone particles, 11 is electrospinning process, 12 is polycaprolactone nanofibers (PCL nanofibers), 13 is biotin-modified polycaprolactone nanofibers, 14 is streptavidin, and 15 is mixed cell membrane functionalized bone repair matrix (HM / PCL).

[0074] Performance Characterization

[0075] (1) The morphology of the hybrid cell membrane was observed by TEM

[0076] The hybrid cell membrane (HM) prepared in step (3) of Example 1 was subjected to TEM detection, and the results are as follows: Figure 2 As shown. Figure 2 The results showed that the TEM image of the hybrid cell membrane (HM) prepared in step (3) of Example 1 showed a distorted spherical vesicle shape of the MM cell membrane with a diameter of about 100 nm.

[0077] (2) The zeta potential of M2, oiM and HM prepared in Example 1 was measured on a Malvern Nano-ZS particle size analyzer.

[0078] The zeta potential of the anti-inflammatory macrophage membrane (M2) prepared in step (1) of Example 1, the osteogenic differentiated bone marrow mesenchymal stem cell membrane (oiM) prepared in step (2), and the mixed cell membrane (HM) prepared in step (3) were measured using a Malvern Nano-ZS particle size analyzer. The results are as follows: Figure 3 As shown, 4 is an anti-inflammatory macrophage membrane (M2), 5 is a bone-differentiated bone marrow mesenchymal stem cell membrane (oiM), and 7 is a mixed cell membrane (HM). Figure 3 The mid-potential analysis showed that the surfaces of M2, oiM and HM prepared in Example 1 were all negatively charged.

[0079] The above physical and chemical characterization results strongly verified that M2, oiM and HM prepared in Example 1 were nanoscale extracellular membrane vesicles.

[0080] (3) SEM observation of the morphology and diameter of PCL nanofibers and HM / PCL prepared in Example 1

[0081] The PCL nanofibers prepared in step (4) of Example 1 and the mixed cell membrane functionalized bone repair matrix (HM / PCL) prepared in step (7) were subjected to SEM testing, and the results are as follows: Figure 4 As shown (where 12 is polycaprolactone nanofiber (PCL nanofiber) and 15 is hybrid cell membrane functionalized bone repair matrix (HM / PCL)). Figure 4 The results showed that the diameter of PCL nanofibers was 313.79±4.69nm, and the diameter of HM / PCL group fibers was 368.76±5.90nm. The diameter of PCL nanofibers increased after the coating formed by the mixed cell membrane, indicating that the mixed cell membrane was successfully coated on the PCL nanofibers to form the HM / PCL bone repair matrix.

[0082] Effects of mixed cell membrane functionalized bone repair matrix on macrophage polarization

[0083] Macrophages were cultured at a rate of 3.0×10 4 Pieces / cm 2The cells were inoculated at a density of 100 μg / mL onto the PCL nanofibers prepared in Example 1, M2 / PCL prepared in Comparative Example 1, oiM / PCL prepared in Comparative Example 2, and HM / PCL bone repair matrices prepared in Example 1, and the cell culture samples were collected after culturing for 3 days and 7 days under the same culture conditions. Total RNA was extracted using Trizol reagent, and cDNA was obtained using reverse transcription reagent. The expression of inflammation-related genes was detected by quantitative polymerase chain reaction.

[0084] The results showed that when the macrophages were cultured for 3 days, the M2 / PCL group in Example 1 and the HM / PCL group in Example 1 were more able to inhibit the expression of CD86 and inducible nitric oxide synthase (iNOS), while the oiM / PCL group in Example 2 and the HM / PCL group in Example 1 were more able to promote the expression of CD163 and arginine (Arg). When the macrophages were cultured for 7 days, the M2 / PCL group in Comparative Example 1, the oiM / PCL group in Comparative Example 2 and the HM / PCL group in Example 1 were all able to inhibit the expression of proinflammatory genes tumor necrosis factor-α (TNF-α) (p<0.05), interleukin-1β (IL-1β) (p<0.05) and chemokine receptor 7 (CCR7) (p<0.05). Compared with the PCL group, the M2 / PCL group and the HM / PCL group had a more significant ability to inhibit the expression of all genes. At the same time, the M2 / PCL group, oiM / PCL group and HM / PCL group were able to promote the expression of anti-inflammatory genes interleukin 10 (IL-10) (p<0.05), CD206 (p<0.05), transforming growth factor-β (TGF-β) (p<0.05) and interleukin-1 receptor antagonist (IL-1ra) (p<0.05). Compared with the PCL group, the oiM / PCL group and HM / PCL group were more capable of promoting the expression of all genes. This shows that HMP has the advantages of both the M2 / PCL group and the oiM / PCL group in regulating the phenotype of macrophages, and better activates the polarization of macrophages. Specifically, Figure 5As shown (12 is PCL electrospinning matrix, 15 is HM functionalized bone repair (HM / PCL) matrix, 16 is M2 functionalized bone repair matrix (M2 / PCL), 17 is oiM functionalized bone repair matrix (oiM / PCL)), among which A is interleukin-6 (IL-6) gene expression; B is tumor necrosis factor-α (TNF-α) gene expression; C is interleukin-1β (IL-1β) gene expression; D is cxc chemokine receptor 7 (CCR7) gene expression; E is interleukin-10 (IL-10) gene expression; F is CD206 gene expression; G is transforming growth factor-β1 (TGF-β1); H is interleukin-1ra (IL-1ra) gene expression.

[0085] In vivo evaluation of hybrid cell membrane functionalized bone repair matrix on bone defect repair

[0086] (1) Establishment of rat skull defect model: 8-week-old SD rats were anesthetized with 3% sodium pentobarbital solution at a dose of 30 mg / kg. The hair on the surface of the skull was removed with a hair pusher, and a 3-4 cm wound was made along the middle of the skull with a scalpel. A 5 mm drill was used to drill a 5 mm critical defect on both sides of the sagittal suture. The cell membrane functionalized bone repair matrix of the PCL group, M2 / PCL group, oiM / PCL group and HM / PCL group was superimposed to 5 layers, and different cell membrane-coated scaffolds with a diameter of 5 mm were made with a punch, and the scaffolds were transplanted into the defect site. Skull samples were removed 4 and 8 weeks after surgery, and the bone defect repair ability was evaluated after fixation with 4% paraformaldehyde.

[0087] (2) The skull graft samples were measured using a micro-CT scanner with a resolution of 19 μm and an X-ray source of 70 kVP, 114 μA. Three-dimensional reconstructed images were generated and analyzed based on the micro-CT dataset. A cylindrical region with a diameter of 4.0 mm within the defect was selected as the volume of interest. The three-dimensionally reconstructed new bone volume (BV), total bone volume (TV), bone volume fraction (BV / TV), and bone mineral density (BMD) were directly evaluated using μ-CT v6.1 analysis software.

[0088] The results showed that the Micro-CT results after 4 weeks of implantation in rat skull defects showed that the boundary between the new bone and the implanted material was blurred in the material transplantation site of the M2 / PCL group, oiM / PCL group and HM / PCL group, and bone formation was observed in the middle of the HM / PCL scaffold transplantation. At 8 weeks of repair, the M2 / PCL group, oiM / PCL group and HM / PCL group had better bone repair in the bone defect. This indicates that the cell membrane-modified material has a better bone formation effect, and the dual-function HM / PCL scaffold has a better repair effect. From the BV / TV and BMD analysis at 4 and 8 weeks, the M2 / PCL group, oiM / PCL group and HM / PCL group were higher than the self-repair group and PCL group. Among them, there was no significant difference between the M2 / PCL group and the oiM / PCL group, and both were lower than the HM / PCL group, indicating that both M2 / PCL and oiM / PCL cell membrane materials can promote bone tissue repair, but the HM / PCL group has both immune regulation and bone repair effects, and the bone integration effect is more significant. Figure 6 As shown in the figure, A is the micro-CT reconstruction image; B is the bone volume fraction (bone volume / total volume, BV / TV); C is the bone mineral density (bone mineral density, BMD). The self-repair group did not add other materials, and the materials added in the transplantation group included 12 polycaprolactone nanofibers (PCL nanofibers), 15 mixed cell membrane functionalized bone repair matrix (HM / PCL), 16 anti-inflammatory macrophage membrane functionalized bone repair matrix (M2 / PCL), and 17 bone-differentiated bone marrow mesenchymal stem cell membrane functionalized bone repair matrix (oiM / PCL).

[0089] Similarly, the above test was performed on the mixed cell membrane functionalized bone repair matrix (HM / PCL) prepared in Example 2 and Example 3, and the results were basically the same as those in Example 1.

[0090] In summary, the present invention discloses a method for preparing a mixed cell membrane functionalized bone repair matrix that regulates an inflammatory microenvironment. A mixed cell membrane formed by an anti-inflammatory macrophage membrane (M2) and an osteogenic differentiated bone marrow mesenchymal stem cell membrane (oiM) is formed into a coating on PCL nanofibers using a biotin-streptomycin method to obtain an HM / PCL bone repair matrix. Compared with M2 / PCL and oiM / PCL formed by simply using an anti-inflammatory macrophage membrane (M2) and an osteogenic differentiated bone marrow mesenchymal stem cell membrane (oiM) as a coating, the mixed cell membrane functionalized bone repair matrix (HM / PCL) can regulate the anti-inflammatory phenotype of macrophages and promote the osteogenic differentiation of MSCs. More importantly, the mixed cell membrane functionalized bone repair matrix contains specific proteins on the surface of anti-inflammatory macrophage membranes and osteogenic differentiated bone marrow mesenchymal stem cell membranes, and promotes bone integration by regulating the inflammatory microenvironment of the bone defect in a rat skull defect model. The present invention proposes a new method for regulating the behavior of macrophages and mesenchymal stem cells using two membrane-functionalized bone repair matrices, which can be expanded to the synergistic effect of various cell membranes and provides a promising option for the preparation of bone regeneration materials.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a mixed cell membrane functionalized bone repair matrix for regulating inflammatory microenvironment, characterized in that: The preparation method comprises the following steps: (1) Preparation of biotin-modified polycaprolactone nanofibers: A 0.1-1.0 mM biotin solution, a 0.1-1.0 M 1-(3-dimethylaminopropyl)-3-ethylcarbamide ester solution, and a 0.1-1.0 M N-hydroxysuccinimide solution were mixed, and then polycaprolactone nanofibers were added and reacted at 37°C for 2-6 h to allow the carboxyl group of biotin to react with the amino group of the polycaprolactone nanofibers to obtain biotin-modified polycaprolactone nanofibers; (2) Preparation of mixed cell membrane: The anti-inflammatory macrophage membrane and osteogenic differentiated bone marrow mesenchymal stem cell membrane were mixed in equal amounts, ultrasonically treated for 15-30 min, and extruded through a 100-400 nm polycarbonate porous membrane for 10-20 times to obtain a mixed cell membrane; (3) Preparation of biotin-modified mixed cell membrane: Mix a 0.1-1.0 mM biotin solution, a 0.1-1.0 M 1-(3-dimethylaminopropyl)-3-ethylcarbamide ester solution, and a 0.1-1.0 M N-hydroxysuccinimide solution, add the mixed cell membrane and treat overnight at 4°C to allow the carboxyl group of biotin to react with the amino group of the mixed cell membrane to obtain a biotin-modified mixed cell membrane; (4) The biotin-modified polycaprolactone nanofibers prepared in step (1), the biotin-modified mixed cell membrane prepared in step (3), and streptavidin are mixed to form a mixture, and the mixture is subjected to coupling reaction for 2 to 6 hours to obtain a mixed cell membrane functionalized bone repair matrix that regulates the inflammatory microenvironment.

2. The preparation method according to claim 1, characterized in that: In step (1), the polycaprolactone nanofibers are prepared as follows: pure polycaprolactone particles are dissolved in hexafluoroisopropanol, and transported at a speed of 1 mL / h through a jet pump, a 16-22 G passivated stainless steel needle, and a 15 kV high-voltage power supply, and collected on aluminum foil; The aluminum foil is wrapped on an open cage drum collector with a rotation speed of no more than 1000 rpm.

3. The preparation method according to claim 1, characterized in that: In step (1), the ratio of biotin, 1-(3-dimethylaminopropyl)-3-ethylcarbamide ester, N-hydroxysuccinimide and the surface area of ​​polycaprolactone nanofibers is 1.22:0.96:0.58:1~4.82:0.40:0.11:1, mg:mg:mg:cm 2 .

4. The preparation method according to claim 1, characterized in that: In step (2), the anti-inflammatory macrophage membrane is prepared according to the following method: (1) Using RAW264.7 as the macrophage cell line, macrophages were cultured in conditioned medium containing 10-100 ng / mL interleukin-4 for 7-14 days to obtain anti-inflammatory macrophages; (2) The anti-inflammatory macrophage suspension was obtained by cell scraping, added to a low-osmotic lysis buffer containing protease inhibitors, cooled in an ice bath for 15-30 min, and placed in liquid nitrogen and a 37°C water bath for repeated freezing and thawing to lyse the cells. The anti-inflammatory macrophage membrane precipitate was obtained by gradient centrifugation. The anti-inflammatory macrophage membrane precipitate was resuspended in triple-distilled water and extruded through a 100-400 nm polycarbonate porous membrane for 10-20 times to obtain the anti-inflammatory macrophage membrane.

5. The preparation method according to claim 1, characterized in that: In step (2), the osteogenic differentiated bone marrow mesenchymal stem cell membrane is prepared according to the following method: (1) A solution containing 1 mM sodium pyruvate and 10 -7 Bone marrow mesenchymal stem cells were cultured in osteogenic induction medium containing 100 μM dexamethasone, 50 μg / mL vitamin C, and 10 mM β-glycerophosphate for 7 to 14 days to obtain osteogenic differentiated bone marrow mesenchymal stem cells; (2) Obtain osteogenic differentiated bone marrow mesenchymal stem cell suspension by cell scraping, add it to a low osmotic lysis buffer containing protease inhibitors, cool it in an ice bath for 15-30 min, and place it in liquid nitrogen and a 37°C water bath for repeated freezing and thawing to lyse and break the cells. Obtain osteogenic differentiated bone marrow mesenchymal stem cell membrane precipitate by gradient centrifugation, resuspend the osteogenic differentiated bone marrow mesenchymal stem cell precipitate in triple distilled water, and extrude it through a 100-400 nm polycarbonate porous membrane for 10-20 times to obtain osteogenic differentiated bone marrow mesenchymal stem cell membrane.

6. The preparation method according to claim 4 or 5, characterized in that: The repeated freezing and thawing specifically includes: rapidly freezing the cell suspension in liquid nitrogen for 10 min, and then rapidly thawing in a 37°C water bath for 10 min; The gradient centrifugation is specifically as follows: the broken cells are centrifuged at 800-1000 rpm for 5-10 min at 4°C to obtain a supernatant; the supernatant is centrifuged at 300-4000 rpm for 10-20 min at 4°C; and the supernatant is centrifuged again at 13000-15000 rpm for 20-30 min at 4°C.

7. The preparation method according to claim 1, characterized in that: In step (3), the mass ratio of biotin, 1-(3-dimethylaminopropyl)-3-ethylcarbamide ester, N-hydroxysuccinimide and mixed cell membrane is 1.22:0.96:0.58:100~4.82:0.40:0.11:

100.

8. The preparation method according to claim 1, characterized in that: In step (4), the surface area ratio of the biotin-modified polycaprolactone nanofibers to the biotin-modified mixed cell membrane is 1:100, cm 2 :mg; The concentration of streptavidin in the mixture is 0.1-1.0 mM.

9. A mixed cell membrane functionalized bone repair matrix for regulating inflammatory microenvironment prepared according to the preparation method according to any one of claims 1 to 8.

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