Biomimetic mineralized cell membrane vesicle cross-linked polyacrylamide hydrogel, and preparation method and application thereof

Abstract

This invention discloses a biomimetic mineralized cell membrane vesicle crosslinked polyacrylamide hydrogel, its preparation method, and its applications, relating to the field of biomedical technology. The method includes: extracting and purifying cell membrane vesicles (CMVs) from cells; incubating CMVs with DSPE-PEG-AM to obtain olefin double-bond functionalized CMV-AM; mixing CMV-AM with acrylamide, an initiator, and a catalyst, and then initiating polymerization to obtain a cell membrane vesicle crosslinked polyacrylamide hydrogel; finally, immersing the hydrogel in a mineralization induction solution to obtain the biomimetic mineralized cell membrane vesicle crosslinked polyacrylamide hydrogel. This hydrogel exhibits good biocompatibility, mechanical properties, and osteogenic activity. The preparation process is mild and controllable, and it can be used to prepare materials for bone defect repair, osteochondral regeneration, or osteoporotic bone repair.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a biomimetic mineralized cell membrane vesicle crosslinked polyacrylamide hydrogel, its preparation method, and its application. Background Technology

[0002] Bone tissue is mainly composed of 50-70% inorganic minerals (primarily hydroxyapatite, HAp), 20-40% organic matrix (primarily type I collagen), and water. While traditional hydrogels possess good biocompatibility and a three-dimensional network structure, they suffer from poor mechanical properties, low osteogenic activity, and insufficient mineralization capacity. Inspired by the mineralization process of natural bone with matrix vesicles at its core, biomimetic mineralizing hydrogels have emerged, aiming to mimic the chemical composition, structural layers, mechanical properties, and osteogenic induction capacity of bone. Biomimetic mineralizing hydrogels have been widely applied in: bone defect repair, osteochondral regeneration, osteoporotic bone repair, and other bone-related diseases (such as osteosarcoma and tendon-bone interface injuries).

[0003] Existing biomimetic mineralized hydrogel construction strategies mainly include reaction-diffusion mineralization, metal-ligand coordination-induced mineralization, enzyme-catalyzed mineralization, light-mediated mineralization, stress-mediated mineralization, and inorganic-organic copolymerization mineralization. Current biomimetic mineralized hydrogels still face several challenges: 1) Insufficient biomimetic properties: Although the stiffness of the hydrogel increases after mineralization, it lacks the ability to mimic the hierarchical structure and dynamic mechanical properties of natural bone. 2) Limited osteogenic induction capacity: There is a lack of comprehensive simulation of the structural state of the natural bone extracellular matrix (ECM). Osteogenic differentiation is induced only by adding additional growth factors or ions, and the actual retained active components of the material lack systematic evaluation. 3) Complex mineralization process: Some mineralized hydrogel construction strategies involve adding enzymes such as alkaline phosphatase to assist in the mineralization process. However, enzymes may become inactive or unstable during the process, leading to prolonged preparation cycles and difficulty in obtaining stable structures.

[0004] Therefore, there is an urgent need to develop a novel hydrogel material with excellent mechanical properties, high osteogenic activity, biomimetic mineralization under mild conditions, and controllable preparation process. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a biomimetic mineralized cell membrane vesicle crosslinked polyacrylamide hydrogel, its preparation method, and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a method for preparing biomimetic mineralized cell membrane vesicle crosslinked polyacrylamide hydrogel, specifically including the following steps: S1: Extraction and purification of cell membrane vesicles (CMVs); S2: Cell membrane vesicles were mixed with DSPE-PEG-AM and incubated to obtain a solution of olefin double bond functionalized cell membrane vesicles (CMV-AM); S3: Under anaerobic conditions, a solution of olefin double bond-functionalized cell membrane vesicles (CMV-AM) was mixed with acrylamide (AM) monomer, and an initiator and catalyst were added. After initiation polymerization, cell membrane vesicle crosslinked polyacrylamide hydrogel was obtained. S4: The cell membrane vesicle cross-linked polyacrylamide hydrogel was immersed in a mineralization induction solution to obtain a biomimetic mineralized cell membrane vesicle cross-linked polyacrylamide hydrogel.

[0007] Further, in step S1, the cell membrane vesicles are prepared by the following method: S11: Cell culture: The ATDC5 chondrogenic cell line was seeded in DMEM medium (containing 1% penicillin and antibiotics and 10% fetal bovine serum) and cultured in a CO2 cell incubator at 37°C. After the cells reached 95% confluence, the cells were collected. S12: Preparation before extraction: Prepare the cell membrane protein extraction kit in advance. Take an equal volume of membrane protein extraction reagent A according to the volume of cells collected in step S11, and add protease inhibitor at a volume ratio of 100:1 before use. S13: Sample Pretreatment: Wash the cells collected in step S11 twice with pre-cooled PBS, centrifuge at 500g for 5 min at 4°C, and discard the residual PBS; then, on ice, centrifuge at 2 × 10⁻⁶ cells per 10⁻⁶ cells. 7 Add 1 mL of pre-cooled membrane protein extraction reagent A containing protease inhibitors to each cell; S14: Cell disruption by vortexing: The cells that have been treated with membrane protein extraction reagent A in step S13 are vortexed vigorously for 1 minute using a vortex mixer in the following sequence: vortex for 10 seconds, pause for 10 seconds, vortex for 10 seconds, pause for 10 seconds. Then, place the cells on ice for 2 minutes. Repeat this vortexing-placement step 4-5 times. A small number of cells are then taken for trypan blue staining to assess the degree of cell disruption. If the degree of cell disruption is less than 80%, increase the number of vigorous vortexing cycles until the degree of cell disruption is greater than or equal to 80%. S15: Centrifuge the cells disrupted in step S14 at 700g for 10 min at 4℃, collect the supernatant to obtain a solution containing cell membrane fragments; centrifuge the collected supernatant at 16000g for 30 min at 4℃ to precipitate cell membrane fragments, and collect the supernatant to obtain cell cytoplasmic proteins. S16: Pass cell membrane fragments sequentially through polycarbonate membranes with pore sizes of 400 nm and 200 nm, squeeze repeatedly for 5-6 cycles, then centrifuge at 16000g for 30 min at 4℃, collect the precipitate, and obtain cell membrane vesicles.

[0008] Further, in step S2, the olefin double-bond functionalized cell membrane vesicle (CMV-AM) solution is prepared by the following method: S21: Surface modification: Take a certain amount of cell membrane vesicles, add DSPE-PEG-AM to obtain a mixture; the concentration of DSPE-PEG-AM is 2% wt / wt (mass fraction). S22: Incubation: Incubate the mixture at 37°C with shaking at 500g for 0.5h. This temperature facilitates the insertion of DSPE into the lipid bilayer. S23: Purification: The incubated mixture was centrifuged at 16000g for 30 min at 4℃ and washed twice with PBS to remove unbound DSPE-PEG-AM, yielding a solution of olefin double-bond functionalized cell membrane vesicles (CMV-AM).

[0009] Further, in step S3, the cell membrane vesicle crosslinked polyacrylamide hydrogel is prepared by the following method: S31: Preparation of pregelation solution: In an anaerobic glove box, add olefin double bond functionalized cell membrane vesicle (CMV-AM) solution to a centrifuge tube, then add acrylamide (AM) monomer, and vortex at 2000 rpm for 5 seconds until completely dissolved; the ratio of the olefin double bond functionalized cell membrane vesicle solution to acrylamide is 2:0.5 in mL:g. S32: Initiating polymerization: Ammonium persulfate (APS) initiator and 2M tetramethylethylenediamine (TMEDA) catalyst are added sequentially to the pregel solution. The mixture is immediately vortexed at 2500 rpm for 5 seconds and then quickly transferred to a polytetrafluoroethylene (PTFE) mold. Polymerization is carried out at room temperature for 30 minutes to obtain cell membrane vesicle crosslinked polyacrylamide hydrogel. The mass ratio of ammonium persulfate to acrylamide in step S31 is 1:10. The ratio of ammonium persulfate to tetramethylethylenediamine is 0.05:2 in g:μL.

[0010] Further, in step S4, the biomimetic mineralized cell membrane vesicle cross-linked polyacrylamide hydrogel is prepared by the following method: the cell membrane vesicle cross-linked polyacrylamide hydrogel is immersed in a mineralization induction solution for 14 days to induce mineralization. The mineralization induction solution is αMEM medium containing 10 mM β-glycerophosphate sodium.

[0011] The present invention also provides a biomimetic mineralized cell membrane vesicle crosslinked polyacrylamide hydrogel prepared by the above method.

[0012] Furthermore, the interior of this biomimetic mineralized cell membrane vesicle crosslinked polyacrylamide hydrogel contains uniformly distributed cell membrane vesicles and in-situ mineralized hydroxyapatite mineralization precursor amorphous calcium phosphate (ACP).

[0013] The present invention also provides an application of a biomimetic mineralized cell membrane vesicle crosslinked polyacrylamide hydrogel, which is used in the preparation of bone repair materials.

[0014] Furthermore, the bone repair material is used for bone defect repair, osteochondral regeneration, or osteoporotic bone repair.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Stable material structure: In this invention, olefin double bond-functionalized cell membrane vesicles (CMV-AM) participate in the construction of the polyacrylamide network as a nano-crosslinking agent, forming a uniform and stable crosslinked structure, which helps to improve the mechanical properties of the hydrogel. At the same time, the subsequent biomimetic mineralization further enhances the stiffness of the material, making it closer to the mechanical properties of natural bone tissue.

[0016] (2) High biomimetic mineralization activity: The cell membrane vesicles are derived from chondrogenic / osteoblast-related cells and retain abundant natural bioactive molecules (such as membrane proteins and receptors), which can provide a biomimetic microenvironment for cell growth and effectively induce bone tissue regeneration. Experiments have confirmed that inorganic mineral elements such as calcium and phosphorus can be detected in the mineralized hydrogel, which has good mineralization ability.

[0017] (3) Mild preparation conditions: The preparation process of this invention is mild (room temperature, neutral pH) and does not involve harsh chemical conditions. The three-dimensional network structure of the hydrogel is stable and retains the biological activity of cell membrane vesicles, which is expected to regulate cell function.

[0018] (4) Uniform mineral distribution: The hydrogel network structure formed by CMV-AM crosslinking is uniform, providing uniform nucleation sites for subsequent mineralization. Combined with the immersion mineralization method, a biomimetic mineralized hydrogel with uniform mineral distribution can be obtained.

[0019] (5) The preparation process is simple and controllable: The present invention uses conventional technologies such as vortex crushing, membrane extrusion and free radical polymerization, which are easy to operate, mild and easy to achieve large-scale production. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the mechanism of the embodiments and comparative examples of the present invention, wherein: Figure 1 (a) is a schematic diagram of the mechanism of olefin double bond functionalized cell membrane vesicles (CMV-AM) in Example 1; Figure 1 (b) is a schematic diagram of the mechanism of the polyacrylamide hydrogel precursor solution in Comparative Example 1. Figure 1 (c) is a schematic diagram of the mechanism of the polyacrylamide hydrogel (PAM) formed after crosslinking in Comparative Example 2; Figure 1 (d) is a schematic diagram of the mechanism of cell membrane vesicle crosslinked polyacrylamide hydrogel (CMV-H) in Example 1; Figure 2 The images show physical representations of the polyacrylamide hydrogel precursor solution, polyacrylamide hydrogel (PAM), and cell membrane vesicle crosslinked polyacrylamide hydrogel (CMV-H) of the present invention, wherein: Figure 2 (a) is a physical image of the polyacrylamide hydrogel precursor solution; Figure 2 (b) is a physical image of the polyacrylamide hydrogel (PAM) formed after cross-linking; Figure 2 (c) is a physical image of cell membrane vesicle cross-linked polyacrylamide hydrogel (CMV-H); Figure 3 The images shown are scanning electron microscope (SEM) images of the polyacrylamide hydrogel (PAM) and cell membrane vesicle crosslinked polyacrylamide hydrogel (CMV-H) of the present invention, wherein: Figure 3 (a) is a low-magnification scanning electron microscope (SEM) image of polyacrylamide hydrogel (PAM); Figure 3 (b) is a high-magnification scanning electron microscope (SEM) image of polyacrylamide hydrogel (PAM); Figure 3 (c) is a low-magnification scanning electron microscope (SEM) image of cell membrane vesicle cross-linked polyacrylamide hydrogel (CMV-H); Figure 3 (d) is a medium-magnification scanning electron microscope (SEM) image of cell membrane vesicle cross-linked polyacrylamide hydrogel (CMV-H); Figure 3 (e) is a high-magnification scanning electron microscope (SEM) image of cell membrane vesicle cross-linked polyacrylamide hydrogel (CMV-H); Figure 4 This is a three-dimensional laser confocal image showing the distribution of cell membrane vesicles within the cell membrane vesicle crosslinked polyacrylamide hydrogel (CMV-H) of the present invention. Figure 5 The image shown is a scanning electron microscope (SEM-EDS) image of the biomimetic mineralized cell membrane vesicle crosslinked polyacrylamide hydrogel (M-CMV-H) according to an embodiment of the present invention, wherein: Figure 5 (a) is a TEM negative staining image; Figure 5 (b) is the characteristic X-ray energy spectrum of phosphorus in the K series; Figure 5 (c) is the characteristic X-ray energy spectrum of carbon in the K series; Figure 5 (d) is the overlap diagram of the energy spectra of each element; Figure 5 (e) is the characteristic X-ray energy spectrum of oxygen in the K series; Figure 5 (f) is the characteristic X-ray elemental spectrum of calcium in the K series; Figure 5 (g) is the X-ray elemental energy spectrum intensity diagram; Figure 6 The Fourier transform infrared (FITR) spectrum of the biomimetic mineralized cell membrane vesicle crosslinked polyacrylamide hydrogel (M-CMV-H) according to an embodiment of the present invention is shown. Detailed Implementation

[0021] To make the objectives and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0022] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0023] In the following examples, the cell membrane protein extraction kit was purchased from Wuhan Sanying Biotechnology Co., Ltd., brand name Proteintech, catalog number PK10015; it consisted of membrane protein extraction reagent A, protease inhibitor (100×), and 0.4% trypan blue solution. The polycarbonate membrane was brand name Avanti, catalog number 610006. The mini liposome extruder was brand name Avanti, catalog number 610000.

[0024] DMEM medium (brand: Gibco, catalog number: C11995500BT, mainly containing high glucose, L-glutamine, phenol red, and sodium pyruvate).

[0025] αMEM medium (brand: Gibco, catalog number: C12571500BT, mainly contains non-essential amino acids, sodium pyruvate, lipoic acid, vitamin B12, biotin and ascorbic acid, and does not contain FBS).

[0026] Example 1 This invention provides a method for preparing biomimetic mineralized cell membrane vesicle crosslinked polyacrylamide hydrogel, specifically including the following steps: S1: Extraction and purification of cell membrane vesicles (CMVs), specifically: S11: Cell culture: The ATDC5 chondrogenic cell line was seeded in DMEM medium (containing 1% penicillin and antibiotics and 10% fetal bovine serum) and cultured in a CO2 cell incubator at 37°C. After the cells reached 95% confluence, the cells were collected. S12: Preparation before extraction: Prepare the cell membrane protein extraction kit in advance. Take an equal volume of membrane protein extraction reagent A according to the volume of cells collected in step S11, and add protease inhibitor (100×) at a volume ratio of 100:1 before use. S13: Sample Pretreatment: Wash the cells collected in step S11 twice with pre-cooled PBS, centrifuge at 500g for 5 min at 4°C, and discard the residual PBS; then, on ice, centrifuge at 2 × 10⁻⁶ cells per 10⁻⁶ cells. 7 Add 1 mL of pre-cooled membrane protein extraction reagent A containing protease inhibitors to each cell; S14: Cell disruption by vortexing: The cells that have been treated with membrane protein extraction reagent A in step S13 are vortexed vigorously for 1 minute using a vortex mixer in the following sequence: vortex for 10 seconds, pause for 10 seconds, vortex for 10 seconds, pause for 10 seconds. Then, place the cells on ice for 2 minutes. Repeat this vortexing-placement step 4-5 times. A small number of cells are then taken for trypan blue staining to assess the degree of cell disruption. If the degree of cell disruption is less than 80%, increase the number of vigorous vortexing cycles until the degree of cell disruption is greater than or equal to 80%. S15: Centrifuge the cells disrupted in step S14 at 700g for 10 min at 4℃, collect the supernatant to obtain a solution containing cell membrane fragments; centrifuge the collected supernatant at 16000g for 30 min at 4℃ to precipitate cell membrane fragments, and collect the supernatant to obtain cell cytoplasmic proteins. S16: Using an extrusion method (small liposome extruder), cell membrane fragments were sequentially passed through polycarbonate membranes with pore sizes of 400 nm and 200 nm, and repeatedly extruded for 5-6 cycles. The mixture was then centrifuged at 16000 g for 30 min at 4 °C, and the precipitate was collected to obtain cell membrane vesicles (CMVs).

[0027] S2: CMVs were mixed with DSPE-PEG-AM and incubated to obtain a solution of olefin double-bond functionalized cell membrane vesicles (CMV-AM), specifically: S21: Surface modification: Take a certain amount of cell membrane vesicles (200 μL), add DSPE-PEG-AM (to make its mass fraction 2% wt / wt), and obtain a mixture; S22: Incubation: Incubate the mixture at 37°C with shaking at 500g for 0.5h. This temperature facilitates the insertion of DSPE into the lipid bilayer. S23: Purification: The incubated mixture was centrifuged at 16000g for 30 min at 4℃, and washed twice with PBS to remove unbound DSPE-PEG-AM, yielding a solution of olefin double-bond functionalized cell membrane vesicles (CMV-AM). A schematic diagram of the mechanism is shown below. Figure 1 (a).

[0028] S3: Preparation of cell membrane vesicle crosslinked polyacrylamide hydrogel (CMV-H), specifically: S31: Preparation of pre-gelation solution: In an oxygen-free glove box, add 2 mL of CMV-AM solution (containing approximately 1.5 × 10⁻⁶ ppm) to a 5 mL centrifuge tube.12 Add 0.5 g (7 mmol) of acrylamide (AM) monomer to CMVs (particles / mL), and vortex at 2000 rpm for 5 seconds until completely dissolved. S32: Initiation of polymerization: 0.05 g (0.2 mmol) of initiator APS and 2 μL of 2 M catalyst TMEDA were added sequentially to the pregel solution. The mixture was immediately vortexed vigorously at 2500 rpm for 5 seconds, and then quickly transferred to a polytetrafluoroethylene mold. Polymerization was carried out at room temperature for 30 min to obtain cell membrane vesicle crosslinked polyacrylamide hydrogel (CMV-H). See the schematic diagram of the mechanism. Figure 1 (d).

[0029] S4: The cell membrane vesicle cross-linked polyacrylamide hydrogel was immersed in a mineralization induction solution (DMEM containing 50 μM ascorbic acid, 10 mM β-glycerol phosphate and 100 nM dexamethasone) for 14 days to induce mineralization, resulting in a biomimetic mineralized cell membrane vesicle cross-linked polyacrylamide hydrogel (M-CMV-H).

[0030] Comparative Example 1: Uncrosslinked polyacrylamide hydrogel precursor solution Adjust the pH to 11 by adding NaOH dropwise to 10 mL of deionized water. Then add 2.5 g of acrylamide monomer and 0.25 g of ammonium persulfate (APS), and add 10 μL of tetramethylethylenediamine (TMEDA) as an initiator. No cross-linking agent is added. After mixing, the precursor solution is obtained. See the schematic diagram of the mechanism. Figure 1 (b).

[0031] Comparative Example 2: Polyacrylamide hydrogel (PAM) The pH of 10 mL of deionized water was adjusted to 11 by adding NaOH dropwise. Then, 2.5 g of acrylamide monomer and 0.25 g of ammonium persulfate (APS) were added and mixed thoroughly. Next, 10 μL of tetramethylethylenediamine (TMEDA) was added as an initiator and 6 mg of methylenebisacrylamide (BIS) as a crosslinking agent. Nitrogen gas was bubbled through the hydrogel precursor solution to remove oxygen during the process. The solution was then left to stand at room temperature for 30 min to obtain a polyacrylamide hydrogel. A schematic diagram of the mechanism is shown below. Figure 1 (c).

[0032] Experimental Example 1: Physicochemical Characterization of Cell Membrane Vesicle Crosslinked Polyacrylamide Hydrogel (CMV-H) 1. Observation of hydrogel state (1) Experimental conditions: The above uncrosslinked polyacrylamide hydrogel precursor solution, polyacrylamide hydrogel (PAM) and cell membrane vesicle crosslinked polyacrylamide hydrogel (CMV-H) were placed in glass bottles at room temperature. The fluidity of the materials before and after crosslinking was observed with the naked eye and photographs were taken. (2) Results: such as Figure 2The results showed that the uncrosslinked polyacrylamide hydrogel precursor solution was fluid and tilted as the glass bottle was tilted, while the crosslinked polyacrylamide hydrogel (PAM) and cell membrane vesicle crosslinked polyacrylamide hydrogel (CMV-H) did not tilt as the bottle was tilted.

[0033] 2. Morphological observation (1) Experimental conditions: Cross-linked polyacrylamide hydrogel (PAM) and cell membrane vesicle cross-linked polyacrylamide hydrogel (CMV-H) were soaked in PBS and swollen overnight, then placed at -80℃ for 12 h for pathological freezing, and then frozen in liquid nitrogen for 5 minutes before quenching the samples to obtain the cross-section. The quenched samples were placed in a freeze dryer and freeze-dried for 24 h. They were then fixed on the sample stage, sputtered with gold, and then their morphology and structure were observed using a scanning electron microscope.

[0034] (2) Results: such as Figure 3 The results show significant differences in the internal microstructure and skeletal morphology between polyacrylamide hydrogel (PAM) and cell membrane vesicle crosslinked polyacrylamide hydrogel (CMV-H). PAM hydrogel exhibits a typical uniform honeycomb porous structure formed after freeze-drying, while CMV-H hydrogel shows numerous interwoven connection structures between the pore walls (see...). Figure 3 (a)-3(b)). Overall, the introduction of cell membrane vesicles significantly altered the microstructure of the PAM hydrogel, transforming it from a regular, smooth honeycomb structure into a coarser, fibrous, and more interconnected three-dimensional network (see [link to article]). Figure 3 (c)-3(e)), this structure is more conducive to subsequent mineralization and cell adhesion. It can be seen that the biomimetic mineralized hydrogel synthesis method used in this embodiment is feasible.

[0035] 3. Fluorescent staining analysis of the distribution of cell membrane vesicles (CMVs) in cell membrane vesicle cross-linked polyacrylamide hydrogel (CMV-H). (1) Experimental conditions: Cell membrane vesicles (CMVs) were labeled with PKH26 and incubated to form PKH26-labeled olefin double bond-functionalized cell membrane vesicles (CMV-AM). The CMV-AM was further prepared into a cross-linked polyacrylamide hydrogel containing fluorescently labeled cell membrane vesicles (CMV-H). The uncross-linked PKH26-labeled CMV-AM was washed away with PBS. The distribution of fluorescently labeled cell membrane vesicles in the material was photographed and analyzed by laser confocal imaging.

[0036] (2) Results: such as Figure 4 The results showed abundant red fluorescence signals distributed within the hydrogel matrix, confirming that CMVs were successfully immobilized within the polymer network. The fluorescence signals were uniformly distributed in both the XY and Z-axis planes, indicating that the CMVs were uniformly embedded in the three-dimensional hydrogel, rather than being confined to the surface, and no obvious aggregation was observed.

[0037] 4. Elemental Analysis (1) Experimental conditions: Carbon, oxygen, phosphorus and calcium elements of the biomimetic mineralized cell membrane vesicle crosslinked polyacrylamide hydrogel (M-CMV-H) prepared in Example 1 were analyzed by energy dispersive spectroscopy (EDS).

[0038] (2) Results: The energy spectrum results are as follows Figure 5 Carbon, oxygen, phosphorus, and calcium are uniformly distributed within the mineralized hydrogel scaffold. Carbon and oxygen primarily originate from the hydrogel matrix, while phosphorus comes from naturally occurring phospholipid phosphate groups in cell membrane vesicles and calcium phosphate minerals deposited during mineralization. Calcium corresponds to deposited calcium phosphate minerals. The high co-localization of Ca / P suggests that mineralization deposition is the main source of increased phosphorus. The high spatial overlap between calcium and phosphorus indicates that calcium phosphate forms on the hydrogel framework, rather than as isolated mineral aggregates. Furthermore, EDS spectra show characteristic peaks for C, O, P, and Ca, confirming the successful integration of calcium phosphate minerals into the hydrogel.

[0039] 5. Infrared spectroscopy analysis (1) Experimental conditions: The molecular structure and chemical composition of the biomimetic mineralized cell membrane vesicle crosslinked polyacrylamide hydrogel (M-CMV-H) prepared in Example 1 were analyzed by Fourier transform infrared spectroscopy (FITR).

[0040] (2) Results: The results are as follows Figure 6 The study showed that the mineralized hydrogel retained characteristic organic matrix peaks, including a broad O–H / N–H stretching vibration peak (~3181 cm⁻¹). -1 C–H stretching vibration peak (~2928 cm⁻¹) -1 ) and amide I / II peaks (~1649 and 1603 cm⁻¹) -1 This indicates that the hydrogel network was maintained after mineralization. More importantly, at 1131, 1053, and 518 cm⁻¹, the hydrogel network was preserved. -1 A characteristic phosphate absorption peak appeared at 1412 cm⁻¹, confirming the formation of calcium phosphate minerals. -1 The carbonate peaks at the specified locations indicate partial carbonate substitution within the apatite lattice, suggesting the formation of bone-like carbonated hydroxyapatite, rather than stoichiometric hydroxyapatite. These results collectively demonstrate successful biomimetic mineralization within the hydrogel.

[0041] In summary, the biomimetic mineralized cell membrane vesicle crosslinked polyacrylamide hydrogel (M-CMV-H) of this embodiment has mild preparation conditions, controllable preparation process, and stable material structure, which can improve the preparation process of traditional mineralized hydrogels and enhance material stability. The biomimetic mineralized hydrogel of this embodiment has high mineralization activity and uniform mineral distribution. Both energy dispersive spectroscopy (EDS) and Fourier transform infrared spectroscopy (FITR) analysis show that calcium phosphate minerals have been successfully integrated into the hydrogel, and that calcium phosphate is formed on the hydrogel framework rather than as isolated mineral aggregates, indicating that successful biomimetic mineralization has been achieved within the hydrogel.

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a biomimetic mineralized cell membrane vesicle crosslinked polyacrylamide hydrogel, characterized in that: Specifically, the steps include the following: S1: Extraction and purification of cell membrane vesicles; S2: Cell membrane vesicles were mixed with DSPE-PEG-AM and incubated to obtain a cell membrane vesicle solution with olefin double bonds functionalized. S3: Under anaerobic conditions, a cell membrane vesicle solution with olefin double bond functionalization is mixed with acrylamide monomer, an initiator and a catalyst are added, and the mixture is polymerized to obtain a cell membrane vesicle crosslinked polyacrylamide hydrogel. S4: The cell membrane vesicle cross-linked polyacrylamide hydrogel was immersed in a mineralization induction solution to obtain a biomimetic mineralized cell membrane vesicle cross-linked polyacrylamide hydrogel.

2. The preparation method according to claim 1, characterized in that: In step S1, the cell membrane vesicles are prepared by the following method: S11: Cell culture: The ATDC5 chondrogenic cell line was seeded in DMEM medium containing 1% penicillin and 10% fetal bovine serum and cultured in a CO2 cell incubator at 37°C. After the cells grew to 95% confluence, the cells were collected. S12: Preparation before extraction: Prepare the cell membrane protein extraction kit in advance. Take an equal volume of membrane protein extraction reagent A according to the volume of cells collected in step S11, and add protease inhibitor at a volume ratio of 100:1 before use. S13: Sample Pretreatment: Wash the cells collected in step S11 twice with pre-cooled PBS, centrifuge at 500g for 5 min at 4°C, and discard the residual PBS; then, on ice, centrifuge at 2 × 10⁻⁶ cells per 10⁻⁶ cells. 7 Add 1 mL of pre-cooled membrane protein extraction reagent A containing protease inhibitors to each cell; S14: Cell disruption by vortexing: The cells that have been treated with membrane protein extraction reagent A in step S13 are vortexed vigorously for 1 minute using a vortex mixer in the following sequence: vortex for 10 seconds, pause for 10 seconds, vortex for 10 seconds, pause for 10 seconds. Then, place the cells on ice for 2 minutes. Repeat this vortexing-placement step 4-5 times. A small number of cells are then taken for trypan blue staining to assess the degree of cell disruption. If the degree of cell disruption is less than 80%, increase the number of vigorous vortexing cycles until the degree of cell disruption is greater than or equal to 80%. S15: Centrifuge the cells disrupted in step S14 at 700g for 10 min at 4℃ and collect the supernatant; centrifuge the collected supernatant at 16000g for 30 min at 4℃ and collect the precipitate to obtain the extracted cell membrane fragments. S16: Pass cell membrane fragments sequentially through polycarbonate membranes with pore sizes of 400 nm and 200 nm, squeeze repeatedly for 5-6 cycles, then centrifuge at 16000g for 30 min at 4℃, collect the precipitate, and obtain cell membrane vesicles.

3. The preparation method according to claim 1, characterized in that: In step S2, the olefin double-bond functionalized cell membrane vesicle solution is prepared by the following method: S21: Surface modification: Take a certain amount of cell membrane vesicles, add DSPE-PEG-AM to obtain a mixture; the concentration of DSPE-PEG-AM is 2% wt / wt; S22: Incubation: Incubate the mixture at 37°C with shaking at 500g for 0.5h; S23： Purification: The incubated mixture was centrifuged at 16000g for 30 min at 4℃ and washed twice with PBS to remove unbound DSPE-PEG-AM, yielding a cell membrane vesicle solution with olefin double bonds.

4. The preparation method according to claim 1, characterized in that: In step S3, the cell membrane vesicle crosslinked polyacrylamide hydrogel is prepared by the following method: S31: Preparation of pregelation solution: In an anaerobic glove box, add olefin double bond functionalized cell membrane vesicle solution to a centrifuge tube, then add acrylamide monomer, and vortex at 2000 rpm for 5 seconds until completely dissolved; the ratio of olefin double bond functionalized cell membrane vesicle solution to acrylamide is 2:0.5 in mL:g. S32: Initiating polymerization: Add the initiator ammonium persulfate and the 2M catalyst tetramethylethylenediamine sequentially to the pregel solution, immediately vortex vigorously at 2500 rpm for 5 seconds to mix, and quickly transfer to a polytetrafluoroethylene mold. Polymerize at room temperature for 30 minutes to obtain cell membrane vesicle crosslinked polyacrylamide hydrogel; the mass ratio of ammonium persulfate to acrylamide in step S31 is 1:10; the ratio of ammonium persulfate to tetramethylethylenediamine is 0.05:2 in g:μL.

5. The preparation method according to claim 1, characterized in that: In step S4, the biomimetic mineralized cell membrane vesicle cross-linked polyacrylamide hydrogel is prepared by the following method: the cell membrane vesicle cross-linked polyacrylamide hydrogel is immersed in a mineralization induction solution for 14 days to induce mineralization. The mineralization induction solution is αMEM medium containing 10 mM β-glycerophosphate sodium.

6. A biomimetic mineralized cell membrane vesicle crosslinked polyacrylamide hydrogel, characterized in that: It is prepared by the preparation method according to any one of claims 1-5.

7. The biomimetic mineralized cell membrane vesicle crosslinked polyacrylamide hydrogel according to claim 6, characterized in that: Its interior contains uniformly distributed cell membrane vesicles and amorphous calcium phosphate, a mineralization precursor of hydroxyapatite generated in situ.

8. An application of the biomimetic mineralized cell membrane vesicle crosslinked polyacrylamide hydrogel as described in claim 6 or 7, characterized in that: The hydrogel is used in the preparation of bone repair materials.

9. The application according to claim 8, characterized in that: The bone repair material is used for bone defect repair, osteochondral regeneration, or osteoporotic bone repair.

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