A ROS and piezoelectric dual-responsive biomimetic vesicle loaded with Mn-doped hollow mesoporous ceria-quercetin composite hydrogel and a preparation method thereof

Abstract

The application provides a ROS and piezoelectric dual-response biomimetic vesicle loaded Mn-doped hollow mesoporous cerium dioxide-quercetin composite hydrogel and a preparation method thereof. The composite hydrogel comprises a hyaluronic acid-sulfur ketal hydrogel matrix, tetragonal barium titanate nanoparticles and Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles coated by bone marrow mesenchymal stem cell-derived biomimetic vesicles. The composite hydrogel can respond to degradation and release functional components under an active oxygen environment, generate a piezoelectric response through barium titanate, remove active oxygen free radicals through Mn-doped hollow mesoporous cerium dioxide and achieve quercetin slow-release delivery. The application has ROS response, piezoelectric stimulation, antioxidant regulation, biomimetic delivery and drug slow-release functions and can be used for the preparation of bone tissue repair, bone defect filling and oxidative stress microenvironment regulation materials.

Description

Technical Field

[0001] This invention belongs to the field of bone tissue engineering materials and nanocomposite hydrogel technology, specifically involving a multifunctional composite hydrogel material and its preparation method that combines ROS response, piezoelectric response, biomimetic vesicle delivery and drug sustained release functions. It can be used as a biomedical material for bone defect repair, bone tissue regeneration, and regulation of oxidative stress and inflammatory microenvironment. Background Technology

[0002] Bone defects, bone degeneration, and bone trauma are common diseases in clinical orthopedics, often accompanied by local tissue damage, increased inflammation, elevated oxidative stress, and decreased bone regeneration capacity. For large-area bone defects, infected bone defects, or complex bone injuries, relying solely on the body's own repair capabilities is insufficient for complete bone tissue regeneration. Therefore, developing novel bone repair materials that simultaneously provide structural support and microenvironment regulation is of great significance.

[0003] Hydrogels, due to their excellent water content, biocompatibility, injectability, and three-dimensional network structure similar to the natural extracellular matrix, are widely used in bone tissue engineering scaffolds, drug delivery systems, and cell carriers. Existing hydrogel materials can typically provide some spatial support in bone defect areas and create a basic environment for cell adhesion, growth, and nutrient exchange. However, traditional hydrogels are mostly passive fillers and physical supports, lacking the ability to actively respond to the pathological microenvironment of bone injury, and are unable to effectively alleviate the persistent oxidative stress and inflammatory response in the damaged area.

[0004] During bone injury repair, high levels of local reactive oxygen species (ROS) induce oxidative damage in cells, promote the continuous release of inflammatory factors, and inhibit the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, thereby affecting bone regeneration. To address this issue, existing technologies have proposed ROS-responsive hydrogels, which can degrade or undergo structural changes under oxidative stress to release drugs or functional components. However, these materials typically rely primarily on a single chemical response mechanism and lack comprehensive regulatory capabilities over cellular electrophysiological behavior, osteogenic signal transduction, and tissue regeneration processes.

[0005] Bone tissue is a biological tissue with piezoelectric properties, capable of converting mechanical stimulation into electrical signals during bodily movement or stress. These electrical signals can participate in regulating cell adhesion, migration, proliferation, and osteogenic differentiation. Based on this, piezoelectric biomaterials are increasingly being applied in the field of bone tissue repair. Among them, barium titanate nanoparticles possess good piezoelectric properties and biocompatibility, and can generate local electrical signals under mechanical stimulation, ultrasonic stimulation, or physiological micro-stress, thus simulating the electrophysiological microenvironment of natural bone tissue. However, simply introducing piezoelectric materials is often insufficient to remove excess reactive oxygen species (ROS) in the bone injury area, nor is it sufficient to continuously deliver anti-inflammatory or osteoproliferative drugs. Therefore, their repair efficacy in complex pathological microenvironments remains limited.

[0006] Nanozyme materials have attracted attention in the field of oxidative stress regulation due to their ability to mimic the catalytic activity of natural enzymes. Cerium dioxide nanoparticles are based on Ce³⁺. + / Ce 4+ Reversible redox cycles can exhibit activities similar to superoxide dismutase and catalase, thereby scavenging reactive oxygen species. Furthermore, manganese doping can increase oxygen vacancy content, improving electron transfer efficiency and antioxidant activity. Constructing cerium dioxide into a hollow mesoporous structure can also increase its specific surface area and drug loading capacity, providing possibilities for nanoplatforms with both antioxidant regulation and drug delivery functions. However, individual nanozyme particles may still suffer from insufficient dispersion stability, limited local retention time, and insufficient targeting when applied in vivo.

[0007] Quercetin is a natural flavonoid compound with antioxidant, anti-inflammatory, and osteogenic differentiation-promoting biological activities, which can be used to improve the bone injury microenvironment and promote bone repair. However, quercetin suffers from poor water solubility, low bioavailability, rapid in vivo metabolism, and difficulty in maintaining local effective concentrations, limiting its direct application. Therefore, suitable nanocarriers are needed to load quercetin to improve its stability and utilization rate, and achieve sustained release.

[0008] To further improve the biocompatibility, stability, and tissue adaptability of nanomaterials, cell membrane-coated biomimetic delivery technology is increasingly being used to construct biomimetic nanomaterials. Cell membranes derived from bone marrow mesenchymal stem cells can retain some of the cell surface's bioactive components, giving nanoparticles good biocompatibility, immune escape capabilities, and potential tissue tropism that adapts to the bone repair microenvironment. However, existing cell membrane biomimetic delivery systems are mostly used to improve the in vivo stability or targeted delivery performance of nanoparticles. Without responsive hydrogel matrices and functional stimulation-regulating components, their overall ability to regulate the complex microenvironment of bone injury remains limited.

[0009] In summary, existing bone repair materials generally have the following shortcomings: First, traditional hydrogels mainly play a supporting and filling role, lacking the ability to actively regulate high ROS and inflammatory microenvironments; second, ROS-responsive hydrogels mostly rely on a single chemical response, making it difficult to simulate the piezoelectric physiological characteristics of natural bone tissue; third, although piezoelectric materials alone can generate local electrical signals, they cannot effectively solve the problems of oxidative stress and sustained drug delivery; fourth, nanozymes or drug carrier materials, when used alone, may have problems such as insufficient local retention, limited biocompatibility, and insufficient targeting; fifth, current technologies lack composite materials that organically integrate ROS-responsive hydrogels, piezoelectric stimulation, biomimetic vesicle delivery, nanozyme antioxidation, and quercetin sustained release into the same system.

[0010] Therefore, it is necessary to develop a composite hydrogel material that combines ROS-responsive degradation, piezoelectric stimulation regulation, biomimetic delivery, antioxidant free radical scavenging, and quercetin sustained release functions to improve oxidative stress and inflammatory microenvironment in bone injury areas, mimic the electrophysiological characteristics of natural bone tissue, and improve the utilization rate and local effects of active drugs, thereby promoting bone tissue repair and bone defect regeneration. Summary of the Invention

[0011] To address the problems of insufficient antioxidant capacity, low drug utilization, limited local retention effect, difficulty in simulating the piezoelectric microenvironment of natural bone tissue, and unsatisfactory bone repair effect of existing bone tissue repair materials, this invention provides a biomimetic vesicle-loaded Mn-doped hollow mesoporous cerium dioxide-quercetin composite hydrogel with both ROS and piezoelectric response, its preparation method, and its application.

[0012] This invention organically combines hyaluronic acid-thioketone hydrogel, tetragonal barium titanate nanoparticles, biomimetic vesicles derived from bone marrow mesenchymal stem cells, and Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles to construct a composite hydrogel system that combines ROS-responsive degradation, piezoelectric stimulation, biomimetic delivery, antioxidant regulation, and sustained release of quercetin. This improves the oxidative stress and inflammatory microenvironment at bone injury sites and promotes bone tissue repair and regeneration.

[0013] To achieve the above objectives, this invention provides a ROS- and piezoelectric dual-responsive biomimetic vesicle-loaded Mn-doped hollow mesoporous cerium dioxide-quercetin composite hydrogel, comprising a ROS-responsive hydrogel matrix, piezoelectric nanoparticles dispersed in the ROS-responsive hydrogel matrix, and biomimetic vesicle-loaded nanocomposite particles; the ROS-responsive hydrogel matrix is ​​a hyaluronic acid-thioacetate hydrogel; the piezoelectric nanoparticles are tetragonal barium titanate nanoparticles; the biomimetic vesicle-loaded nanocomposite particles comprise biomimetic vesicles derived from bone marrow mesenchymal stem cells and Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles encapsulated by the biomimetic vesicles derived from bone marrow mesenchymal stem cells.

[0014] In one possible implementation, the hyaluronic acid-thioketal hydrogel is formed by reacting hyaluronic acid with a crosslinking component or grafting component containing thioketal bonds. The thioketal bonds can break under reactive oxygen species conditions, giving the composite hydrogel ROS-responsive degradation properties.

[0015] In one possible implementation, the tetragonal barium titanate nanoparticles are used to generate a piezoelectric response under external mechanical stimulation, ultrasonic stimulation, or physiological micro-stress to form a local electrical signal.

[0016] In one possible implementation, the Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles comprise an Mn-doped hollow mesoporous cerium dioxide support and quercetin loaded on the pores and / or surface of the Mn-doped hollow mesoporous cerium dioxide support.

[0017] In one possible implementation, the Mn-doped hollow mesoporous cerium dioxide support has a hollow mesoporous structure and Ce³⁺. + / Ce 4+ It has reversible redox activity and is used to scavenge reactive oxygen free radicals.

[0018] In one possible implementation, the bone marrow mesenchymal stem cell-derived biomimetic vesicles are prepared by breaking, centrifuging and purifying, and extruding bone marrow mesenchymal stem cell membranes, and then coated onto the surface of the Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles to form a core-shell structure.

[0019] In one possible implementation, the biomimetic vesicle-loaded nanocomposite particles have a particle size of 100–250 nm, preferably 130–180 nm; the quercetin loading rate in the composite hydrogel is 10%–30%, and the encapsulation rate is 50%–90%.

[0020] The present invention also provides a method for preparing the above-mentioned composite hydrogel, comprising the following steps: S1. Preparation of hyaluronic acid-thioacetate hydrogel precursor; S2. Preparation of tetragonal barium titanate nanoparticles; S3. Prepare Mn-doped hollow mesoporous cerium dioxide nanoparticles, and load quercetin into the Mn-doped hollow mesoporous cerium dioxide nanoparticles to obtain Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles. S4. Prepare biomimetic vesicles from bone marrow mesenchymal stem cells, and use the biomimetic vesicles to coat the Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles to obtain biomimetic vesicle-loaded nanocomposite particles. S5. The biomimetic vesicle-loaded nanocomposite particles, hyaluronic acid-thioacetate hydrogel precursor, tetragonal barium titanate nanoparticles and photoinitiator are mixed and photocured to obtain the composite hydrogel.

[0021] In one possible implementation, in step S3, the Mn-doped hollow mesoporous cerium dioxide nanoparticles are prepared by the following method: after a hydrothermal reaction of cerium salt, polyvinylpyrrolidone, glacial acetic acid, and ethylene glycol, manganese salt is added to continue the reaction, followed by washing, drying, and calcination to obtain Mn-doped hollow mesoporous cerium dioxide nanoparticles; subsequently, the Mn-doped hollow mesoporous cerium dioxide nanoparticles are dispersed in an aqueous system, mixed with a quercetin solution, and stirred to load quercetin into the Mn-doped hollow mesoporous cerium dioxide nanoparticles.

[0022] In one possible implementation, in step S4, the biomimetic vesicles are prepared by the following method: bone marrow mesenchymal stem cells are collected, washed, lysed or sonicated, centrifuged to remove cell nuclei and unbroken cells, and then ultracentrifuged to obtain cell membrane components; the cell membrane components are mixed with Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles, and extruded together through a polycarbonate membrane to obtain biomimetic vesicle-loaded nanocomposite particles.

[0023] In one possible implementation, in step S5, the photoinitiator is an LAP photoinitiator, and the photocuring is performed by irradiation with 405 nm visible light.

[0024] The present invention also provides the application of the above-mentioned composite hydrogel or the composite hydrogel prepared by the above-mentioned preparation method in the preparation of materials for bone tissue repair, bone defect filling, oxidative stress microenvironment regulation, inflammatory microenvironment regulation or promoting the proliferation of bone marrow mesenchymal stem cells.

[0025] Based on the above technical solution, the present invention provides a ROS-responsive piezoelectric dual-response biomimetic vesicle-loaded Mn-doped hollow mesoporous cerium dioxide-quercetin composite hydrogel. A ROS-responsive three-dimensional network is constructed using hyaluronic acid-thioacetate hydrogel. Tetragonal barium titanate nanoparticles impart piezoelectric responsiveness to the material. Mn-doped hollow mesoporous cerium dioxide is used to load quercetin and scavenge reactive oxygen species. Bone marrow mesenchymal stem cell-derived biomimetic vesicles enhance the biocompatibility and delivery stability of the nanoparticles. This solution addresses the problems of insufficient antioxidant regulation, low drug utilization, lack of piezoelectric stimulation, and insufficient multifunctional synergistic repair capabilities in existing bone repair materials.

[0026] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The present invention uses hyaluronic acid-thioketene hydrogel as ROS-responsive hydrogel matrix. Thioketene bonds can be broken in the reactive oxygen environment, causing the hydrogel to undergo responsive degradation, which is conducive to the release of functional nanoparticles and quercetin in the microenvironment of bone injury oxidative stress.

[0027] (2) The present invention introduces tetragonal barium titanate nanoparticles, which enable the composite hydrogel to generate a piezoelectric response under mechanical stimulation, ultrasonic stimulation or physiological micro-stress, forming a local electrical signal, which is beneficial to simulate the piezoelectric microenvironment of natural bone tissue and promote cell proliferation and bone tissue repair.

[0028] (3) This invention uses Mn-doped hollow mesoporous cerium dioxide as a functional carrier, which has a hollow mesoporous structure and Ce³⁺. + / Ce 4+ It exhibits reversible redox activity, which can enhance quercetin loading capacity and help scavenge reactive oxygen free radicals, thereby improving the oxidative stress state in bone injury areas.

[0029] (4) The present invention loads quercetin onto the pores and / or surface of a Mn-doped hollow mesoporous cerium dioxide support, which can improve the problems of poor water solubility, low bioavailability and rapid in vivo metabolism of quercetin, and realize the continuous delivery and local action of quercetin.

[0030] (5) The present invention uses biomimetic vesicles derived from bone marrow mesenchymal stem cells to coat Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles to form a core-shell structure, which helps to improve the biocompatibility, stability and adaptability of nanoparticles to the bone repair microenvironment.

[0031] (6) The present invention integrates ROS-responsive hydrogel, piezoelectric nanoparticles, nanoenzyme antioxidant carrier, quercetin sustained-release system and biomimetic vesicle delivery system into the same composite hydrogel to achieve the synergistic effect of ROS-responsive release, piezoelectric stimulation, antioxidant regulation, drug sustained release and biomimetic delivery, thereby improving the bone tissue repair effect. Attached Figure Description

[0032] Figure 1 This is a scanning electron microscope image of the HA-TK hydrogel.

[0033] Figure 2 The energy dispersive X-ray spectrum of the HA-TK hydrogel.

[0034] Figure 3 This is a scanning electron microscope image of a ROS- and piezoelectric dual-response hydrogel.

[0035] Figure 4 The energy dispersive X-ray spectrum of the ROS and piezoelectric dual-response hydrogel is shown.

[0036] Figure 5This is a transmission electron microscope image of barium titanate nanoparticles.

[0037] Figure 6 This is a transmission electron microscope image of Mn-doped hollow mesoporous cerium dioxide nanoparticles.

[0038] Figure 7 The energy dispersive X-ray spectrum of Mn-doped hollow mesoporous cerium dioxide nanoparticles.

[0039] Figure 8 Transmission electron microscope image of Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles.

[0040] Figure 9 Transmission electron microscopy image of Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles coated with biomimetic vesicles derived from bone marrow mesenchymal stem cells.

[0041] Figure 10 The in vitro drug release curve of Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles is shown.

[0042] Figure 10-1 The UV spectrum and standard curve of quercetin.

[0043] Figure 10-2 The drug release curves and drug loading results of Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles are shown in the figure.

[0044] Figure 11 This is a UV-Vis absorption spectrum.

[0045] Figure 12 The graph shows the rheological properties of the composite hydrogel.

[0046] Figure 13 X-ray photoelectron spectra of the ROS and piezoelectric dual-response hydrogel @mNVs@Mn-HCeO2 / Que composite system. Detailed Implementation

[0047] This invention provides a ROS- and piezoelectric dual-response biomimetic vesicle-loaded Mn-doped hollow mesoporous cerium dioxide-quercetin composite hydrogel. This hydrogel is synergistically constructed from multifunctional components, including: 1. ROS-responsive hydrogel matrix: mainly composed of hyaluronic acid-thioacetate (HA-TK) hydrogel, which can undergo responsive degradation in a reactive oxygen environment to achieve the controlled release of functional components.

[0048] 2. Piezoelectric nanoparticles: Tetragonal barium titanate (t-BaTiO3) nanoparticles are selected, which can generate local electrical signals under mechanical stimulation, ultrasound or physiological micro-stress, simulating the piezoelectric microenvironment of natural bone tissue.

[0049] 3. Biomimetic vesicle-loaded nanocomposite particles: Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles are encapsulated by biomimetic vesicles derived from bone marrow mesenchymal stem cells, which have the functions of drug delivery, antioxidant regulation and biocompatibility enhancement.

[0050] This composite hydrogel achieves synergistic effects through multiple mechanisms: ROS-responsive degradation promoting release, piezoelectric stimulation regulating cell behavior, Mn-HCeO2 antioxidant scavenging of reactive oxygen species, sustained-release delivery of quercetin, and biomimetic vesicles enhancing tissue targeting and stability. This invention, by organically integrating multiple functional components, not only improves the bone defect microenvironment but also enhances bone tissue repair and regeneration.

[0051] The specific embodiments of the present invention are used to demonstrate the preparation method, structural characteristics, drug loading and release performance, and biocompatibility evaluation of hydrogels, and do not constitute a limitation on the scope of protection of the present invention.

[0052] Example 1: Preparation of composite hydrogel

[0053] This embodiment provides a method for preparing a ROS- and piezoelectric dual-response biomimetic vesicle-loaded Mn-doped hollow mesoporous cerium dioxide-quercetin composite hydrogel. The method sequentially prepares HA-TK hydrogel precursors, tetragonal barium titanate nanoparticles, Mn-doped hollow mesoporous cerium dioxide nanoparticles, Mn-HCeO2 / Que nanocomposite particles, and biomimetic vesicles derived from bone marrow mesenchymal stem cells. Subsequently, the functional components are combined and photocured to form the composite hydrogel.

[0054] 1. Preparation of HA-TK hydrogel precursor

[0055] Weigh 200 mg of hyaluronic acid and dissolve it in 10 mL of deionized water. Stir until the hyaluronic acid is completely dissolved to obtain a hyaluronic acid solution. Then, add 300 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 200 mg of N-hydroxysuccinimide to the hyaluronic acid solution and activate it at 45 °C for 1 h to activate the carboxyl groups in the hyaluronic acid molecules.

[0056] 100 mg of thioketene crosslinking agent was added to the activated system, and the mixture was stirred continuously at 45 °C for 24 h to allow hyaluronic acid to react with the crosslinking component containing thioketene bonds, forming a hyaluronic acid-thioketene hydrogel precursor with ROS responsiveness.

[0057] After the reaction was complete, the resulting product was placed in a dialysis bag and dialyzed with deionized water for 3 days, changing the water 3-4 times a day to remove unreacted small molecules, cross-linking agent residues, and byproducts. After dialysis, the sample was freeze-dried to obtain the HA-TK hydrogel precursor.

[0058] The resulting HA-TK hydrogel has a three-dimensional porous network structure, such as Figure 1 As shown; its elemental composition is as follows Figure 2 As shown, this porous network structure facilitates the subsequent loading of piezoelectric nanoparticles and biomimetic vesicle-loaded nanocomposite particles, and provides a spatial basis for cell adhesion, growth, and nutrient exchange.

[0059] 2. Preparation of Tetragonal Barium Titanate Nanoparticles

[0060] 17.018 g of titanium butoxide was added to 20 mL of ethanol, stirred until homogeneous, and then 7 mL of ammonium hydroxide solution was added to obtain a titanium source precursor solution. Separately, 23.660 g of Ba(OH)₂·8H₂O was weighed and dissolved in 25 mL of deionized water to obtain a transparent barium source solution.

[0061] The barium source solution was slowly added to the titanium source precursor solution while continuously stirring to form a homogeneous suspension. The resulting suspension was then transferred to a 100 mL polytetrafluoroethylene-lined reactor and subjected to hydrothermal reaction at 200 °C for 48 h.

[0062] After the reaction was completed, the reaction system was cooled to room temperature. The obtained product was repeatedly washed with deionized water and anhydrous ethanol, and then dried under vacuum at 80 °C for 24 h to obtain BaTiO3 particles. Subsequently, the obtained BaTiO3 particles were placed in a muffle furnace, heated to 900 °C at a heating rate of 5 °C / min and held at that temperature for 4 h, and then cooled to 800 °C at a heating rate of 2 °C / min and held at that temperature for 4 h. After cooling, tetragonal barium titanate nanoparticles were obtained.

[0063] The morphology of the obtained tetragonal barium titanate nanoparticles is as follows: Figure 5 As shown, these particles, dispersed as piezoelectric nanoparticles in a hydrogel matrix, can generate piezoelectric responses under mechanical stimulation, ultrasonic stimulation, or physiological micro-stress, thereby providing the composite hydrogel with the ability to simulate the electrical signal microenvironment of natural bone tissue.

[0064] 3. Preparation of Mn-doped hollow mesoporous cerium dioxide nanoparticles

[0065] Weigh 1 g of cerium nitrate hexahydrate and add it to a mixed solution consisting of 10 mL of deionized water and 20 mL of ethylene glycol. Stir well at room temperature. Then add 1 mL of glacial acetic acid dropwise and slowly add 0.8 g of polyvinylpyrrolidone. Continue stirring until all components are fully dissolved to obtain the cerium source reaction system.

[0066] The resulting mixed solution was transferred to a 50 mL polytetrafluoroethylene-lined reactor and subjected to hydrothermal reaction at 180 °C for 22 h. After the reaction was completed and cooled to room temperature, manganese chloride solution was added to the system, and the hydrothermal reaction was continued for another 2 h to introduce manganese into the cerium dioxide structure.

[0067] After the reaction, the resulting product was washed twice, sequentially with ethanol, methanol, and deionized water by centrifugation, to remove unreacted raw materials and impurities. The precipitate was then vacuum-dried at 50 °C to obtain precursor powder. The precursor powder was placed in a calcination apparatus and calcined at 500 °C for 2 h to obtain Mn-doped hollow mesoporous cerium dioxide nanoparticles, denoted as Mn-HCeO2.

[0068] The obtained Mn-HCeO2 nanoparticles exhibit a hollow mesoporous structure, such as Figure 6 As shown; its EDS results are as follows Figure 7 As shown, the sample contains Ce, Mn, and O elements, indicating that Mn was successfully introduced into the cerium dioxide system. These Mn-HCeO2 nanoparticles possess a high specific surface area and porous structure, making them suitable as a quercetin carrier, while also being based on Ce³⁺. + / Ce 4+ The reversible redox properties and the oxygen vacancy regulation ability brought by Mn doping can be used to scavenge reactive oxygen free radicals.

[0069] 4. Preparation of Mn-HCeO2 / Que nanocomposite particles

[0070] A certain amount of Mn-HCeO2 nanoparticles were weighed and dispersed in ultrapure water, and then ultrasonically treated to form a uniform suspension. Subsequently, quercetin solution was added to the suspension, and the mixture was stirred overnight at room temperature in the dark to allow quercetin to be fully adsorbed and loaded onto the pores and / or surface of the Mn-HCeO2 nanoparticles.

[0071] After stirring, the reaction system was centrifuged at 8000 rpm for 10 min, the supernatant was discarded and the precipitate was collected. After washing, the precipitate yielded Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles, denoted as Mn-HCeO2 / Que.

[0072] The morphology of the obtained Mn-HCeO2 / Que nanocomposite particles is as follows: Figure 8 As shown. By Figure 8 It is evident that the quercetin particles retain a relatively intact hollow mesoporous structure after loading, indicating that Mn-HCeO2 can serve as a nanocarrier for quercetin, providing a structural basis for the efficient loading and subsequent sustained release of quercetin.

[0073] 5. Preparation of biomimetic vesicles derived from bone marrow mesenchymal stem cells

[0074] Log-phase bone marrow mesenchymal stem cells were collected, washed three times with PBS, and centrifuged to collect the cell pellet. The resulting cell pellet was resuspended in lysis buffer containing protease inhibitors and the cells were disrupted by sonication.

[0075] The ultrasonic disruption conditions were: 400 W power, 3 s operation time, 5 s interval, repeated 10–15 times. After ultrasonic disruption, the system was centrifuged at 10,000 g for 20 min at 4 ℃ to remove cell nuclei and undisrupted cells.

[0076] The supernatant was collected and ultracentrifuged at 100,000 g for 60 min to obtain the cell membrane component. The cell membrane component was then resuspended in washing buffer and ultracentrifuged again at 100,000 g for 30 min to obtain the purified bone marrow mesenchymal stem cell membrane component.

[0077] The cell membrane component is used to subsequently construct biomimetic vesicles, enabling the surface of the nanocomposite particles to possess bioactive components derived from bone marrow mesenchymal stem cell membranes, thereby improving the biocompatibility and stability of the nanoparticles.

[0078] 6. Preparation of mNVs@Mn-HCeO2 / Que nanocomposite particles

[0079] The Mn-HCeO2 / Que nanocomposite particles obtained in step 4 are mixed with the bone marrow mesenchymal stem cell membrane components obtained in step 5 to ensure that the cell membrane components are in full contact with the nanocomposite particles.

[0080] The mixture was then repeatedly extruded through polycarbonate membranes with pore sizes of 800 nm, 400 nm, and 200 nm, with each pore size condition being repeated 15 times. Through this stepwise membrane extrusion process, the cell membrane components were reassembled and coated onto the surface of the Mn-HCeO2 / Que nanocomposite particles, resulting in Mn-HCeO2 / Que nanocomposite particles coated with biomimetic vesicles derived from bone marrow mesenchymal stem cells, denoted as mNVs@Mn-HCeO2 / Que.

[0081] The morphology of the obtained mNVs@Mn-HCeO2 / Que nanocomposite particles is as follows: Figure 9 As shown. By Figure 9 As can be seen, a distinct membrane layer forms around the particles, exhibiting a typical core-shell structure, indicating that the biomimetic vesicles derived from bone marrow mesenchymal stem cells are successfully coated on the surface of Mn-HCeO2 / Que nanocomposite particles.

[0082] 7. Construction of ROS and piezoelectric dual-response composite hydrogels

[0083] Weigh 80 mg of LAP photoinitiator and add it to 20 mL of PBS solution to prepare a 0.4% (w / v) LAP solution. Add the mNVs@Mn-HCeO2 / Que nanocomposite particles obtained in step 6 to the LAP solution and disperse them uniformly.

[0084] Subsequently, 2 g of HA-TK hydrogel precursor and 1 mg of tetragonal barium titanate nanoparticles were added to the system. The resulting mixture was ultrasonically dispersed in an ultrasonic water bath for 15 min to ensure uniform mixing of the HA-TK hydrogel precursor, tetragonal barium titanate nanoparticles, and mNVs@Mn-HCeO2 / Que nanocomposite particles.

[0085] After dispersion, the mixture was cured under 405 nm visible light irradiation for 2 min to obtain a biomimetic vesicle-loaded Mn-doped hollow mesoporous cerium dioxide-quercetin composite hydrogel with ROS and piezoelectric dual response.

[0086] The scanning electron microscope image of the obtained composite hydrogel is shown below. Figure 3 As shown, the energy-dispersive X-ray spectrum is as follows: Figure 4 As shown. By Figure 3 It is evident that the composite hydrogel maintains a continuous and stable three-dimensional porous network structure; Figure 4 It is evident that piezoelectric components such as Ba and Ti are present in the sample, indicating that tetragonal barium titanate nanoparticles have been successfully introduced into the composite hydrogel system.

[0087] In addition, the rheological properties of the composite hydrogel are as follows: Figure 12 As shown, the results indicate that the composite hydrogel possesses stable gelation properties and mechanical structure; the X-ray photoelectron spectroscopy results of the composite system are as follows. Figure 13 As shown, characteristic element peaks such as C, N, O, Ce, Mn, Ba, and Ti are displayed, further proving that the HA-TK hydrogel matrix, tetragonal barium titanate nanoparticles, Mn-HCeO2 / Que nanocomposite particles, and biomimetic vesicles have been successfully composited.

[0088] Through the above steps, this embodiment successfully prepared a composite hydrogel that combines ROS-responsive degradation, piezoelectric response, biomimetic delivery, antioxidant regulation, and quercetin sustained release functions.

[0089] Example 2: Characterization of the structure and physicochemical properties of the composite hydrogel

[0090] In this embodiment, the HA-TK hydrogel, ROS and piezoelectric dual-response composite hydrogel, tetragonal barium titanate nanoparticles, Mn-HCeO2 nanoparticles, Mn-HCeO2 / Que nanocomposite particles, and mNVs@Mn-HCeO2 / Que nanocomposite particles prepared in Example 1 were used as test samples. Their morphology, structure, elemental composition, spectral characteristics, rheological properties, and surface elemental composition were characterized and analyzed to verify the successful construction of the composite hydrogel and the effective combination of each functional component.

[0091] 1. SEM and EDS characterization

[0092] After the freeze-dried HA-TK hydrogel and ROS-piezoelectric dual-response hydrogel samples were sputtered with gold, their microstructure was observed using scanning electron microscopy, and their elemental composition was analyzed using energy-dispersive X-ray spectroscopy.

[0093] like Figure 1 As shown, the HA-TK hydrogel exhibits a continuous three-dimensional porous network structure with a relatively uniform pore distribution. This structure facilitates water retention, nutrient exchange, cell adhesion, and growth, providing a structural basis for its subsequent use as a scaffold material in bone tissue engineering.

[0094] like Figure 2 As shown, the HA-TK hydrogel sample mainly contains elements such as C and O, indicating that the obtained hydrogel has the basic elemental composition characteristics of hyaluronic acid-based hydrogels.

[0095] like Figure 3 As shown, after introducing tetragonal barium titanate nanoparticles and mNVs@Mn-HCeO2 / Que nanocomposite particles, the ROS and piezoelectric dual-response composite hydrogel still maintains a continuous and stable three-dimensional porous structure, indicating that the introduction of functional nanoparticles does not destroy the hydrogel network structure.

[0096] like Figure 4 As shown, C, O, Ba, and Ti elemental signals were detected in the ROS-piezoelectric dual-response composite hydrogel sample. The Ba and Ti elements originated from tetragonal barium titanate nanoparticles, indicating that the piezoelectric nanoparticles were successfully incorporated into the hydrogel system.

[0097] 2. TEM characterization

[0098] The morphology of tetragonal barium titanate nanoparticles, Mn-HCeO2 nanoparticles, Mn-HCeO2 / Que nanocomposite particles and mNVs@Mn-HCeO2 / Que nanocomposite particles was observed using transmission electron microscopy.

[0099] like Figure 5As shown, the prepared tetragonal barium titanate nanoparticles have a relatively regular particle morphology and can be dispersed in the HA-TK hydrogel matrix as a piezoelectric functional component, providing a piezoelectric response basis for the composite hydrogel.

[0100] like Figure 6 As shown, the Mn-HCeO2 nanoparticles exhibit a distinct hollow structure and mesoporous characteristics. This hollow mesoporous structure helps to increase the specific surface area and drug loading space of the material, providing a carrier basis for subsequent quercetin loading.

[0101] like Figure 7 As shown, Ce, Mn, and O elements were detected in Mn-HCeO2 nanoparticles, and the elemental distribution was relatively uniform, indicating that Mn was successfully introduced into the cerium dioxide nanostructure. Mn doping helps to regulate the oxygen vacancy structure and electron transfer performance of CeO2, thereby enhancing its antioxidant capacity.

[0102] like Figure 8 As shown, the Mn-HCeO2 / Que nanocomposite particles obtained after quercetin loading still maintain a relatively intact hollow mesoporous structure, indicating that the quercetin loading process did not significantly damage the Mn-HCeO2 support structure.

[0103] like Figure 9 As shown, after being coated with biomimetic vesicles derived from bone marrow mesenchymal stem cells, the mNVs@Mn-HCeO2 / Que nanocomposite particles exhibit a typical core-shell structure, and a membrane-like coating layer can be observed on the outer layer of the particles, indicating that the biomimetic vesicles were successfully coated on the surface of the Mn-HCeO2 / Que nanocomposite particles.

[0104] 3. Ultraviolet-Vis absorption spectroscopy characterization

[0105] The absorption spectroscopic analysis of quercetin and related nanocomposite systems was performed using ultraviolet-visible spectrophotometry, and the results are as follows: Figure 11 As shown.

[0106] Depend on Figure 11 It is known that quercetin possesses corresponding UV-Vis absorption characteristics; the observation of quercetin-related absorption characteristics in the Mn-HCeO2 / Que nanocomposite system indicates that quercetin has been successfully loaded into Mn-HCeO2 nanoparticles. This result corroborates the TEM observations, demonstrating that Mn-HCeO2 can serve as a quercetin carrier, enabling the loading of quercetin.

[0107] 4. Rheological property characterization

[0108] The rheological properties of HA-TK hydrogel and ROS / piezoelectric dual-response hydrogel were tested using a rotational rheometer. The test results are as follows: Figure 12 As shown.

[0109] Frequency scanning results show that, within the test frequency range, the storage modulus G' of the hydrogel sample is always higher than the loss modulus G'', indicating that both the HA-TK hydrogel and the ROS-piezoelectric dual-response composite hydrogel exhibit typical gel properties.

[0110] Amplitude scanning results show that the ROS-piezoelectric dual-response composite hydrogel can maintain a stable network structure within a certain strain range, indicating that the hydrogel still has good structural integrity and mechanical stability after the introduction of tetragonal barium titanate nanoparticles and mNVs@Mn-HCeO2 / Que nanocomposite particles.

[0111] The above results demonstrate that the composite hydrogel prepared by this invention can meet the basic requirements of bone tissue engineering materials for gelation performance and structural stability.

[0112] 5. XPS characterization

[0113] The surface elemental composition of the ROS and piezoelectric dual-response hydrogel @mNVs@Mn-HCeO2 / Que composite system was analyzed by X-ray photoelectron spectroscopy. The results are as follows: Figure 13 As shown.

[0114] Depend on Figure 13 It can be seen that characteristic element peaks such as C, N, O, Ce, Mn, Ba, and Ti exist in the composite system. Among them, C, N, and O elements mainly originate from the HA-TK hydrogel matrix and biomimetic vesicle membrane components; Ce element originates from cerium dioxide nanoparticles; Mn element indicates successful manganese doping; Ba and Ti elements originate from tetragonal barium titanate nanoparticles.

[0115] XPS results further demonstrate that the HA-TK hydrogel matrix, tetragonal barium titanate piezoelectric nanoparticles, Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles, and biomimetic vesicles derived from bone marrow mesenchymal stem cells have been successfully integrated into the same composite hydrogel system.

[0116] In summary, this embodiment demonstrates, through characterization techniques such as SEM, EDS, TEM, UV-Vis, rheology, and XPS, that the present invention successfully constructed a ROS-piezoelectric dual-response biomimetic vesicle-loaded Mn-doped hollow mesoporous cerium dioxide-quercetin composite hydrogel with a three-dimensional porous structure, multi-component composite characteristics, stable gel properties, and a biomimetic core-shell nanostructure.

[0117] Example 3: Evaluation of drug loading and in vitro release performance

[0118] In this embodiment, the Mn-HCeO2 / Que nanocomposite particles prepared in Example 1 were used as the test object to evaluate their quercetin loading performance and in vitro release performance, so as to verify the feasibility of Mn-doped hollow mesoporous cerium dioxide nanoparticles as quercetin delivery carriers, as well as the sustained release and ROS-responsive release characteristics of the nanocomposite system.

[0119] 1. Establishment of the quercetin standard curve

[0120] To determine the loading capacity of Mn-HCeO2 nanoparticles for quercetin, a standard curve for quercetin was established using ultraviolet-visible spectrophotometry.

[0121] Accurately weigh a certain amount of quercetin standard and prepare quercetin standard solutions of different concentration gradients using ethanol. Measure the absorbance of each concentration standard solution at the characteristic absorption wavelength of quercetin, and plot a standard curve with quercetin concentration on the x-axis and absorbance value on the y-axis.

[0122] like Figure 10-1 As shown, within the test concentration range, there is a good linear relationship between quercetin concentration and absorbance, indicating that the established standard curve can be used for subsequent quantitative determination of quercetin loading, encapsulation efficiency and release.

[0123] 2. Determination of drug loading and encapsulation efficiency

[0124] A certain amount of Mn-HCeO2 nanoparticles were weighed and dispersed in ultrapure water, and then subjected to ultrasonic treatment to form a uniform suspension. Subsequently, a pre-prepared quercetin solution was added to the suspension, and the mixture was stirred overnight at room temperature in the dark to allow quercetin to fully enter the hollow mesoporous structure of the Mn-HCeO2 nanoparticles and adsorb onto their pores and / or surface.

[0125] After the reaction was complete, the system was centrifuged at 8000 rpm for 10 min, and the supernatant was collected. The concentration of unloaded quercetin in the supernatant was determined by UV-Vis spectrophotometry, and based on... Figure 10-1 The standard curve shown is used to calculate the mass of unloaded quercetin, thus obtaining the mass of quercetin loaded in the material.

[0126] Drug loading rate is calculated using the following formula: DLC (%) = Drug mass in loaded material / Total mass of drug-loaded material × 100%.

[0127] Drug encapsulation efficiency is calculated using the following formula: EE (%) = Mass of drug in the loaded material / Mass of drug initially added × 100%.

[0128] like Figure 10-2As shown, the drug loading rate of Mn-HCeO2 / Que nanocomposite particles was 18.7%, and the encapsulation efficiency was 74.8%. These results indicate that Mn-HCeO2 nanoparticles possess high quercetin loading capacity, and their hollow mesoporous structure provides a large drug-containing space, which is beneficial for improving the delivery efficiency and local utilization of quercetin.

[0129] 3. In vitro drug release experiment

[0130] The in vitro drug release performance of Mn-HCeO2 / Que nanocomposite particles was evaluated using dialysis. A certain amount of Mn-HCeO2 / Que nanocomposite particles were placed in a dialysis bag, and the dialysis bag was placed in a release medium for in vitro drug release experiments.

[0131] The experiment included both a standard PBS buffer environment and a simulated oxidative stress environment to compare the release behavior of quercetin under different microenvironmental conditions. At predetermined time points, a certain volume of the release solution was collected, and an equal volume of fresh release medium was added simultaneously to maintain a constant total volume of the release system.

[0132] The concentration of quercetin in the release solution at each time point was determined by ultraviolet-visible spectrophotometry, and based on... Figure 10-1 The standard curve shown is used to calculate the release amount and cumulative release rate of quercetin at each time point, and to plot the in vitro drug release curve.

[0133] like Figure 10 and Figure 10-2 As shown, the Mn-HCeO2 / Que nanocomposite particles exhibit a relatively obvious sustained-release characteristic. In the initial stage of release, no obvious burst release phenomenon was observed; as the release time increased, quercetin was gradually and continuously released, indicating that the hollow mesoporous structure of Mn-HCeO2 can play a role in stabilizing the loading and continuous release of quercetin.

[0134] Furthermore, under simulated oxidative stress, the quercetin release rate was higher than that under normal PBS conditions, indicating that the nanocomposite system can respond to ROS stimulation and achieve accelerated drug release. This phenomenon may be related to the oxidative stress environment promoting changes in the material's microenvironment and the responsive degradation of the hydrogel, thereby accelerating the outward diffusion of quercetin from the mesoporous structure.

[0135] In summary, the results of this embodiment demonstrate that Mn-HCeO2 / Que nanocomposite particles possess high drug loading capacity and excellent sustained-release properties, and can achieve ROS-responsive release under simulated oxidative stress conditions. This property facilitates the sustained release of quercetin from the composite hydrogel in the high-ROS microenvironment of bone injury, thereby exerting antioxidant, anti-inflammatory, and bone-repair-promoting effects.

[0136] Example 4: Biocompatibility Evaluation

[0137] In this embodiment, bone marrow mesenchymal stem cells were used as the evaluation object. The CCK-8 assay was used to detect the effect of mNVs@Mn-HCeO2 / Que nanocomposite particles on the viability and proliferation capacity of bone marrow mesenchymal stem cells, so as to evaluate the cell compatibility and biosafety of the composite system of the present invention.

[0138] 1. BMSCs culture and treatment

[0139] Log-phase bone marrow mesenchymal stem cells were collected and prepared into a concentration of 1×10⁻⁶ after trypsin digestion. 5 Cell suspension of cells / mL.

[0140] According to 1×10 per hole 4 To achieve a seeding density of 100 μL of cells, the cell suspension was seeded into each well of a 96-well cell culture plate. The plates were then incubated at 37 °C in a 5% CO2 incubator for 24 h to allow the cells to adhere fully.

[0141] After the cells adhered, the culture medium was replaced with serum-free medium containing 1% BSA, and the cells were cultured for another 12 hours to synchronize them.

[0142] The experiment was divided into the following two groups: (1) Control group: BMSCs; (2) Experimental group: BMSCs+mNVs@Mn-HCeO2 / Que.

[0143] A certain concentration of mNVs@Mn-HCeO2 / Que nanocomposite particles was added to the experimental group, while an equal volume of culture medium was added to the control group. Cells were then cultured for 0, 1, 3, and 7 days for subsequent cell viability assays.

[0144] 2. CCK-8 cell viability assay

[0145] After the predetermined incubation time is reached, add 10 μL of CCK-8 working solution to each well and place the culture plate in a 37 ℃ incubator for another 2 h.

[0146] After incubation, the absorbance of each well was measured at 450 nm using a microplate reader. Cell proliferation rate was calculated based on the measured OD values ​​using the following formula: Cell proliferation rate (%) = (OD value of experimental group - OD value of blank group) ÷ (OD value of control group - OD value of blank group) × 100%.

[0147] The effect of mNVs@Mn-HCeO2 / Que nanocomposite particles on the viability and proliferation of BMSCs was evaluated by comparing the cell proliferation rates of the control group and the experimental group under different culture times.

[0148] 3. Results Analysis

[0149] CCK-8 assay results showed that bone marrow mesenchymal stem cells treated with mNVs@Mn-HCeO2 / Que nanocomposite particles maintained high cell viability at all culture time points, and no obvious cytotoxicity was observed.

[0150] The cell proliferation rate results at different culture times are shown in the table below:

[0151] As shown in the table above, the cell activity of the experimental group and the control group was basically similar in the early stage of culture, namely 0 days and 1 day, indicating that the mNVs@Mn-HCeO2 / Que nanocomposite particles did not produce significant toxic side effects on bone marrow mesenchymal stem cells.

[0152] With prolonged culture time, the cell proliferation rate in the experimental group was significantly higher than that in the control group. Specifically, after 3 days of culture, the cell proliferation rate in the experimental group was 191.0%, higher than the 159.3% in the control group; after 7 days of culture, the cell proliferation rate in the experimental group was 234.7%, higher than the 200.3% in the control group.

[0153] The above results indicate that the mNVs@Mn-HCeO2 / Que nanocomposite particles constructed in this invention have good cell compatibility, no significant cytotoxicity to bone marrow mesenchymal stem cells, and can promote the proliferation of bone marrow mesenchymal stem cells.

[0154] Analysis suggests that the biomimetic vesicle-encapsulated structure derived from bone marrow mesenchymal stem cells can enhance the interaction between nanocomposite particles and cells. Mn-HCeO2 nanoparticles have antioxidant regulatory capabilities, and quercetin has antioxidant, anti-inflammatory, and osteogenic-related biological activities. These factors work together to improve the cell growth microenvironment, thereby promoting cell proliferation.

[0155] Therefore, the ROS-piezoelectric dual-response biomimetic vesicle-loaded Mn-doped hollow mesoporous cerium dioxide-quercetin composite hydrogel of the present invention has good biocompatibility and cell compatibility, and is suitable as a bone tissue engineering scaffold material and drug delivery material for bone defect repair.

[0156] VI. Comprehensive Analysis of Results from Examples

[0157] Combined with the experimental results and figures from Examples 1 to 4 ( Figures 1 to 13 The technical performance of the ROS and piezoelectric dual-response biomimetic vesicle-loaded Mn-doped hollow mesoporous cerium dioxide-quercetin composite hydrogel constructed in this invention can be comprehensively evaluated as follows: 1. Hydrogel network structure and multi-component composite characteristics SEM results ( Figure 1 , Figure 3 The results showed that both the HA-TK hydrogel and the composite hydrogel exhibited a continuous and stable three-dimensional porous network structure, providing a spatial basis for cell adhesion, nutrient exchange and functional component loading.

[0158] EDS analysis ( Figure 2 , Figure 4 It has been confirmed that tetragonal barium titanate nanoparticles and Mn-HCeO2 / Que nanocomposite particles have been successfully introduced into the hydrogel system to achieve multi-component composite.

[0159] 2. Morphology and functional properties of nanoparticles

[0160] TEM results ( Figures 5 to 9 )show: Tetragonal barium titanate nanoparticles are regularly shaped and can provide piezoelectric properties; Mn-HCeO2 nanoparticles exhibit a distinct hollow mesoporous structure, and Mn element has been successfully doped. Quercetin retains its hollow mesoporous structure after loading, proving that the drug loading process does not damage the carrier structure; Biomimetic vesicles were successfully coated onto the surface of Mn-HCeO2 / Que nanocomposite particles, forming a core-shell structure that enhances the biocompatibility and stability of the material.

[0161] 3. Drug loading and release performance

[0162] The drug loading rate reached 18.7%, and the encapsulation efficiency was 74.8%, demonstrating that the Mn-HCeO2 carrier has a high loading capacity. Figure 10-2 ).

[0163] In vitro release experiments showed that the composite particles were released slowly under normal PBS conditions, while the release rate was significantly accelerated under simulated oxidative stress conditions, achieving ROS-responsive drug release. Figure 10 , Figure 10-2 ).

[0164] This demonstrates that the material can achieve continuous and responsive drug release in the high ROS microenvironment of bone injury, thereby improving treatment efficacy.

[0165] 4. Physicochemical properties

[0166] Ultraviolet-visible absorption spectrum ( Figure 11 The absorption characteristics of quercetin and the nanocomposite system were confirmed, indicating that the drug loading was successful.

[0167] Rheological testing ( Figure 12 The results show that the composite hydrogel has a high storage modulus G', and G' is greater than the loss modulus G'', indicating that the hydrogel has good gelation properties and structural stability.

[0168] XPS analysis ( Figure 13 The results showed characteristic peaks of C, N, O, Ce, Mn, Ba and Ti, further verifying the successful composite of HA-TK hydrogel, piezoelectric nanoparticles, Mn-HCeO2 / Que and biomimetic vesicles.

[0169] 5. Biocompatibility and cell proliferation promotion

[0170] The CCK-8 experiment results showed that the mNVs@Mn-HCeO2 / Que nanocomposite particles had no obvious toxicity to BMSCs, and the cell proliferation rate of the experimental group reached 234.7% after 7 days of culture, which was significantly higher than that of the control group (200.3%), demonstrating that the material has good biocompatibility and promotes cell proliferation.

[0171] 6. Overall technical effects

[0172] The composite hydrogel of this invention achieves a multi-mechanism synergistic effect of ROS-responsive release, piezoelectric stimulation, antioxidant regulation, drug sustained release, and biomimetic delivery.

[0173] The components complement each other: HA-TK hydrogel provides microenvironment responsive support; tetragonal barium titanate nanoparticles provide piezoelectric signals; Mn-HCeO2 provides antioxidant capacity and loads quercetin; and biomimetic vesicles improve biocompatibility and local stability.

[0174] This composite hydrogel can improve the bone injury microenvironment, promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, and provide an effective functional material platform for bone tissue repair and bone defect treatment.

[0175] Conclusion: Through comprehensive analysis of various structural, physicochemical, drug loading, and biological experimental results, this invention successfully constructed a multifunctional composite hydrogel system, achieving synergistic functions of ROS response, piezoelectric stimulation, antioxidant regulation, drug sustained release, and biomimetic delivery, providing an efficient and controllable bone repair material solution for bone tissue engineering and regenerative medicine.

[0176] In summary, the ROS- and piezoelectric dual-responsive biomimetic vesicle-loaded Mn-doped hollow mesoporous cerium dioxide-quercetin composite hydrogel provided by this invention achieves a multi-mechanism synergistic effect through the organic combination of HA-TK hydrogel network, tetragonal barium titanate nanoparticles, Mn-doped hollow mesoporous cerium dioxide carrier, quercetin, and biomimetic vesicles derived from bone marrow mesenchymal stem cells. This composite hydrogel simultaneously possesses ROS-responsive release, piezoelectric stimulation, antioxidant regulation, drug sustained release, and biomimetic delivery functions, thereby effectively improving the bone injury microenvironment, promoting the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, and providing a highly efficient functionalized material solution for bone tissue repair and bone defect treatment. This invention is applicable to bone tissue engineering scaffolds, drug delivery systems, and microenvironment regulation materials, and has significant innovation and practical value.

[0177] The embodiments disclosed in this specification are only used to explain the principles and applications of the present invention and should not be considered as limiting the scope of protection of the present invention. Those skilled in the art can make various equivalent transformations or modifications without departing from the spirit and scope of the present invention. The scope of protection of the present invention should be determined by the appended claims and their equivalent technical features.

Claims

1. A biomimetic vesicle-supported Mn-doped hollow mesoporous cerium dioxide-quercetin composite hydrogel with both ROS and piezoelectric response, characterized in that... It includes a ROS-responsive hydrogel matrix, piezoelectric nanoparticles dispersed in the ROS-responsive hydrogel matrix, and biomimetic vesicle-loaded nanocomposite particles. The ROS-responsive hydrogel matrix is ​​a hyaluronic acid-thioacetate hydrogel. The piezoelectric nanoparticles are tetragonal barium titanate nanoparticles. The biomimetic vesicle-loaded nanocomposite particles include biomimetic vesicles derived from bone marrow mesenchymal stem cells and Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles coated by the biomimetic vesicles derived from bone marrow mesenchymal stem cells.

2. The composite hydrogel according to claim 1, characterized in that, The hyaluronic acid-thioketal hydrogel is formed by the reaction of hyaluronic acid with cross-linked or grafted components containing thioketal bonds. The thioketal bonds can be broken in an active oxygen environment, giving the composite hydrogel ROS-responsive degradation properties.

3. The composite hydrogel according to claim 1, characterized in that, The tetragonal barium titanate nanoparticles are used to generate a piezoelectric response under external mechanical stimulation, ultrasonic stimulation, or physiological micro-stress to form a local electrical signal.

4. The composite hydrogel according to claim 1, characterized in that, The Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles comprise a Mn-doped hollow mesoporous cerium dioxide support and quercetin loaded onto the pores and / or surface of the Mn-doped hollow mesoporous cerium dioxide support.

5. The composite hydrogel according to claim 4, characterized in that, The Mn-doped hollow mesoporous cerium dioxide support has a hollow mesoporous structure and Ce³⁺. + / Ce 4+ It has reversible redox activity and is used to scavenge reactive oxygen free radicals.

6. The composite hydrogel according to claim 1, characterized in that, The biomimetic vesicles derived from bone marrow mesenchymal stem cells are prepared by breaking, centrifuging and purifying bone marrow mesenchymal stem cell membranes and extruding them, and are coated on the surface of the Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles to form a core-shell structure.

7. The composite hydrogel according to claim 1, characterized in that, The biomimetic vesicle-loaded nanocomposite particles have a particle size of 100–250 nm, preferably 130–180 nm; the quercetin loading rate in the composite hydrogel is 10%–30%, and the encapsulation rate is 50%–90%.

8. A method for preparing the composite hydrogel according to any one of claims 1 to 7, characterized in that, Includes the following steps: S1. Preparation of hyaluronic acid-thioacetate hydrogel precursor; S2. Preparation of tetragonal barium titanate nanoparticles; S3. Prepare Mn-doped hollow mesoporous cerium dioxide nanoparticles, and load quercetin into the Mn-doped hollow mesoporous cerium dioxide nanoparticles to obtain Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles. S4. Prepare biomimetic vesicles from bone marrow mesenchymal stem cells, and use the biomimetic vesicles to coat the Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles to obtain biomimetic vesicle-loaded nanocomposite particles. S5. The biomimetic vesicle-loaded nanocomposite particles, hyaluronic acid-thioacetate hydrogel precursor, tetragonal barium titanate nanoparticles and photoinitiator are mixed and photocured to obtain the composite hydrogel.

9. The preparation method according to claim 8, characterized in that, In step S3, the Mn-doped hollow mesoporous cerium dioxide nanoparticles are prepared by the following method: After hydrothermal reaction of cerium salt, polyvinylpyrrolidone, glacial acetic acid and ethylene glycol, manganese salt was added to continue the reaction. After washing, drying and calcination, Mn-doped hollow mesoporous cerium dioxide nanoparticles were obtained. Subsequently, the Mn-doped hollow mesoporous cerium dioxide nanoparticles were dispersed in an aqueous system, mixed with a quercetin solution, and stirred to load quercetin into the Mn-doped hollow mesoporous cerium dioxide nanoparticles.

10. The preparation method according to claim 8, characterized in that, In step S4, the biomimetic vesicles are prepared by the following method: Bone marrow mesenchymal stem cells were collected, washed, lysed or sonicated, centrifuged to remove cell nuclei and unbroken cells, and then subjected to ultracentrifugation to obtain cell membrane components. The cell membrane component was mixed with Mn-doped hollow mesoporous cerium dioxide-quercetin nanocomposite particles and extruded together through a polycarbonate membrane to obtain biomimetic vesicle-loaded nanocomposite particles.

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