A functional hydrogel material for repairing trilayer osteochondral by promoting chondrogenesis and osteogenesis through traditional Chinese medicine and physical stimulation respectively and a preparation method thereof

Abstract

This invention discloses a three-layered functional hydrogel material for osteochondral repair that promotes chondrogenesis through traditional Chinese medicine and physical stimulation, and its preparation method. Specifically, the method includes: 1) preparing a PCL electrospun membrane using electrospinning technology, and forming an HA coating on its surface using a hydrothermal method with biomimetic mineralization; 2) constructing the HA coating on the surface of black phosphorus sheets and preparing cryptotanshinone nanopowder using in-situ mineralization and spraying methods, and mixing it with BMSCs in a porous GelMA solution; 3) preparing MCSN nanoparticles loaded with baicalein using a hydrothermal template method and ultrasonic impregnation process; 4) dispersing barium titanate nanowires, drug-loaded MCSN, and BMSCs in a SilMA / HAMA solution; 5) pouring the GelMA mixture into a mold and pre-curing it, placing a plasma-treated mineralized electrospun membrane on top, then pouring the SilMA / HAMA mixture and completely curing it to obtain the desired hydrogel. This composite material has a three-layer structure, matching the needs of osteochondral repair.

Description

Technical Field

[0001] This invention relates to the field of osteochondral repair gel preparation technology, specifically to a three-layer osteochondral repair functional hydrogel material that promotes cartilage-osteogenesis through traditional Chinese medicine and physical stimulation, and its preparation method. Background Technology

[0002] Osteocartilage defects are a common orthopedic condition, often caused by trauma, osteoarthritis, or tumor resection. Because articular cartilage lacks blood vessels and nerves, and chondrocytes have limited proliferative capacity, it is almost impossible for it to repair itself once damaged. Natural osteocartilage tissue has a complex layered gradient structure, consisting of a cartilage layer, a calcified cartilage layer, and a subchondral bone layer, from superficial to deep. Each layer differs significantly in composition and function. The calcified cartilage layer, acting as an interface unit, plays a crucial role in mechanical buffering and isolating different microenvironments. Therefore, an ideal osteocartilage repair strategy should mimic this layered structure to achieve synchronous regeneration of cartilage and bone tissue.

[0003] Current clinical treatments mainly include microfracture surgery and osteochondral transplantation, but both have significant drawbacks: microfracture surgery produces fibrocartilage with poor mechanical properties; osteochondral transplantation has limited donor sites, carries the risk of immune rejection, and makes long-term stable repair difficult. Tissue engineering has developed various osteochondral repair scaffolds, but existing technologies still suffer from the following shortcomings: monophasic or bilayer scaffolds often neglect the isolating function of the calcification layer, leading to interference between the microenvironments of the upper and lower layers; insufficient interfacial bonding strength, making interlayer separation easy; lack of effective physical stimulus response; difficulty in achieving controlled drug release in specific zones; and complex preparation processes with poor reproducibility.

[0004] In osteochondral tissue engineering research, achieving targeted differentiation induction of the cartilage and bone layers has always been a core challenge. Studies have shown that piezoelectric materials can generate electrical signals under physiological stress, promoting stem cell differentiation through electromechanical transduction; photothermal effects can regulate cell behavior through gentle temperature increases; and active ingredients in traditional Chinese medicine, such as cryptotanshinone and baicalin, have anti-inflammatory and cartilage / osteogenic effects. However, current technologies have not yet organically integrated the above physical stimuli with the activity of traditional Chinese medicine, nor have they been able to apply them in sections to different layers for precise regulation: cartilage repair requires the synergy of piezoelectric / photothermal stimulation and cartilage-promoting traditional Chinese medicine, while bone repair requires the synergy of piezoelectric / photothermal stimulation and bone-promoting traditional Chinese medicine, and the two types of stimulation need to be spatially isolated to avoid mutual interference. If simply mixed, it may lead to the cartilage layer mistakenly receiving osteogenic signals and the bone layer mistakenly receiving cartilage-promoting signals, ultimately resulting in repair failure.

[0005] Therefore, developing a functional repair material that can simulate the three-layer structure of cartilage layer-calcification layer-subchondral bone layer, achieve precise regional regulation of physical stimulation and active ingredients of traditional Chinese medicine, and avoid crosstalk between interlayer signals has become a key technical problem that urgently needs to be solved in the field of osteochondral tissue engineering. Summary of the Invention

[0006] The purpose of this invention is to address the problem of poor repair effects of traditional two-layer osteochondral repair by providing a functional hydrogel material for three-layer osteochondral repair that promotes cartilage-osteogenesis through traditional Chinese medicine and physical stimulation, and its preparation method.

[0007] This invention is achieved using the following technical solution: A method for preparing a three-layered osteochondral repair functional hydrogel material that promotes cartilage-osteogenesis through traditional Chinese medicine and physical stimulation, comprising the following steps: 1) PCL (polycaprolactone) electrospun membranes were prepared using electrospinning technology, and HA (hydroxyapatite) coatings were formed on the surface of the PCL electrospun membranes by biomimetic mineralization using a hydrothermal method to obtain a mid-layer mineralized electrospun membrane. 2) An HA coating was constructed on the surface of black phosphorus sheets using in-situ mineralization, and cryptotanshinone nanopowder was prepared by spraying. The HA-modified black phosphorus sheets, cryptotanshinone nanopowder and BMSCs (bone marrow mesenchymal stem cells) were mixed in a porous GelMA (methacrylamide gelatin) solution to obtain the lower layer casting solution. 3) MCSN (mesoporous calcium silicate) nanoparticles were prepared by hydrothermal template method and baicalin was loaded by ultrasonic impregnation process to obtain drug-loaded MCSN nanoparticles. 4) Barium titanate nanowires, the drug-loaded MCSN nanoparticles and BMSCs are dispersed in a solution containing SilMA (methacrylamide silk fibroin) and HAMA (methacrylamide hyaluronic acid) to obtain the upper casting solution. 5) The lower layer casting solution is poured into the mold and pre-cured to form a lower layer gel. A plasma-treated middle layer mineralized electrospun membrane is placed on top of the lower layer gel. The upper layer casting solution is then poured in and completely cured to obtain a three-layer functional hydrogel material for osteochondral repair.

[0008] Further, in step 1), the preparation process of the PCL electrospun membrane includes: preparing a PCL electrospun solution with a mass fraction of 6-11 wt%, and preparing a nanofiber membrane by electrospinning under the conditions of a spinning voltage of 15-30 kV and a receiving distance of 15-25 cm; the biomimetic mineralization process includes: placing the PCL electrospun membrane in a biomimetic mineralization solution system and hydrothermally treating it at 60-80℃ for 48-120 h to allow HA crystals to grow in situ on the fiber surface.

[0009] Furthermore, in step 2), the construction of the HA coating on the surface of the black phosphorus sheet specifically includes: using black phosphorus with a diameter of 200-500 nm, a thickness of 1-10 nm, and 2-10 layers as raw material, dispersing it at a concentration of 0.1-0.5 mg / mL in a biomimetic mineralization solution system with a pH of 7.4, and mineralizing it in a water bath at 37-45℃ for 48-72 h; The preparation process of the cryptotanshinone nanopowder includes: dissolving cryptotanshinone raw material and functional adjuvants together in anhydrous ethanol to prepare a solution with a total solid content of 1-5 mg / mL; removing the solvent by spray drying technology to obtain cryptotanshinone nanopowder with a particle size of 100-300 nm; wherein the mass ratio of cryptotanshinone to adjuvants is 5:1 to 10:1; the functional adjuvants include 0.5-1.0 wt% polyvinylpyrrolidone (PVP, film-forming aid), 0.2-0.5 wt% soybean lecithin (surfactant), and 0.1-0.3 wt% trehalose (lyophilization protectant); the spray drying process parameters are: feed concentration 1-5 mg / mL, inlet temperature 80-100°C, outlet temperature 40-50°C, atomization pressure 0.5-0.7 MPa, and feed rate 2-5 mL / min; The preparation process of the lower layer casting solution includes: under sterile, light-protected conditions, mixing 7-12 wt% porous GelMA solution, 0.05-0.25 wt% LAP photoinitiator, 0.1-0.5 wt% HA-modified black phosphorus sheets, and 0.01-0.05 wt% cryptotanshinone nanoparticles in a water bath at 50-65℃ to form a mixed solution; after the solution cools to 35-37℃, adding P3-P5 generation BMSCs suspension until the final cell density is 5-10 × 10⁻⁶ cells / year. 6 The mixture was prepared by centrifugation to remove bubbles after mixing the cells / mL of the solution to obtain the lower layer casting solution.

[0010] Furthermore, the formulation of the biomimetic mineralization solution system is as follows: 0.80 wt% NaCl, 0.035 wt% NaHCO3, 0.022 wt% KCl, 0.023 wt% K2HPO4·3H2O, 0.030 wt% MgCl2·6H2O, 0.028 wt% CaCl2, 0.007 wt% Na2SO4, 0.01 wt% gelatin, 0.005 wt% sodium alginate, 0.005 wt% collagen peptides, and 0.001 wt% silk fibroin, with the remainder being deionized water.

[0011] Further, in step 3), the preparation of MCSN nanoparticles using the hydrothermal template method includes: dissolving 1.0-2.0 wt% CTAB (hexadecyltrimethylammonium bromide) template agent in a mixed solvent of deionized water and anhydrous ethanol at 25-35°C and under magnetic stirring, wherein the volume ratio of deionized water to anhydrous ethanol is 3:1; adding 0.25-1.5 wt% calcium nitrate tetrahydrate, 0.9-2.0 wt% tetraethyl orthosilicate, 0.05-0.25 wt% triethanolamine, and 0.01-0.075 wt% polyethylene glycol; and using 20-28... Adjust the pH of the mixed solution to 10.5-11.5 with wt% ammonia. Continue stirring at 35-45°C for 1-3 hours to allow the silicon and calcium sources to fully pre-hydrolyze and self-assemble into an ordered mesoscopic structure. Transfer the mixture to a high-pressure reactor with a filling density of 60-80%. Heat to 100-200°C at a heating rate of 1-5°C / min and maintain the temperature for 12-36 hours. After the reaction, allow it to cool naturally to room temperature. Collect the precipitate by centrifugation at 8000-13000 rpm for 5-15 minutes. Wash the precipitate 3-6 times with deionized water and anhydrous ethanol to remove residual solvent and unreacted substances. Dry the washed product under vacuum at 50-70°C for 8-12 hours, then heat to 500-650°C at a heating rate of 1-5°C / min and calcine for 4-8 hours to obtain mesopores with a pore size of 3-6 nm and a specific surface area of ​​400-600 m². 2 / g, MCSN nanoparticles with a particle size of 80-200nm; The ultrasonic impregnation process for loading baicalein specifically includes: dissolving 0.1-0.5 wt% baicalein in anhydrous ethanol or a mixed solvent of anhydrous ethanol and dimethyl sulfoxide in a volume ratio of 9:1 to 13:1, and ultrasonicating for 2-5 minutes to aid dissolution; adding MCSN nanoparticles at a baicalein to MCSN nanoparticle mass ratio of 1:1 to 1:4, and performing ultrasonic impregnation under light-protected conditions, with an ultrasonic power of 150-250 W, a frequency of 35-45 kHz, a time of 20-40 min, and a temperature controlled at 15-20℃; centrifuging after ultrasonication (centrifugation speed of 500-750 rpm, time of 3-5 min), washing, and freeze-drying for 6-12 h, repeating 2-3 times to obtain the drug-loaded MCSN nanoparticles.

[0012] Further, in step 4), the preparation process of the upper casting solution includes: preparing a prepolymer solution containing SilMA, HAMA, and LAP photoinitiators in a water bath at 50-70℃ under sterile, light-protected conditions; cooling to 30-37℃; dispersing barium titanate nanowires with a diameter of 50-200 nm and a length of 2-10 μm, drug-loaded MCSN nanoparticles, and P3-P5 generation BMSCs in the prepolymer solution; mixing thoroughly; and centrifuging to remove bubbles to obtain the upper casting solution; in the upper casting solution, the concentration of SilMA is 4-7.5 wt%, the concentration of HAMA is 4-7.5 wt%, the concentration of LAP is 0.1-0.25 wt%, and the density of BMSCs is 2 × 10⁻⁶. 6 Up to 1×10 7 The concentration of barium titanate nanowires was 0.05-0.5 wt%, and the concentration of drug-loaded MCSN nanoparticles was 0.02-0.1 wt%.

[0013] Furthermore, in step 5), the pouring height of both the lower layer pouring solution and the upper layer pouring solution is 1-3mm; The pre-curing conditions are: 405 nm, light intensity 5-15 mW / cm². 2 Irradiate under ultraviolet light for 5-15 seconds; the complete curing conditions are: 405 nm, light intensity 5-10 mW / cm². 2 Expose to ultraviolet light for 20-40 seconds.

[0014] Furthermore, in step 5), the intermediate mineralized electrospun membrane is treated with oxygen plasma at a power of 50-100 W for 30-60 seconds and an oxygen flow rate of 20-50 sccm. Its diameter or side length is 2-5 mm smaller than the cross-section of the mold. It is placed in the central area above the lower gel, with a 1-2.5 mm wide annular gap reserved around it. The upper casting solution is poured into the annular gap and above the intermediate mineralized electrospun membrane. After solidification, a composite hydrogel structure is formed in which the intermediate mineralized electrospun membrane is embedded in the interior and the edges are directly connected by the upper and lower gels.

[0015] A three-layered functional hydrogel material for osteochondral repair that promotes cartilage-osteogenesis through traditional Chinese medicine and physical stimulation is prepared by the above-mentioned method. The three-layered functional hydrogel material for osteochondral repair consists of an upper layer that protects against piezoelectricity and promotes cartilage formation through traditional Chinese medicine, a middle layer that mimics calcified cartilage layer with mineralized electrospun film, and a lower layer that has photothermal and traditional Chinese medicine-induced osteogenic functions.

[0016] Furthermore, the three-layer osteochondral repair functional hydrogel material can induce osteogenic formation through photothermal stimulation in conjunction with the active ingredients of traditional Chinese medicine; the photothermal stimulation uses an 808 nm near-infrared laser as the light source, with an irradiation power density of 0.5-1.5 W / cm².2 The irradiation time is 5-15 minutes per session. The irradiation parameters are adjusted to maintain the internal temperature of the material at 40-43°C. The irradiation frequency is once a day or once every two days, for 1-2 weeks.

[0017] The core design of the three-layer osteochondral repair functional hydrogel material of this invention lies in: The upper cartilage-inducing layer is loaded with bone marrow mesenchymal stem cells, barium titanate nanowires, and MCSNs loaded with baicalin. The barium titanate nanowires possess both piezoelectric and photothermal effects, generating electrical signals and mildly increasing temperature under physiological stress or near-infrared light irradiation. This synergistic effect with the chondrogenic effect of baicalin achieves highly efficient directed cartilage differentiation. The necessity of this layer design lies in the fact that simple chemical induction cannot simulate the mechanical microenvironment of cartilage development, while simple physical stimulation lacks specific signaling molecules; only through the synergy of both can the natural cartilage development process be simulated to the greatest extent.

[0018] The lower osteogenic induction layer is loaded with bone marrow mesenchymal stem cells, HA-modified black phosphorus, and cryptotanshinone nanoparticles. The piezoelectric / photothermal effect of barium titanate synergizes with the osteogenic effect of cryptotanshinone, while the calcium and silicon ions generated from the degradation of calcium silicate also promote osteoogenesis, forming a triple synergistic osteogenic induction. The necessity of this layer design lies in the fact that bone tissue regeneration requires the combined action of mechanoelectric stimulation, the participation of bioactive ions, and specific osteogenic signaling molecules; a single approach is unlikely to achieve the desired effect.

[0019] The intermediate isolation layer employs a biomimetic mineralized polycaprolactone electrospun membrane to mimic the barrier function of the calcified cartilage layer. Its necessity lies in the fact that without this isolation layer, the cartilage-promoting signals (baicalin, piezoelectric signals) in the upper layer and the osteogenic signals (cryptoshinone, calcium-silicon ions, photothermal signals) in the lower layer would diffuse and interfere with each other, leading to disordered cell differentiation fate. Simultaneously, this layer achieves good binding with the upper and lower gel layers through HA mineralization, and its membrane size is smaller than the gel layer. After embedding, it is directly connected to the gel on all sides, significantly enhancing the interfacial bonding strength and preventing interlayer separation.

[0020] In summary, this invention is the first to apply piezoelectric / photothermal dual physical stimulation and the synergistic application of active ingredients from traditional Chinese medicine to osteochondral repair. Through a three-layer structure design, it achieves functional zoning, signal isolation, and interface enhancement, solving key problems in existing technologies such as the difficulty in synergistic effects of physical stimulation and chemical induction, interlayer signal interference, and insufficient interfacial bonding strength. The preparation process is simple and controllable, providing an innovative strategy with greater clinical translational potential for osteochondral defect repair.

[0021] Compared with the prior art, the present invention has the following advantages: 1) This invention is the first to organically integrate barium titanate nanowires, hydroxyapatite-modified black phosphorus sheets, cryptotanshinone nanoparticles, baicalin-loaded mesoporous calcium silicate nanoparticles, biomimetic mineralized polycaprolactone electrospun membranes, and bone marrow mesenchymal stem cells. Through a combination of multiple processes including electrospinning, hydrothermal mineralization, spray drying, hydrothermal template, ultrasonic impregnation, and stepwise photocrosslinking casting, a functional hydrogel material with a three-layer biomimetic structure for bone and cartilage repair is constructed. The selection of raw materials and the combination of processes mentioned above are original to this invention. Currently, there are no reports on the application of piezoelectric effects to promote cartilage growth, photothermal effects to promote bone growth, and the synergistic application of active ingredients from traditional Chinese medicine in bone and cartilage repair.

[0022] 2) The "upper-middle-lower" three-layer biomimetic composite hydrogel scaffold constructed in this invention simulates the hyaline cartilage layer, calcified cartilage layer, and subchondral bone layer of natural osteochondral tissue, respectively. The upper layer is a SilMA / HAMA hydrogel loaded with bone marrow mesenchymal stem cells, barium titanate nanowires, and cryptotanshinone nanoparticles, simulating the microenvironment of the cartilage layer; the middle layer is a biomimetic mineralized polycaprolactone electrospun membrane, the surface of which is coated with hydroxyapatite in situ using a hydrothermal method, simulating the dense structure, barrier function, and osteoconductive interface of the natural calcified cartilage layer; the lower layer is a porous GelMA hydrogel loaded with bone marrow mesenchymal stem cells, hydroxyapatite-modified black phosphorus sheets, and mesoporous calcium silicate nanoparticles loaded with baicalin, simulating the microenvironment of the subchondral bone layer. The three-layer structure is integrally formed through a stepwise photocrosslinking process. The electrospun membrane is smaller than the gel layer. After embedding, the edges are directly connected by the upper and lower gel layers to form an integral structure. This not only achieves precise biomimicry of the layered structure of natural osteochondral, but also significantly enhances the interfacial bonding strength and overall mechanical stability, solving the technical problem of interlayer separation that is prone to occur in existing multilayer scaffolds.

[0023] 3) This invention achieves precise regional regulation at the physical stimulation level: The upper layer introduces barium titanate nanowires, utilizing their piezoelectric effect to generate local electrical signals under the subtle mechanical stimulation of physiological activities, promoting the differentiation of bone marrow mesenchymal stem cells towards cartilage through electromechanical transduction; the lower layer introduces hydroxyapatite-modified black phosphorus sheets, utilizing their near-infrared photothermal response characteristics to achieve a gentle temperature rise of 40-43℃ with the assistance of an in vitro photothermal device, synergistically promoting the expression of osteogenic genes through the photothermal effect. This design applies the piezoelectric and photothermal effects to the cartilage and bone layers respectively, enabling physical stimulation to precisely target the repair area, overcoming the shortcomings of simple chemical induction in simulating the microenvironment of tissue developmental mechanics.

[0024] 4) This invention also achieves precise regional regulation at the level of active ingredients in traditional Chinese medicine: the upper layer is loaded with cryptotanshinone nanoparticles, utilizing their anti-inflammatory, antioxidant, and cartilage matrix-promoting pharmacological effects, forming a dual synergistic effect of "physical-traditional Chinese medicine" with the piezoelectric effect of barium titanate, to achieve efficient cartilage-directed differentiation; the lower layer is loaded with mesoporous calcium silicate nanoparticles loaded with baicalin, utilizing the anti-inflammatory, antioxidant, and bone-differentiation-promoting effects of baicalin, while the calcium and silicon ions generated by the degradation of mesoporous calcium silicate can also promote osteoogenesis, forming a triple synergistic effect of "physical-traditional Chinese medicine-ions" with the photothermal effect of black phosphorus flakes, to achieve efficient osteogenic-directed differentiation. This design organically combines the chemical induction and physical stimulation of active ingredients in traditional Chinese medicine, enabling the upper and lower layers to have differentiated synergistic mechanisms, solving the problem of limited cartilage / osteogenic effects of single methods in existing technologies.

[0025] 5) The design of the intermediate biomimetic mineralized polycaprolactone electrospun membrane in this invention features several innovations: First, the surface hydroxyapatite coating simulates the mineralized matrix components of the natural calcified cartilage layer, endowing it with excellent osteoconductive properties and biocompatibility. Second, the dense fiber network structure simulates the physical barrier function of the calcified cartilage layer, effectively isolating the upper cartilage-promoting signals (cryptotanshinone, piezoelectric signals) from the lower osteogenic signals (baicalin, calcium-silicon ions, photothermal signals), avoiding mutual diffusion interference that could lead to disordered cell differentiation fate. Third, the membrane size is smaller than that of the gel layer, allowing the upper and lower gel layers to connect directly at the edges, forming a "sandwich" integral structure that ensures both signal isolation and mechanical integration. This layer's dual biomimicry of the structure and function of the natural calcified cartilage layer solves the key problems of existing double-layer scaffolds neglecting the function of the calcified layer and interlayer signal interference.

[0026] 6) This invention constructs a functionally synergistic, precisely zoned, and structurally biomimetic osteochondral repair system through a holistic three-layer design: the upper layer achieves efficient cartilage differentiation promotion through piezoelectric effect and synergy with cryptotanshinone; the lower layer achieves efficient bone differentiation promotion through photothermal effect and synergy with baicalin and calcium-silicon ions; and the middle layer achieves signal isolation and interface enhancement through a biomimetic mineralized electrospun membrane. Each of the three layers performs its specific function while synergistically enhancing each other, integrating multiple functions such as physical stimulation, traditional Chinese medicine activity, bioactive ions, and biomimetic interfaces into a single scaffold. This enables precise temporal and spatial control of the fate of cartilage and bone cells, providing a more clinically translational innovative strategy for the functional repair of osteochondral defects.

[0027] 7) Innovative Photothermal Osteogenesis Promotion of Hydroxyapatite-Modified Black Phosphorus Sheets: This invention is the first to apply hydroxyapatite-modified black phosphorus sheets to the underlying osteochondral repair layer. Black phosphorus sheets possess excellent near-infrared photothermal response characteristics, enabling gentle heating to promote osteoogenesis with the assistance of an in vitro photothermal device. The in-situ grown hydroxyapatite coating on its surface enhances the biocompatibility and stability of black phosphorus, slowing its degradation rate. Furthermore, the osteoconductivity of hydroxyapatite itself synergizes with the photothermal effect of black phosphorus, jointly promoting osteogenic differentiation in the underlying layer. This design solves the problems of excessively rapid degradation and insufficient biocompatibility when black phosphorus is used alone.

[0028] 8) Innovative Piezoelectric Effect for Autonomous Cartilage Development: This invention introduces barium titanate nanowires into the upper layer, utilizing their piezoelectric effect to automatically generate local electrical signals under the minute mechanical stimulation produced by joint physiological activities. The innovation of this design lies in the fact that piezoelectric stimulation requires no external equipment assistance and can be achieved autonomously during the patient's daily activities, avoiding the inconvenience of repeated external interventions; simultaneously, the synergistic effect of the piezoelectric signal and the chemical induction of cryptotanshinone more closely resembles the mechanochemical coupling microenvironment during natural cartilage development, significantly improving cartilage differentiation efficiency.

[0029] 9) Stepwise Photocrosslinking Casting Process and Structural Integration Innovation: This invention employs a stepwise photocrosslinking casting process to achieve integrated molding of a three-layer structure. The specific innovation lies in: firstly, the lower layer of GelMA mixed solution is pre-cured for a short time to form the bottom gel, at which point the gel maintains a certain strength but its surface remains reactive; then, a mineralized electrospun membrane treated with oxygen plasma is bonded to the central region of the pre-cured layer. Plasma treatment introduces polar groups onto the surface of the electrospun membrane, significantly enhancing its interfacial bonding with the hydrophilic hydrogel; finally, the upper layer of SilMA / HAMA mixed solution is cast and completely cured, allowing the electrospun membrane to be firmly embedded within the two gel layers. This process ensures that the edges of the electrospun membrane are directly connected by the upper and lower gel layers to form an integral structure, guaranteeing both signal isolation functionality and significantly enhancing interfacial bonding strength.

[0030] 10) Innovation in the selection of polycaprolactone electrospun membrane as the intermediate isolation layer: This invention selects polycaprolactone electrospun membrane as the substrate for the intermediate isolation layer. Its innovation lies in the following: polycaprolactone possesses good biocompatibility and an adjustable degradation rate; its electrospun fiber membrane has high porosity and a dense network structure, effectively mimicking the physical barrier function of natural calcified cartilage layers; simultaneously, polycaprolactone has moderate mechanical properties, providing sufficient support without being overly rigid and damaging the upper and lower gel layers; its surface is easily coated with a hydroxyapatite layer in situ using a hydrothermal method, achieving biomimetic mineralization modification. This material selection addresses multiple requirements, including barrier function, mechanical matching, interfacial bonding, and biomimetic modification, representing a dual biomimetic approach to the structure and function of calcified cartilage layers.

[0031] 11) Innovative Dual Function of Isolation and Interface Reinforcement in Biomimetic Mineralized Electrospun Membranes: This invention modifies polycaprolactone electrospun membranes with hydroxyapatite biomimetic mineralization, giving them dual functions: Firstly, the hydroxyapatite coating imparts excellent osteoconductivity and biocompatibility to the electrospun membrane, making its interface bonding with the upper and lower hydrogel layers stronger; secondly, the dense fiber network structure of the electrospun membrane itself acts as a physical barrier, effectively isolating the cartilage-promoting signals in the upper layer from the osteogenic signals in the lower layer, preventing mutual diffusion and interference. Simultaneously, the membrane size is smaller than the gel layer, allowing the upper and lower gel layers to connect directly at the edges, forming a "sandwich" integral structure that ensures both signal isolation and mechanical integration. This design solves the key problems of existing double-layer scaffolds neglecting the function of the calcified layer and interlayer signal interference.

[0032] 12) Innovative Nanostructuring and Regional Application of Active Ingredients from Traditional Chinese Medicine: This invention is the first to apply two active ingredients from traditional Chinese medicine, cryptotanshinone and baicalein, to the upper and lower layers of osteochondral repair, respectively, and to achieve nanostructuring using spray drying and mesoporous calcium silicate loading technologies. Its innovation lies in the following: Cryptotanshinone nanopowder, through the synergistic effect of functional additives such as polyvinylpyrrolidone, soybean lecithin, and trehalose, yields nanomedicines with controllable particle size and good dispersibility, facilitating uniform dispersion and slow release in the upper hydrogel; baicalein achieves efficient loading and sustained release through the high specific surface area and ordered mesoporous structure of mesoporous calcium silicate. This design organically combines the chemical induction and physical stimulation of active ingredients from traditional Chinese medicine, enabling the upper and lower layers to possess differentiated synergistic mechanisms. Simultaneously, the nanostructuring technology significantly improves the bioavailability and sustained-release performance of the traditional Chinese medicine. Attached Figure Description

[0033] Figure 1 This is a schematic diagram illustrating the preparation process of a three-layer osteochondral repair material that promotes cartilage-osteogenesis through traditional Chinese medicine and physical stimulation, as described in an embodiment of the present invention.

[0034] Figure 2 The results of CCK-8 in the upper and lower gel layers of the present invention after 7 days of BMSCs culture. Examples 1 and 3 of the present invention are shown.

[0035] Figure 3 The image shows fluorescence images of BMSCs within the composite gel (corresponding to Example 1).

[0036] Figure 4 PCR results for chondrogenic and osteogenic differentiation of cells (corresponding to Example 1 and Example 1).

[0037] Figure 5 The results of Col II immunofluorescence staining of BMSCs are shown (a corresponds to Example 3, b corresponds to Example 1). Detailed Implementation

[0038] The present invention will now be described in further clarity and detail with reference to the accompanying drawings and specific examples.

[0039] Comparative Example 1 1) Preparation of the lower layer (promoting osteogenic functional units): Preparation of the lower layer casting solution: Under sterile, light-protected conditions and a 60°C water bath, a mixed solution was prepared using 10 wt% porous GelMA solution and 0.15 wt% LAP photoinitiator. After the solution cooled to 37°C, BMSCs (P4 generation) suspension was added until the final cell density was 8 × 10⁻⁶ cells / mL. 6 cells / mL. Gently pipette 30 times to disperse evenly, then centrifuge at 600 rpm for 4 min to remove bubbles, and store at 4℃ for later use.

[0040] 3) Preparation of the upper layer (facilitating chondrogenic functional units): Preparation of the upper layer casting solution: Under sterile, light-protected conditions and in a 60°C water bath, prepare a prepolymer solution containing 6 wt% SilMA, 6 wt% HAMA, and 0.15 wt% photoinitiator LAP. After the solution cools to 35°C, add a suspension of BMSCs (P4 generation) until the final cell density is 6 × 10⁻⁶ cells / mL. 6 cells / mL. Gently pipette to mix thoroughly, then centrifuge at 600 rpm for 4 min to remove bubbles, and store at 4℃ for later use.

[0041] 4) Casting and molding of the double-layer composite hydrogel: a. Pour the lower layer casting solution into the mold to a height of 2 mm, and place it under light at 405 nm and an intensity of 10 mW / cm². 2 a. Pre-curing is performed by irradiating the membrane under ultraviolet light for 10 seconds to form a bottom layer gel with appropriate strength. b. The upper layer casting solution is poured into the annular gap above and around the electrospun membrane to a height of 2 mm, and then placed under a light source at 405 nm and an intensity of 8 mW / cm². 2 The material was completely cured by irradiation with ultraviolet light for 30 seconds, resulting in a double-layer composite hydrogel material.

[0042] 5) Mechanical property testing: The compressive modulus of the composite hydrogel was tested using a universal testing machine. The results showed that its compressive modulus was approximately 245 ± 32 kPa, indicating good mechanical support properties. Biocompatibility testing: Upper and lower hydrogels encapsulating BMSCs were prepared separately. After 7 days of culture, the cell viability rates were 86.7% (upper layer) and 85.9% (lower layer), respectively, indicating good cell compatibility. Animal experiments: A rabbit knee joint osteochondral defect model was constructed, and the prepared bilayer composite hydrogel material was implanted into the defect area. Imaging and histological evaluations were performed 8 weeks post-surgery. Micro-CT results showed that a small amount of new bone tissue was scattered in the subchondral bone defect area, exhibiting a weak osteogenic tendency. However, the trabecular structure was sparse and disordered, with a low bone volume fraction, failing to form a continuous subchondral bone plate structure. MRI results showed a significant difference in signal intensity between the cartilage defect area and the surrounding normal cartilage. New tissue filling was incomplete, predominantly low-signal fibrous tissue, without the typical high-signal characteristics of cartilage. Histological staining (Safranin O-Fixed Green staining) revealed that the defect area was mainly filled with disordered fibrous tissue, with extremely weak or even negative Safranin O staining, and no hyaline cartilage-like extracellular matrix deposition. No clear stratification was formed between the upper and lower layers; the entire defect area exhibited uniform fibrous repair, with no clear distinction between the cartilage and bone layers. Col II immunohistochemical staining was negative, confirming the lack of functional cartilage matrix formation.

[0043] Example 1 like Figure 1 As shown, the preparation process of a three-layer osteochondral repair functional hydrogel material that promotes cartilage-osteogenesis through traditional Chinese medicine and physical stimulation is as follows: 1) Preparation of the intermediate layer (mineralized electrospun membrane): First, a PCL electrospinning solution with a mass fraction of 8wt% was prepared. Under the conditions of spinning voltage of 20 kV and receiving distance of 20 cm, nanofiber membranes were prepared by electrospinning technology. Subsequently, the PCL electrospun membrane was placed in a biomimetic mineralization solution system (formulation: 0.80 wt% NaCl, 0.035 wt% NaHCO3, 0.022 wt% KCl, 0.023 wt% K2HPO4·3H2O, 0.030 wt% MgCl2·6H2O, 0.028 wt% CaCl2, 0.007 wt% Na2SO4, 0.01 wt% gelatin, 0.005 wt% sodium alginate, 0.005 wt% collagen peptides and 0.001 wt% silk fibroin, with the remainder being deionized water), and hydrothermally treated at 70°C for 72 h to allow HA crystals to grow in situ on the fiber surface, thus obtaining a biomimetic mineralized PCL electrospun membrane.

[0044] 2) Preparation of the lower layer (promoting bone function units): a. HA-modified black phosphorus flakes: Black phosphorus flakes with a diameter of approximately 300 nm and a thickness of approximately 5 nm were dispersed at a concentration of 0.25 mg / mL in the above-mentioned biomimetic mineralization solution with a pH of approximately 7.4 and mineralized in a 40°C water bath for 60 h to obtain HA-modified black phosphorus flakes. b. Cryptotanshinone nanopowder: Cryptotanshinone raw material and functional adjuvants (cryptoshinone to adjuvant mass ratio 8:1, adjuvants containing 0.8 wt% polyvinylpyrrolidone, 0.3 wt% soybean lecithin and 0.2 wt% trehalose) were dissolved together in anhydrous ethanol to prepare a solution with a total solid content of 3 mg / mL. Cryptotanshinone nanopowder with a particle size of approximately 200 nm was collected by spray drying technology (inlet temperature 90°C, outlet temperature 45°C, atomization pressure 0.6 MPa, feed rate 3 mL / min). c. Preparation of the lower layer casting solution: Under sterile, light-protected conditions and in a 60°C water bath, a mixed solution was prepared using 10 wt% porous GelMA solution, 0.15 wt% LAP photoinitiator, and 0.3 wt% HA-modified black phosphorus sheets and 0.03 wt% cryptotanshinone nanoparticles. After the solution cooled to 37°C, a suspension of BMSCs (P4 generation) was added until the final cell density reached 8 × 10⁻⁶ cells / mL. 6 cells / mL. Gently pipette 30 times to disperse evenly, then centrifuge at 600 rpm for 4 min to remove bubbles, and store at 4℃ for later use.

[0045] 3) Preparation of the upper layer (facilitating cartilage functional units): a. Preparation of baicalin-loaded MCSN: Using a hydrothermal template method, 1.5 wt% CTAB was dissolved in a mixed solvent of deionized water and anhydrous ethanol (volume ratio 3:1) under magnetic stirring at 30°C. Then, 0.8 wt% calcium nitrate tetrahydrate, 1.5 wt% tetraethyl orthosilicate, 0.15 wt% triethanolamine, and 0.05 wt% PEG-400 were added sequentially. The pH was adjusted to 11.0 using ammonia, and stirring continued for 2 hours. The mixed solution was transferred to a high-pressure reactor and reacted at 150°C at a rate of 3°C / min for 24 hours. After centrifugation, washing, and drying, the product was calcined in a muffle furnace at 550°C at a rate of 2°C / min for 6 hours to remove the template, yielding mesoporous pores with a diameter of approximately 5 nm and a specific surface area of ​​approximately 500 m². 2a. MCSN nanoparticles with a particle size of approximately 150 nm. b. Ultrasonic impregnation for drug loading: 0.3 wt% baicalin was dissolved in a mixed solvent of anhydrous ethanol and dimethyl sulfoxide (volume ratio 12:1). After ultrasonic dissolution, MCSN nanoparticles were added at a drug-to-carrier mass ratio of 1:3. Ultrasonic impregnation was performed under light-protected conditions (ultrasonic power 200 W, frequency 40 kHz, time 30 min, temperature 18°C). After impregnation, the precipitate was collected by centrifugation, rapidly washed with anhydrous ethanol, and freeze-dried for 8 h. This process was repeated twice to obtain MCSN loaded with baicalin. c. Preparation of the upper layer casting solution: A prepolymer solution containing 6 wt% SilMA, 6 wt% HAMA, and 0.15 wt% photoinitiator LAP was prepared under sterile, light-protected conditions in a 60°C water bath. After the solution cooled to 35°C, 0.2 wt% barium titanate nanowires (approximately 100 nm in diameter and 5 μm in length), 0.05 wt% baicalein-loaded MCSN, and BMSCs (P4 generation) suspension were added until the final cell density reached 6 × 10⁻⁶ cells / year. 6 cells / mL. Gently pipette to mix thoroughly, then centrifuge at 600 rpm for 4 min to remove bubbles, and store at 4℃ for later use.

[0046] 4) Casting and molding of the three-layer composite hydrogel: a. Inject the lower layer casting solution into the mold to a height of 2 mm, and place it under light at 405 nm and an intensity of 10 mW / cm². 2 a) Pre-curing was performed by irradiating the electrospun PCL membrane under ultraviolet light for 10 seconds to form a bottom gel with appropriate strength. b) The mineralized PCL electrospun membrane prepared in step 1) was cut into a circle 3 mm smaller than the cross-section of the mold, and treated with oxygen plasma (treatment power 80 W, treatment time 45 seconds, oxygen flow rate 30 sccm) to make its surface hydrophilic. Then the electrospun membrane was flatly attached to the central area of ​​the upper surface of the pre-cured GelMA layer, leaving an annular gap of about 1.5 mm wide around its edges. c) The upper casting solution was poured into the upper part of the electrospun membrane and the annular gap around it, with a casting height of 2 mm, and placed under 405 nm light intensity 8 mW / cm. 2 After being exposed to ultraviolet light for 30 seconds, the material is completely cured, resulting in a three-layer composite hydrogel material with an electrospun membrane embedded inside and the edges directly connected by gel.

[0047] 5) Mechanical property testing: The compressive modulus of the composite hydrogel was tested using a universal testing machine. The results showed that its compressive modulus was approximately 342±19 kPa, indicating good mechanical support properties. Biocompatibility testing: Upper and lower hydrogels encapsulating BMSCs were prepared separately. After 7 days of culture, the cell viability rates were 92.7% (upper layer) and 91.1% (lower layer), respectively. Furthermore, fluorescence staining showed very few dead cells, indicating that the material has good cell compatibility (e.g., ...). Figure 2 , Figure 3 (As shown). Gene expression related to chondrogenic and osteogenic differentiation was detected in the upper and lower cell layers, respectively. The results showed that, compared to Comparative Example 1, the expression of genes related to chondrogenic differentiation in the upper cell layer and osteogenic differentiation in the lower cell layer was significantly upregulated. Figure 4 Immunofluorescence staining of the upper cells with ColII showed strong green fluorescence, indicating high ColII expression levels. Figure 5 Animal experiments: A rabbit knee joint osteochondral defect model was constructed, and the three-layer composite hydrogel material prepared above was implanted into the defect area. Starting from the 3rd day after implantation, the defect area was irradiated with 808 nm near-infrared laser (power density 1.0 W / cm²). 2 The irradiation time was 10 minutes (maintaining the internal temperature of the scaffold at approximately 42°C), once daily for two consecutive weeks. Eight weeks post-surgery, samples were harvested for imaging and histological evaluation. Micro-CT results showed that the subchondral bone plate was intact and continuous, the trabecular structure was dense and regularly arranged, and the newly formed bone tissue was well integrated with the host bone. MRI results showed that the signal intensity of the newly formed cartilage tissue was similar to that of the surrounding normal cartilage, the cartilage layer thickness was uniform, and the surface was smooth and flat. Histological staining (Safranin O-Fix Green staining) revealed that the regenerated tissue exhibited a typical hyaline cartilage-like appearance, with normal chondrocyte morphology and orderly arrangement, and abundant proteoglycans in the extracellular matrix; Col II immunohistochemical staining was strongly positive, indicating that the main component of the newly formed cartilage matrix was Col II. These results demonstrate that this three-layer composite hydrogel material successfully achieved integrated regeneration and repair of osteochondrocytes and cartilage through the synergistic effect of the active ingredients of traditional Chinese medicine and physical stimulation.

[0048] Example 2 1) Preparation of the intermediate layer (mineralized electrospun membrane): First, a PCL electrospinning solution with a mass fraction of 8wt% was prepared. Under the conditions of spinning voltage of 20 kV and receiving distance of 20 cm, nanofiber membranes were prepared by electrospinning technology. Subsequently, the PCL electrospun membrane was placed in a biomimetic mineralization solution system (formulation: 0.80 wt% NaCl, 0.035 wt% NaHCO3, 0.022 wt% KCl, 0.023 wt% K2HPO4·3H2O, 0.030 wt% MgCl2·6H2O, 0.028 wt% CaCl2, 0.007 wt% Na2SO4, 0.01 wt% gelatin, 0.005 wt% sodium alginate, 0.005 wt% collagen peptides and 0.001 wt% silk fibroin, with the remainder being deionized water), and hydrothermally treated at 70°C for 72 h to allow HA crystals to grow in situ on the fiber surface, thus obtaining a biomimetic mineralized PCL electrospun membrane.

[0049] 2) Preparation of the lower layer (promoting bone function units): a. HA-modified black phosphorus flakes: Black phosphorus flakes with a diameter of approximately 300 nm and a thickness of approximately 5 nm were dispersed at a concentration of 0.25 mg / mL in the above-mentioned biomimetic mineralization solution with a pH of approximately 7.4 and mineralized in a 40°C water bath for 60 h to obtain HA-modified black phosphorus flakes. b. Cryptotanshinone nanopowder: Cryptotanshinone raw material and functional adjuvants (cryptoshinone to adjuvant mass ratio 8:1, adjuvants containing 0.8 wt% polyvinylpyrrolidone, 0.3 wt% soybean lecithin and 0.2 wt% trehalose) were dissolved together in anhydrous ethanol to prepare a solution with a total solid content of 3 mg / mL. Cryptotanshinone nanopowder with a particle size of approximately 200 nm was collected by spray drying technology (inlet temperature 90°C, outlet temperature 45°C, atomization pressure 0.6 MPa, feed rate 3 mL / min). c. Preparation of the lower layer casting solution: Under sterile, light-protected conditions and in a 60°C water bath, a mixed solution was prepared using 10 wt% porous GelMA solution, 0.15 wt% LAP photoinitiator, and 0.3 wt% HA-modified black phosphorus sheets and 0.03 wt% cryptotanshinone nanoparticles. After the solution cooled to 37°C, a suspension of BMSCs (P4 generation) was added until the final cell density reached 8 × 10⁻⁶ cells / mL. 6 cells / mL. Gently pipette 30 times to disperse evenly, then centrifuge at 600 rpm for 4 min to remove bubbles, and store at 4℃ for later use.

[0050] 3) Preparation of the upper layer (facilitating cartilage functional units): a. Preparation of baicalin-loaded MCSN: Using a hydrothermal template method, 1.5 wt% CTAB was dissolved in a mixed solvent of deionized water and anhydrous ethanol (volume ratio 3:1) under magnetic stirring at 30°C. Then, 0.8 wt% calcium nitrate tetrahydrate, 1.5 wt% tetraethyl orthosilicate, 0.15 wt% triethanolamine, and 0.05 wt% PEG-400 were added sequentially. The pH was adjusted to 11.0 using ammonia, and stirring continued for 2 hours. The mixed solution was transferred to a high-pressure reactor and reacted at 150°C at a rate of 3°C / min for 24 hours. After centrifugation, washing, and drying, the product was calcined in a muffle furnace at 550°C at a rate of 2°C / min for 6 hours to remove the template, yielding mesoporous pores with a diameter of approximately 5 nm and a specific surface area of ​​approximately 500 m². 2a. MCSN nanoparticles with a particle size of approximately 150 nm. b. Ultrasonic impregnation for drug loading: 0.3 wt% baicalin was dissolved in a mixed solvent of anhydrous ethanol and dimethyl sulfoxide (volume ratio 12:1). After ultrasonic dissolution, MCSN nanoparticles were added at a drug-to-carrier mass ratio of 1:3. Ultrasonic impregnation was performed under light-protected conditions (ultrasonic power 200 W, frequency 40 kHz, time 30 min, temperature 18°C). After impregnation, the precipitate was collected by centrifugation, rapidly washed with anhydrous ethanol, and freeze-dried for 8 h. This process was repeated twice to obtain MCSN loaded with baicalin. c. Preparation of the upper layer casting solution: A prepolymer solution containing 6 wt% SilMA, 6 wt% HAMA, and 0.15 wt% photoinitiator LAP was prepared under sterile, light-protected conditions in a 60°C water bath. After the solution cooled to 35°C, 0.2 wt% barium titanate nanowires (approximately 100 nm in diameter and 5 μm in length), 0.05 wt% baicalein-loaded MCSN, and BMSCs (P4 generation) suspension were added until the final cell density reached 6 × 10⁻⁶ cells / year. 6 cells / mL. Gently pipette to mix thoroughly, then centrifuge at 600 rpm for 4 min to remove bubbles, and store at 4℃ for later use.

[0051] 4) Casting and molding of the three-layer composite hydrogel: a. Inject the lower layer casting solution into the mold to a height of 2 mm, and place it under light at 405 nm and an intensity of 10 mW / cm². 2 a) Pre-curing was performed by irradiating the electrospun PCL membrane under ultraviolet light for 10 seconds to form a bottom gel with appropriate strength. b) The mineralized PCL electrospun membrane prepared in step 1) was cut into a circle 3 mm smaller than the cross-section of the mold, and treated with oxygen plasma (treatment power 80 W, treatment time 45 seconds, oxygen flow rate 30 sccm) to make its surface hydrophilic. Then the electrospun membrane was flatly attached to the central area of ​​the upper surface of the pre-cured GelMA layer, leaving an annular gap of about 1.5 mm wide around its edges. c) The upper casting solution was poured into the upper part of the electrospun membrane and the annular gap around it, with a casting height of 2 mm, and placed under 405 nm light intensity 8 mW / cm. 2 After being exposed to ultraviolet light for 30 seconds, the material is completely cured, resulting in a three-layer composite hydrogel material with an electrospun membrane embedded inside and the edges directly connected by gel.

[0052] 5) Compared to Example 1, this example reduces the photothermal treatment of the defect area. Animal experiment: A rabbit knee joint osteochondral defect model was constructed, and the three-layer composite hydrogel material prepared above was implanted into the defect area. Imaging and histological evaluations were performed 8 weeks post-surgery. Micro-CT results showed that the subchondral bone plate was intact and continuous, the trabecular structure was dense and regularly arranged, and the newly formed bone tissue was well integrated with the host bone. MRI results showed that the signal intensity of the newly formed cartilage tissue was similar to that of the surrounding normal cartilage, the cartilage layer thickness was uniform, and the surface was smooth and flat. Histological staining (Safranin O-Fixed Green staining) revealed that the regenerated tissue exhibited a typical hyaline cartilage-like appearance, with normal chondrocyte morphology and orderly arrangement, and abundant proteoglycan content in the extracellular matrix; Col II immunohistochemical staining was strongly positive, indicating that the main component of the newly formed cartilage matrix was Col II. The above results indicate that this three-layer composite hydrogel material successfully achieved integrated regeneration and repair of osteochondral through the synergistic effect of the active ingredients of traditional Chinese medicine and physical stimulation. Micro-CT results showed that new bone tissue filled the subchondral bone region, and the trabecular structure was relatively dense. However, compared with Example 1, the bone volume fraction of the new bone tissue was lower, and the trabecular arrangement was slightly looser, indicating that the osteogenic repair effect was not as good as that of Example 1 with the synergistic effect of photothermal stimulation. MRI results showed that the signal intensity of the new cartilage tissue was close to that of the surrounding normal cartilage, the cartilage layer thickness was relatively uniform, and the surface smoothness was acceptable, indicating that the chondrogenic function of the upper layer was not significantly affected. Histological staining (Safranin O-Fix Green staining) showed that the regenerated tissue was mainly hyaline cartilage, the chondrocyte morphology was relatively normal and the arrangement was orderly, and the extracellular matrix was rich in proteoglycan deposition; Col II immunohistochemical staining was positive, confirming the effectiveness of the synergistic induction of chondrogenic effect by the upper layer of traditional Chinese medicine baicalein and piezoelectric effect. However, in the lower layer, the integration of new bone tissue with the host bone was poor, and the amount of bone matrix generated was less than that in Example 1.

[0053] Example 3 1) Preparation of the intermediate layer (mineralized electrospun membrane): First, a PCL electrospinning solution with a mass fraction of 8wt% was prepared. Under the conditions of spinning voltage of 20 kV and receiving distance of 20 cm, nanofiber membranes were prepared by electrospinning technology. Subsequently, the PCL electrospun membrane was placed in a biomimetic mineralization solution system (formulation: 0.80 wt% NaCl, 0.035 wt% NaHCO3, 0.022 wt% KCl, 0.023 wt% K2HPO4·3H2O, 0.030 wt% MgCl2·6H2O, 0.028 wt% CaCl2, 0.007 wt% Na2SO4, 0.01 wt% gelatin, 0.005 wt% sodium alginate, 0.005 wt% collagen peptides and 0.001 wt% silk fibroin, with the remainder being deionized water), and hydrothermally treated at 70°C for 72 h to allow HA crystals to grow in situ on the fiber surface, thus obtaining a biomimetic mineralized PCL electrospun membrane.

[0054] 2) Preparation of the lower layer (promoting bone function units): a. HA-modified black phosphorus sheets: Black phosphorus sheets with a diameter of approximately 300 nm and a thickness of approximately 5 nm were dispersed at a concentration of 0.25 mg / mL in the above-mentioned biomimetic mineralization solution at a pH of approximately 7.4 and mineralized in a 40°C water bath for 60 h to obtain HA-modified black phosphorus sheets. b. Preparation of the lower layer casting solution: Under sterile, light-protected conditions and in a 60°C water bath, a mixed solution was prepared using 10 wt% porous GelMA solution, 0.15 wt% LAP photoinitiator, and 0.3 wt% HA-modified black phosphorus sheets. After the solution cooled to 37°C, BMSCs (P4 generation) suspension was added until the final cell density was 8 × 10⁻⁶ cells / mL. 6 cells / mL. Gently pipette 30 times to disperse evenly, then centrifuge at 600 rpm for 4 min to remove bubbles, and store at 4℃ for later use.

[0055] 3) Preparation of the upper layer (facilitating chondrocyte functional units): a. Preparation of the upper layer casting solution: Under sterile, light-protected conditions and in a 60°C water bath, a prepolymer solution containing 6wt% SilMA, 6wt% HAMA, and 0.15wt% photoinitiator LAP was prepared. After the solution cooled to 35°C, 0.2wt% barium titanate nanowires (approximately 100 nm in diameter and 5 μm in length) and a suspension of BMSCs (P4 generation) were added until the final cell density was 6 × 10⁻⁶ cells / year. 6 cells / mL. Gently pipette to mix thoroughly, then centrifuge at 600 rpm for 4 min to remove bubbles, and store at 4℃ for later use.

[0056] 4) Casting and molding of the three-layer composite hydrogel: a. Inject the lower layer casting solution into the mold to a height of 2 mm, and place it under light at 405 nm and an intensity of 10 mW / cm².2 a) Pre-curing was performed by irradiating the electrospun PCL membrane under ultraviolet light for 10 seconds to form a bottom gel with appropriate strength. b) The mineralized PCL electrospun membrane prepared in step 1) was cut into a circle 3 mm smaller than the cross-section of the mold, and treated with oxygen plasma (treatment power 80 W, treatment time 45 seconds, oxygen flow rate 30 sccm) to make its surface hydrophilic. Then the electrospun membrane was flatly attached to the central area of ​​the upper surface of the pre-cured GelMA layer, leaving an annular gap of about 1.5 mm wide around its edges. c) The upper casting solution was poured into the upper part of the electrospun membrane and the annular gap around it, with a casting height of 2 mm, and placed under 405 nm light intensity 8 mW / cm. 2 After being exposed to ultraviolet light for 30 seconds, the material is completely cured, resulting in a three-layer composite hydrogel material with an electrospun membrane embedded inside and the edges directly connected by gel.

[0057] 5) Compared to Example 1, this example reduces the introduction of cryptotanshinone powder in both the upper and lower layers and the baicalin-loaded MCSN. Mechanical property testing: The compressive modulus of the composite hydrogel was tested using a universal testing machine. The results showed that its compressive modulus was approximately 308±22 kPa, indicating good mechanical support properties. Material biocompatibility testing: Upper and lower hydrogels loaded with BMSCs were prepared separately. After 7 days of culture, the cell viability rates were 89.4% (upper layer) and 85.9% (lower layer), respectively, indicating that the material has good cell compatibility (e.g., ...). Figure 2 , Figure 3 As shown). Immunofluorescence staining of the upper cells with Col II revealed relatively little green fluorescence, indicating low Col II expression levels. Figure 5Animal experiments: A rabbit knee joint osteochondral defect model was constructed, and the three-layer composite hydrogel material prepared above was implanted into the defect area. Starting from the 3rd day after implantation, the defect area was irradiated with 808 nm near-infrared laser (power density 1.0 W / cm2, irradiation time 10 minutes, maintaining the internal temperature of the scaffold at around 42°C) once daily for 2 weeks. Eight weeks post-operation, samples were taken for imaging and histological evaluation. Micro-CT results showed that a small amount of new bone tissue was visible filling the subchondral bone area, and the trabecular structure was relatively sparse and irregularly arranged, showing a significant difference compared to Example 1. This indicates that relying solely on the photothermal effect of black phosphorus sheets has limited osteogenic induction capacity and unsatisfactory repair effect. MRI results showed that some new tissue was visible filling the cartilage layer defect area, with signal intensity lower than the surrounding normal cartilage. The cartilage layer thickness was uneven, and the surface smoothness was poor, indicating that relying solely on the piezoelectric effect of barium titanate nanowires has weak chondrogenic induction capacity and limited repair effect. Histological staining (Safranin O-Fix Green staining) revealed that the regenerated tissue was mainly fibrous, with a small amount of chondrocyte-like extracellular matrix deposition in local areas. Col II immunohistochemical staining showed weak positivity, with a small positive area and light staining, confirming that although piezoelectric physical stimulation had a certain inducing effect, the effect was far less than that of Example 1, which showed the synergistic effect of traditional Chinese medicine and piezoelectricity. Only sporadic new bone islands were observed in the lower layer, and the amount of bone matrix generated was limited.

[0058] Example 4 1) Preparation of the lower layer (promoting bone function units): a. HA-modified black phosphorus flakes: Black phosphorus flakes with a diameter of approximately 300 nm and a thickness of approximately 5 nm were dispersed at a concentration of 0.25 mg / mL in the above-mentioned biomimetic mineralization solution with a pH of approximately 7.4 and mineralized in a 40°C water bath for 60 h to obtain HA-modified black phosphorus flakes. b. Cryptotanshinone nanopowder: Cryptotanshinone raw material and functional adjuvants (cryptoshinone to adjuvant mass ratio 8:1, adjuvants containing 0.8 wt% polyvinylpyrrolidone, 0.3 wt% soybean lecithin and 0.2 wt% trehalose) were dissolved together in anhydrous ethanol to prepare a solution with a total solid content of 3 mg / mL. Cryptotanshinone nanopowder with a particle size of approximately 200 nm was collected by spray drying technology (inlet temperature 90°C, outlet temperature 45°C, atomization pressure 0.6 MPa, feed rate 3 mL / min). c. Preparation of the lower layer casting solution: Under sterile, light-protected conditions and in a 60°C water bath, a mixed solution was prepared using 10 wt% porous GelMA solution, 0.15 wt% LAP photoinitiator, and 0.3 wt% HA-modified black phosphorus sheets and 0.03 wt% cryptotanshinone nanoparticles. After the solution cooled to 37°C, a suspension of BMSCs (P4 generation) was added until the final cell density reached 8 × 10⁻⁶ cells / mL. 6cells / mL. Gently pipette 30 times to disperse evenly, then centrifuge at 600 rpm for 4 min to remove bubbles, and store at 4℃ for later use.

[0059] 2) Preparation of the upper layer (facilitating cartilage functional units): a. Preparation of baicalin-loaded MCSN: Using a hydrothermal template method, 1.5 wt% CTAB was dissolved in a mixed solvent of deionized water and anhydrous ethanol (volume ratio 3:1) under magnetic stirring at 30°C. Then, 0.8 wt% calcium nitrate tetrahydrate, 1.5 wt% tetraethyl orthosilicate, 0.15 wt% triethanolamine, and 0.05 wt% PEG-400 were added sequentially. The pH was adjusted to 11.0 using ammonia, and stirring continued for 2 hours. The mixed solution was transferred to a high-pressure reactor and reacted at 150°C at a rate of 3°C / min for 24 hours. After centrifugation, washing, and drying, the product was calcined in a muffle furnace at 550°C at a rate of 2°C / min for 6 hours to remove the template, yielding mesoporous pores with a diameter of approximately 5 nm and a specific surface area of ​​approximately 500 m². 2 a. MCSN nanoparticles with a particle size of approximately 150 nm. b. Ultrasonic impregnation for drug loading: 0.3 wt% baicalin was dissolved in a mixed solvent of anhydrous ethanol and dimethyl sulfoxide (volume ratio 12:1). After ultrasonic dissolution, MCSN nanoparticles were added at a drug-to-carrier mass ratio of 1:3. Ultrasonic impregnation was performed under light-protected conditions (ultrasonic power 200 W, frequency 40 kHz, time 30 min, temperature 18°C). After impregnation, the precipitate was collected by centrifugation, rapidly washed with anhydrous ethanol, and freeze-dried for 8 h. This process was repeated twice to obtain MCSN loaded with baicalin. c. Preparation of the upper layer casting solution: A prepolymer solution containing 6 wt% SilMA, 6 wt% HAMA, and 0.15 wt% photoinitiator LAP was prepared under sterile, light-protected conditions in a 60°C water bath. After the solution cooled to 35°C, 0.2 wt% barium titanate nanowires (approximately 100 nm in diameter and 5 μm in length), 0.05 wt% baicalein-loaded MCSN, and BMSCs (P4 generation) suspension were added until the final cell density reached 6 × 10⁻⁶ cells / year. 6 cells / mL. Gently pipette to mix thoroughly, then centrifuge at 600 rpm for 4 min to remove bubbles, and store at 4℃ for later use.

[0060] 3) Casting and molding of the double-layer composite hydrogel: a. Pour the lower layer casting solution into the mold to a height of 2 mm, and place it under light at 405 nm and an intensity of 10 mW / cm². 2 a) Pre-curing is performed by irradiating with ultraviolet light for 10 seconds to form a bottom layer gel with appropriate strength. b) The upper layer casting solution is then poured to a height of 2 mm and placed under 405 nm light with an intensity of 8 mW / cm². 2The material was completely cured by irradiation with ultraviolet light for 30 seconds, resulting in a double-layer composite hydrogel material.

[0061] 4) Mechanical property testing: The compressive modulus of the composite hydrogel was tested using a universal testing machine. The results showed that its compressive modulus was approximately 267.4 ± 28 kPa. Animal experiments: A rabbit knee joint osteochondral defect model was constructed, and the prepared bilayer composite hydrogel material was implanted into the defect area. Starting from the 3rd day after implantation, the defect area was irradiated with 808 nm near-infrared laser (power density 1.0 W / cm2, irradiation time 10 minutes, maintaining the internal temperature of the scaffold at approximately 42°C) once daily for 2 consecutive weeks. Imaging and histological evaluation were performed 8 weeks post-operation. Micro-CT results showed that new bone tissue filled the subchondral bone area, but the new bone tissue abnormally grew into the upper layer, partially occupying the space that should have been filled by cartilage tissue, resulting in an upward displacement of the bone-cartilage interface and disruption of the physiological tide line structure. MRI results showed that although the cartilage layer defect was filled with new tissue, the cartilage signal was mixed with the bone signal, and the interface between the cartilage layer and subchondral bone was blurred, making it difficult to clearly distinguish the normal bone-cartilage boundary. Histological staining (Safranin O-Fix Green staining) revealed that osteogenic and chondrogenic regions were intermingled in the regenerated tissue, with obvious bone tissue intrusion in the upper layer and bone islands forming within the expected cartilage layer; occasional cartilage-like island structures were observed in the lower layer. Due to the lack of a physical barrier and structural transition of the mineralized intermediate layer at the interface between the upper and lower layers, the tideline structure disappeared, and the physiological bone-cartilage interface was significantly shifted, failing to reconstruct the gradient structure of the natural bone-cartilage tissue. Col II immunohistochemical staining positive areas were mainly concentrated in the upper layer, but the distribution was uneven, and cartilage matrix and bone matrix were mixed and coexisted at the interface with bone tissue. The above results indicate that in the absence of a biomimetic intermediate mineralization layer, there is a lack of necessary physical barriers and functional transitions between the upper and lower layers, which leads to mutual interference between osteogenic and chondrogenic differentiation signals, abnormal invasion of bone tissue into the upper layer, upward shift and blurred boundary of the bone-cartilage interface, and failure to reconstruct the gradient structure and clear tide line of natural osteochondral tissue.

Claims

1. A method for preparing a three-layered osteochondral repair material that promotes cartilage-osteogenesis through traditional Chinese medicine and physical stimulation, characterized in that, Includes the following steps: 1) PCL electrospun membranes were prepared using electrospun technology, and HA coatings were formed on the surface of the PCL electrospun membranes by biomimetic mineralization using a hydrothermal method to obtain a mid-layer mineralized electrospun membrane. 2) An HA coating was constructed on the surface of black phosphorus flakes using in-situ mineralization, and cryptotanshinone nanopowder was prepared by spraying. The HA-modified black phosphorus flakes, cryptotanshinone nanopowder and BMSCs were mixed in a porous GelMA solution to obtain the lower layer casting solution. 3) MCSN nanoparticles were prepared by hydrothermal template method and loaded with baicalin by ultrasonic impregnation process to obtain drug-loaded MCSN nanoparticles; 4) Barium titanate nanowires, the drug-loaded MCSN nanoparticles and BMSCs are dispersed in a solution containing SilMA and HAMA to obtain the upper casting solution. 5) The lower layer casting solution is poured into the mold and pre-cured to form a lower layer gel. A plasma-treated middle layer mineralized electrospun membrane is placed on top of the lower layer gel. The upper layer casting solution is then poured in and completely cured to obtain the three-layer functional hydrogel material for osteochondral repair.

2. The preparation method according to claim 1, characterized in that, In step 1), the preparation process of the PCL electrospun membrane includes: preparing a PCL electrospun solution with a mass fraction of 6-11 wt%, and preparing a nanofiber membrane by electrospinning under the conditions of a spinning voltage of 15-30 kV and a receiving distance of 15-25 cm; the biomimetic mineralization process includes: placing the PCL electrospun membrane in a biomimetic mineralization solution system and hydrothermally treating it at 60-80℃ for 48-120 h to allow HA crystals to grow in situ on the fiber surface.

3. The preparation method according to claim 1, characterized in that, In step 2), the construction of the HA coating on the surface of the black phosphorus sheet specifically includes: using black phosphorus with a diameter of 200-500 nm, a thickness of 1-10 nm, and 2-10 layers as raw material, dispersing it at a concentration of 0.1-0.5 mg / mL in a biomimetic mineralization solution system with a pH of 7.4, and mineralizing it in a water bath at 37-45℃ for 48-72 h; The preparation process of the cryptotanshinone nanopowder includes: dissolving cryptotanshinone raw material and functional adjuvants together in anhydrous ethanol to prepare a solution with a total solid content of 1-5 mg / mL; removing the solvent by spray drying technology to obtain cryptotanshinone nanopowder with a particle size of 100-300 nm; wherein the mass ratio of cryptotanshinone to adjuvants is 5:1 to 10:1, and the functional adjuvants include 0.5-1.0 wt% polyvinylpyrrolidone, 0.2-0.5 wt% soybean lecithin, and 0.1-0.3 wt% trehalose; the spray drying process parameters are: feed concentration 1-5 mg / mL, inlet temperature 80-100°C, outlet temperature 40-50°C, atomization pressure 0.5-0.7 MPa, and feed rate 2-5 mL / min; The preparation process of the lower layer casting solution includes: under sterile, light-protected conditions, mixing 7-12 wt% porous GelMA solution, 0.05-0.25 wt% LAP photoinitiator, 0.1-0.5 wt% HA-modified black phosphorus sheets, and 0.01-0.05 wt% cryptotanshinone nanoparticles in a water bath at 50-65℃ to form a mixed solution; after the solution cools to 35-37℃, adding P3-P5 generation BMSCs suspension until the final cell density is 5-10 × 10⁻⁶ cells / year. 6 The mixture was prepared by centrifugation to remove bubbles after mixing the cells / mL of the solution to obtain the lower layer casting solution.

4. The preparation method according to claim 2 or 3, characterized in that, The biomimetic mineralization solution system is formulated as follows: 0.80 wt% NaCl, 0.035 wt% NaHCO3, 0.022 wt% KCl, 0.023 wt% K2HPO4·3H2O, 0.030 wt% MgCl2·6H2O, 0.028 wt% CaCl2, 0.007 wt% Na2SO4, 0.01 wt% gelatin, 0.005 wt% sodium alginate, 0.005 wt% collagen peptides, and 0.001 wt% silk fibroin, with the remainder being deionized water.

5. The preparation method according to claim 1, characterized in that, In step 3), the preparation of MCSN nanoparticles using the hydrothermal template method includes: dissolving 1.0-2.0 wt% CTAB template agent in a mixed solvent of deionized water and anhydrous ethanol at 25-35℃ and under magnetic stirring, wherein the volume ratio of deionized water to anhydrous ethanol is 3:1; adding 0.25-1.5 wt% calcium nitrate tetrahydrate, 0.9-2.0 wt% tetraethyl orthosilicate, 0.05-0.25 wt% triethanolamine, and 0.01-0.075 wt% polyethylene glycol; and using 20-28... Adjust the pH of the mixed solution to 10.5-11.5 with wt% ammonia water, and continue stirring at 35-45°C for 1-3 hours; transfer to a high-pressure reactor with a filling degree of 60-80%, heat to 100-200°C at a heating rate of 1-5°C / min, and maintain the temperature for 12-36 hours; after cooling, centrifuge, wash, and dry, then heat to 500-650°C at a heating rate of 1-5°C / min and calcine for 4-8 hours to obtain mesoporous pores with a pore size of 3-6 nm and a specific surface area of ​​400-600 m². 2 / g, MCSN nanoparticles with a particle size of 80-200 nm; The ultrasonic impregnation process for loading baicalein specifically includes: dissolving 0.1-0.5 wt% baicalein in anhydrous ethanol or a mixed solvent of anhydrous ethanol and dimethyl sulfoxide in a volume ratio of 9:1 to 13:1, and ultrasonicating for 2-5 minutes to aid dissolution; adding MCSN nanoparticles at a baicalein to MCSN nanoparticle mass ratio of 1:1 to 1:4, and performing ultrasonic impregnation under light-protected conditions, with an ultrasonic power of 150-250 W, a frequency of 35-45 kHz, a time of 20-40 min, and a temperature controlled at 15-20℃; centrifuging, washing, and lyophilizing for 6-12 h after ultrasonication, repeating 2-3 times to obtain the drug-loaded MCSN nanoparticles.

6. The preparation method according to claim 1, characterized in that, In step 4), the preparation process of the upper casting solution includes: preparing a prepolymer solution containing SilMA, HAMA, and LAP photoinitiators in a water bath at 50-70℃ under sterile and light-protected conditions; cooling to 30-37℃ and then dispersing barium titanate nanowires with a diameter of 50-200 nm and a length of 2-10 μm, drug-loaded MCSN nanoparticles, and P3-P5 generation BMSCs in the prepolymer solution; mixing thoroughly and then centrifuging to remove bubbles to obtain the upper casting solution; in the upper casting solution, the concentration of SilMA is 4-7.5 wt%, the concentration of HAMA is 4-7.5 wt%, the concentration of LAP is 0.1-0.25 wt%, and the density of BMSCs is 2 × 10⁻⁶. 6 Up to 1×10 7 The concentration of barium titanate nanowires was 0.05-0.5 wt%, and the concentration of drug-loaded MCSN nanoparticles was 0.02-0.1 wt%.

7. The preparation method according to claim 1, characterized in that, In step 5), the pouring height of both the lower layer pouring solution and the upper layer pouring solution is 1-3 mm; The pre-curing conditions are: 405 nm, light intensity 5-15 mW / cm². 2 Irradiate under ultraviolet light for 5-15 seconds; the complete curing conditions are: 405 nm, light intensity 5-10 mW / cm². 2 Expose to ultraviolet light for 20-40 seconds.

8. The preparation method according to claim 1, characterized in that, In step 5), the diameter or side length of the middle mineralized electrospun membrane is 2-5 mm smaller than the cross-section of the mold. It is placed in the central area above the lower gel, with a 1-2.5 mm wide annular gap reserved around it. The upper casting solution is poured into the annular gap and above the middle mineralized electrospun membrane. After solidification, a composite hydrogel structure is formed in which the middle mineralized electrospun membrane is embedded inside and the edges are directly connected by the upper and lower gels.

9. A functional hydrogel material for repairing three-layered osteochondral tissue by promoting cartilage-osteogenesis through traditional Chinese medicine and physical stimulation, characterized in that, The three-layer osteochondral repair functional hydrogel material, prepared by any one of claims 1-8, is composed of an upper layer that protects against piezoelectricity and promotes cartilage development with traditional Chinese medicine, a middle layer that mimics calcified cartilage, and a lower layer that has photothermal and traditional Chinese medicine bone-promoting functions.

10. The functional hydrogel material for three-layer osteochondral repair according to claim 9, characterized in that, The described three-layer osteochondral repair functional hydrogel material can induce osteogenic formation through photothermal stimulation in conjunction with the active ingredients of traditional Chinese medicine; the photothermal stimulation uses an 808 nm near-infrared laser as the light source, with an irradiation power density of 0.5-1.5 W / cm². 2 The irradiation time is 5-15 minutes per session. The irradiation parameters are adjusted to maintain the internal temperature of the material at 40-43°C. The irradiation frequency is once a day or once every two days, for 1-2 weeks.

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