Bionic hydrogel for treating cartilage defect and preparation method thereof

Abstract

The invention discloses a preparation method of bionic hydrogel for treating cartilage defects. The preparation method comprises the following steps: S1, synthesizing lignin-silver nanoparticles (AgLNPs); s2, lignin-silver nanoparticle-pectin-polyacrylic acid hydrogel (AgLNPs-PPA) is prepared, and the lignin-silver nanoparticle-pectin-polyacrylic acid hydrogel (AgLNPs-PPA) is prepared; s3, loading platelet rich plasma (PRP); s4, preparing a mesenchymal stem cell (BMSCs) core-shell microcapsule (ACB) by using a microfluidic technology; and S5, loading (AgLNPs / PPA / PRP / ACB) of the microcapsules. The present invention integrates sustained wet adhesion, enhanced mechanical properties, targeted stem cell delivery, antibacterial protection, and load-triggered therapeutic release. The characteristics jointly establish a comprehensive and responsive scaffold which can support cartilage regeneration in a mechanically active environment.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering, and more specifically, to a biomimetic hydrogel for treating cartilage defects and its preparation method. Background Technology Articular cartilage damage, caused by trauma, degenerative arthritis, or chronic mechanical stress, poses a persistent clinical challenge due to the tissue's avascular nature, low cellularity, and limited regenerative potential. If left untreated, patients may progress to arthritis, leading to chronic knee pain, decreased mobility, and a significant decline in quality of life. Current clinical interventions, including microfractures, autologous chondrocyte transplantation, osteochondral transplantation, and platelet-rich plasma (PRP) injections, only provide temporary symptom relief and often fail to restore the structure or long-term function of natural hyaline cartilage. PRP therapy requires multiple injections to maintain effective concentrations, is invasive, carries a risk of infection, and requires high patient compliance. These limitations have accelerated the need for tissue engineering strategies, particularly those utilizing hydrogels as biomimetic scaffolds to deliver cells and drugs. Hydrogels, with their high water content, tunable physical properties, and ability to mimic the extracellular matrix of natural cartilage, have become attractive candidates for cartilage repair. When combined with stem cell-based, PRP-based therapies, particularly bone marrow-derived mesenchymal stem cells (BMSCs), they provide a promising platform to promote in situ chondrogenesis and functional tissue regeneration.

[0002] However, despite their theoretical advantages, traditional hydrogel-loaded cell systems face significant transformational barriers. Stem cell survival and retention at the transplantation site remain major challenges, as transplanted cells are often lost from the defect site due to insufficient scaffold fixation and inadequate mechanical protection, particularly in dynamic joint environments. Simultaneously, many existing hydrogels lack sufficient tissue adhesion under wet and mechanically active conditions. This insufficient adhesion weakens scaffold stability and limits the effectiveness of local treatment. Furthermore, the mechanical properties of most hydrogel systems differ significantly from natural cartilage, making them unable to provide load-bearing support in the early healing stages. This can lead to premature scaffold deformation, collapse, or delamination, resulting in failure before effective tissue integration. Additionally, the biocompatibility of the materials must be carefully considered in the selection of implants.

[0003] Therefore, developing a hydrogel with good biocompatibility, durable tissue adhesion, mechanical robustness, and intelligent therapeutic delivery remains a key need in cartilage tissue engineering. Summary of the Invention

[0004] The present invention aims to provide a biomimetic hydrogel for treating cartilage defects and its preparation method, which integrates sustained wet adhesion, enhanced mechanical properties, targeted stem cell delivery, antibacterial protection, and load-triggered therapeutic release. These properties collectively create a comprehensive and responsive scaffold capable of supporting cartilage regeneration in mechanically active environments.

[0005] As one of the objectives of this invention, this invention provides a method for preparing a biomimetic hydrogel for treating cartilage defects, comprising the following steps: S1: Synthetic lignin-silver nanoparticles (AgLNPs); S2: Preparation of lignin-silver nanoparticles-pectin-polyacrylic acid hydrogel (AgLNPs-PPA). S3: Loading platelet-rich plasma (PRP), including the following steps: The AgLNPs-PPA hydrogel prepared by S3 was repeatedly washed with sterile PBS buffer and equilibrated in PBS at 4°C for 12–24 h to remove unreacted monomers, initiator residues, or rapidly released Ag. + Sudden release; Place the hydrogel in a sterile well plate, add enough fresh PRP to completely submerge the hydrogel, with a hydrogel to liquid volume ratio of 1:5–1:10, and gently shake at 4°C for 4 hours. S4: Preparation of nucleoshell microcapsules (ACB) of mesenchymal stem cells (BMSCs) using microfluidic technology. S5: Loading of microcapsules (AgLNPs / PPA / PRP / ACB), including the following steps: Under aseptic conditions, the hydrogel prepared by S3 is drawn up with a sterile syringe, inserted and dispersed into the core-shell microcapsules prepared by S4, and thus obtained.

[0006] Preferably, S1 includes the following steps: (1) Preparation of lignin solution A: Weigh 0.5 g of lignin and 0.5 g of sodium hydroxide, add 10 mL of water, and dissolve by sonication; (2) Preparation of silver ammonia solution B: Weigh 33-134 mg of silver nitrate and add 10 mL of water to prepare a silver nitrate solution with a concentration of 3.3-13.4 mg / mL; take 9 mL of silver nitrate solution in a beaker and slowly add 6 mL of 5 M ammonia water to prepare a silver ammonia solution with a concentration of 0.25%-0.99%; (3) Synthesis of lignin-silver nanoparticles: 2.5 mL of solution A was added dropwise to 7.5 mL of solution B and the reaction was carried out at room temperature for 1 h.

[0007] Preferably, S2 includes the following steps: 4 mL of lignin-silver nanoparticle solution, 0.135 g of ammonium persulfate, and 6 mL of water were poured into a beaker and stirred until completely dissolved. Then, 0.27 g of pectin, 2.7 mL of acrylic acid, and 50 μL of polyethylene glycol dimethacrylate were added, and the mixture was sonicated until completely dissolved. Finally, the sample was placed in a N2 atmosphere and reacted at room temperature for 20 minutes to obtain AgLNPs-PPA hydrogel.

[0008] Preferably, in S3, the sample is washed repeatedly with sterile PBS buffer at least three times, for 10 minutes each time.

[0009] Preferably, step S4 includes the following steps: the outer phase is a 2% sodium alginate (ALG) solution, and the inner phase is a 1:1 volume ratio of BMSC culture medium suspension and a solution containing 2% carboxymethyl cellulose (CMC). Both the outer and inner phases are propelled using a syringe pump at a set rate: 30 μl / min for the outer phase and 10 μl / min for the inner phase. At the terminal junction, the inner phase liquid is encapsulated by the outer phase liquid and cleaved by voltage to form microcapsules. The microcapsules are collected using a solution containing 2% calcium chloride, rapidly cross-linked into a hydrogel, washed three times with MEM culture medium, and then added to the culture medium and placed in an incubator for incubation.

[0010] As a second objective of this invention, this invention also provides a biomimetic hydrogel prepared by the above method.

[0011] As a third objective of this invention, this invention also provides the application of the above-mentioned biomimetic hydrogel in the preparation of drugs for treating cartilage defects.

[0012] Compared with the prior art, the present invention has the following beneficial effects: In this invention, pectin and polyacrylic acid (PAA) form an interpenetrating network, and through multiple crosslinking of covalent and non-covalent bonds, the hydrogel acquires excellent mechanical properties. Lignin, a natural aromatic polymer with antioxidant properties, contributes to the structural integrity and oxidative stability of the hydrogel, while pectin enhances biocompatibility and network flexibility. The addition of silver lignin nanoparticles (Ag-LNPs) further introduces a dynamic active mechanism with a dual purpose: exerting antibacterial effects and continuously generating catechol groups in situ. These catechol groups drive a dynamic, mussel-inspired adhesion process with surrounding tissue through reversible covalent and non-covalent interactions. This mechanism enables long-term, repeatable adhesion under physiological conditions, ensuring that the hydrogel is stably anchored in the irregular structure of cartilage defects during joint movement. Compared to traditional adhesives that rely on static bonding, this redox-driven system's active regenerative adhesion function makes it particularly suitable for the mechanically demanding intra-articular environment. Superior adhesion performance and repeatable compression-recovery mechanical properties have been validated through testing.

[0013] To address the challenges of stem cell delivery and retention, hydrogels encapsulate BMSCs in alginate-based core-shell microcapsules, forming a protected delivery carrier that preserves cell viability and enhances immunoisolation. These microcapsules provide a favorable microenvironment for cartilage differentiation through spatial control of transplanted cells. Their integration with the hydrogel scaffold allows for sustained release of therapeutic cells while protecting them from mechanical shearing and inflammatory stress early in the implantation process. Importantly, this hydrogel system is designed to be mechanoresponsive. Physiological joint loads, such as those generated during walking or knee flexion, act as mechanical stimuli triggering the release of the encapsulated therapeutic components. This force-activated release strategy enables the delivery of bioactive agents synchronized with the patient's natural movements, enhancing cellular responses and mimicking the inherent mechanotransmission processes of natural cartilage repair. By translating biomechanical cues into local therapeutic outputs, this approach is closely aligned with the dynamic microenvironment of joint movement. Attached Figure Description

[0014] Figure 1 Scanning electron microscope image of silver lignin nanoparticles prepared in Example 1; Figure 2 This is a scanning electron microscope image of the AgLNPs-PPA hydrogel prepared in Example 1; Figure 3 This is a schematic diagram of the preparation process of the core-shell microcapsules in Example 1; Figure 4 An optical microscope image of the microcapsules prepared in Example 1; Figure 5 This is a schematic diagram of the cyclic compressive strain curve of AgLNPs-PPA hydrogel; Figure 6 The results of cell compatibility testing of hydrogels with different concentrations of AgLNPs; Figure 7 The results of the test on the promotion of chondrocyte proliferation by hydrogel loading of PRP and BMSC; Figure 8 The results of the antibacterial properties test for AgLNPs-PPA hydrogel; Figure 9 Alican staining results for hydrogel-induced chondrocyte differentiation. Detailed Implementation

[0015] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1 This embodiment provides a method for preparing a biomimetic hydrogel for treating cartilage defects, comprising the following steps: 1. Synthesis of lignin-silver nanoparticles (AgLNPs) Preparation of lignin solution A: Weigh 0.5 g of lignin and 0.5 g of sodium hydroxide, add 10 mL of water, and dissolve by sonication.

[0017] Preparation of silver ammonia solution B: Weigh 33.3 mg of silver nitrate and add 10 mL of water to prepare a 3.33 mg / mL silver nitrate solution; take 9 mL of silver nitrate solution in a beaker and slowly add 6 mL of 5 M ammonia water to prepare the silver ammonia solution.

[0018] Synthesis of lignin-silver nanoparticles: 2.5 mL of solution A was added dropwise to 7.5 mL of solution B, and the reaction was carried out at room temperature for 1 h.

[0019] The morphology of silver-lignin nanoparticles was observed using a scanning electron microscope (SEM; JSM 6390, JEOL Ltd., Japan). Before the experiment, the silver-lignin nanoparticle solution was freeze-dried, and the morphology of the dried particles was then observed under a scanning electron microscope. The results are as follows: Figure 1 As shown in the figure, SEM reveals that the lignin-silver nanoparticles are spherical with a particle size of 20-105 nm.

[0020] 2. Preparation of lignin-silver nanoparticles-pectin-polyacrylic acid hydrogel (AgLNPs-PPA) 4 mL of lignin-silver nanoparticle solution, 0.135 g of ammonium persulfate, and 6 mL of water were poured into a beaker and stirred until completely dissolved. Then, 0.27 g of pectin, 2.7 mL of acrylic acid, and 50 μL of polyethylene glycol dimethacrylate were added, and the mixture was sonicated until completely dissolved. Finally, the sample was placed in a N2 atmosphere and reacted at room temperature for 20 minutes to obtain AgLNPs-PPA hydrogel.

[0021] 3. Loading of platelet-rich plasma (PRP) (AgLNPs / PPA / PRP) After the AgLNPs-PPA hydrogel was prepared, it was washed three times with a large amount of sterile PBS buffer for 10 min each time, and then equilibrated in PBS at 4℃ for 12–24 h to remove unreacted monomers, initiator residues, or rapidly released Ag. + Suddenly released.

[0022] Place the hydrogel in a sterile plate and add sufficient fresh PRP to completely submerge the hydrogel (hydrogel to liquid volume ratio 1:5–1:10). Gently shake at 4°C for 4 hours.

[0023] 4. Preparation of BMSC microcapsules (ACB) BMSC-loaded hydrogel core-shell microcapsules were prepared using microfluidic technology and collected into a gel using a 2% calcium chloride solution. The microcapsules were prepared using a microfluidic electrospray device, which consisted of a 300 μm diameter circular capillary inside a 100 μm diameter circular capillary. Specifically, the outer phase was a 2% sodium alginate (ALG) solution, and the inner phase was a 1:1 mixture of BMSCs and a culture medium containing 2% carboxymethyl cellulose (CMC). Both phases were propelled using a syringe pump at a set rate (30 μl / min for the outer phase and 10 μl / min for the inner phase). At the junction, the inner phase liquid was encapsulated by the outer phase liquid and cleaved by voltage to form microcapsules. The microcapsules were collected in a container containing 2% calcium chloride and rapidly crosslinked into a hydrogel (e.g., ...). Figure 3 As shown in the figure, after washing three times with MEM medium, the microcapsules were added to the medium and placed in an incubator for incubation.

[0024] like Figure 4 As shown, the microcapsules prepared in Example 1 were observed under a regular optical microscope (DM205C, Leica), which showed that microfluidic technology prepared microcapsules of uniform size, with a size of 200-300 μm.

[0025] 5. Loading of BMSC microcapsules (AgLNPs / PPA / PRP / ACB) First, based on treatment needs, AgLNPs / PPA hydrogels of appropriate size were prepared. After loading with PRP, microspheres were inserted into and dispersed into the hydrogel using a sterile syringe under aseptic conditions. The hydrogels were then cultured in MEM medium for subsequent in vitro and in vivo experiments.

[0026] Example 2 This embodiment provides a method for preparing a biomimetic hydrogel for treating cartilage defects, comprising the following steps: 1. Synthesis of lignin-silver nanoparticles (AgLNPs) Preparation of lignin solution A: Weigh 0.5 g of lignin and 0.5 g of sodium hydroxide, add 10 mL of water, and dissolve by sonication.

[0027] Preparation of silver ammonia solution B: Weigh 66.6 mg of silver nitrate and add 10 mL of water to prepare a 6.66 mg / mL silver nitrate solution; take 9 mL of silver nitrate solution in a beaker and slowly add 6 mL of 5 M ammonia water to prepare the silver ammonia solution.

[0028] Synthesis of lignin-silver nanoparticles: 2.5 mL of solution A was added dropwise to 7.5 mL of solution B, and the reaction was carried out at room temperature for 1 h.

[0029] 2. Preparation of lignin-silver nanoparticles-pectin-polyacrylic acid hydrogel (AgLNPs-PPA) 4 mL of lignin-silver nanoparticle solution, 0.135 g of ammonium persulfate, and 6 mL of water were poured into a beaker and stirred until completely dissolved. Then, 0.27 g of pectin, 2.7 mL of acrylic acid, and 50 μL of polyethylene glycol dimethacrylate were added, and the mixture was sonicated until completely dissolved. Finally, the sample was placed in a N2 atmosphere and reacted at room temperature for 20 minutes to obtain AgLNPs-PPA hydrogel.

[0030] 3. Loading of platelet-rich plasma (PRP) (AgLNPs / PPA / PRP) After the AgLNPs-PPA hydrogel was prepared, it was washed three times with a large amount of sterile PBS buffer for 10 min each time, and then equilibrated in PBS at 4℃ for 12–24 h to remove unreacted monomers, initiator residues, or rapidly released Ag. + Suddenly released.

[0031] Place the hydrogel in a sterile plate and add sufficient fresh PRP to completely submerge the hydrogel (hydrogel to liquid volume ratio 1:5–1:10). Gently shake at 4°C for 4 hours.

[0032] 4. Preparation of BMSC microcapsules (ACB) BMSC-loaded hydrogel core-shell microcapsules were prepared using microfluidic technology and collected as a gel using a 2% calcium chloride solution. The microcapsules were prepared using a microfluidic electrospray device, which consisted of a 300 μm diameter circular capillary inside a 100 μm diameter circular capillary. Specifically, the outer phase was a 2% sodium alginate (ALG) solution, and the inner phase was a 1:1 mixture of BMSCs and a culture medium containing 2% carboxymethyl cellulose (CMC). Both phases were propelled using a syringe pump at a set rate (30 μl / min for the outer phase and 10 μl / min for the inner phase). At the junction, the inner phase liquid was encapsulated by the outer phase liquid and cleaved by voltage to form microcapsules. The microcapsules were collected in a container containing 2% calcium chloride, rapidly cross-linked into a hydrogel, washed three times with MEM culture medium, and then added to the culture medium for incubation.

[0033] 5. Loading of BMSC microcapsules (AgLNPs / PPA / PRP / ACB) First, based on treatment needs, AgLNPs / PPA hydrogels of appropriate size were prepared. After loading with PRP, microspheres were inserted into and dispersed into the hydrogel using a sterile syringe under aseptic conditions. The hydrogels were then cultured in MEM medium for subsequent in vitro and in vivo experiments.

[0034] Example 3 This embodiment provides a method for preparing a biomimetic hydrogel for treating cartilage defects, comprising the following steps: 1. Synthesis of lignin-silver nanoparticles (AgLNPs) Preparation of lignin solution A: Weigh 0.5 g of lignin and 0.5 g of sodium hydroxide, add 10 mL of water, and dissolve by sonication.

[0035] Preparation of silver ammonia solution B: Weigh 133.2 mg of silver nitrate and add 10 mL of water to prepare a 13.32 mg / mL silver nitrate solution; take 9 mL of silver nitrate solution in a beaker and slowly add 6 mL of 5 M ammonia water to prepare the silver ammonia solution.

[0036] Synthesis of lignin-silver nanoparticles: 2.5 mL of solution A was added dropwise to 7.5 mL of solution B, and the reaction was carried out at room temperature for 1 h.

[0037] 2. Preparation of lignin-silver nanoparticles-pectin-polyacrylic acid hydrogel (AgLNPs-PPA) 4 mL of lignin-silver nanoparticle solution, 0.135 g of ammonium persulfate, and 6 mL of water were poured into a beaker and stirred until completely dissolved. Then, 0.27 g of pectin, 2.7 mL of acrylic acid, and 50 μL of polyethylene glycol dimethacrylate were added, and the mixture was sonicated until completely dissolved. Finally, the sample was placed in a N2 atmosphere and reacted at room temperature for 20 minutes to obtain AgLNPs-PPA hydrogel.

[0038] The morphology of the AgLNPs-PPA hydrogel was observed using a scanning electron microscope (SEM; JSM 6390, JEOL Ltd., Japan). Figure 2 As shown, AgLNPs-PPA hydrogels all exhibit a three-dimensional network porous structure, which is conducive to material exchange.

[0039] 3. Loading of platelet-rich plasma (PRP) (AgLNPs / PPA / PRP) After the AgLNPs-PPA hydrogel was prepared, it was washed three times with a large amount of sterile PBS buffer for 10 min each time, and then equilibrated in PBS at 4℃ for 12–24 h to remove unreacted monomers, initiator residues, or rapidly released Ag. + Suddenly released.

[0040] Place the hydrogel in a sterile plate and add sufficient fresh PRP to completely submerge the hydrogel (hydrogel to liquid volume ratio 1:5–1:10). Gently shake at 4°C for 4 hours.

[0041] 4. Preparation of BMSC microcapsules (ACB) BMSC-loaded hydrogel core-shell microcapsules were prepared using microfluidic technology and collected as a gel using a 2% calcium chloride solution. The microcapsules were prepared using a microfluidic electrospray device, which consisted of a 300 μm diameter circular capillary inside a 100 μm diameter circular capillary. Specifically, the outer phase was a 2% sodium alginate (ALG) solution, and the inner phase was a 1:1 mixture of BMSCs and a culture medium containing 2% carboxymethyl cellulose (CMC). Both phases were propelled using a syringe pump at a set rate (30 μl / min for the outer phase and 10 μl / min for the inner phase). At the junction, the inner phase liquid was encapsulated by the outer phase liquid and cleaved by voltage to form microcapsules. The microcapsules were collected in a container containing 2% calcium chloride, rapidly cross-linked into a hydrogel, washed three times with MEM culture medium, and then added to the culture medium for incubation.

[0042] 5. Loading of BMSC microcapsules (AgLNPs / PPA / PRP / ACB) First, based on treatment needs, AgLNPs / PPA hydrogels of appropriate size were prepared. After loading with PRP, microspheres were inserted into and dispersed into the hydrogel using a sterile syringe under aseptic conditions. The hydrogels were then cultured in MEM medium for subsequent in vitro and in vivo experiments.

[0043] Experimental Example 1: Mechanical Property Testing of Hydrogels The hydrogel prepared in Example 2 was fabricated into cylindrical specimens (7 mm in diameter and 7 mm in height) for compression testing. The mechanical properties of the hydrogel were tested using a universal testing machine (model 5567, Instrand, USA) equipped with a 100 Newton force sensor. The compression test was conducted at a rate of 1 mm / min, achieving a compression ratio of 90%. The test results showed that the AgLNPs-PPA hydrogel exhibited excellent elasticity and toughness. The material remained stable after undergoing complete deformation under high compressive load, and automatically and rapidly returned to its original shape when the compressive load was removed. Figure 5 As shown, the loading-unloading compressive stress-strain curves indicate that the AgLNPs-PAA hydrogel has good recovery properties.

[0044] Experimental Example 2: Cell Compatibility Test of Hydrogels To verify this, different concentrations of AgLNPs-PPA hydrogels prepared in Examples 1-3 (Examples 1-3 correspond to L-AgLNPs-PPA, M-AgLNPs-PPA, and H-AgLNPs-PPA, respectively) were co-cultured with mouse pre-osteoblasts (ATDC-5) in 24-well plates for 72 h. The culture medium was then removed, and the cells were washed with PBS. Cell viability / deadness staining was performed using the Calcein AM / PI kit (Beyotime, Shanghai) to detect ATDC-5 cells. Observation was performed using a Leica scanning microscope (CLSM, TCS SP8, Leica, Germany). The results are as follows: Figure 6 As shown in the figure, the low-concentration hydrogel has good compatibility with ATDC-5 cells. When 13.32 mg / ml silver nitrate was used to prepare the hydrogel, it inhibited the proliferation of ATDC-5 cells. Therefore, medium (6.66 mg / ml) and low (3.33 mg / ml) concentration hydrogels can be selected for in vitro and in vivo studies. Taking into full account the mechanical properties, the hydrogel with the intermediate concentration is preferred (i.e., Example 2).

[0045] Experiment 3: Effects of different components of hydrogel on the proliferation of ATDC-5 cells To test the effects of different hydrogel components on the proliferation of ATDC-5 cells, the hydrogels from Examples 1-3 were co-cultured with ATDC-5 cells in 24-well plates for 72 h. The results showed that, compared with the control group, PPA and AgLNPs-PPA hydrogels exhibited good compatibility. Loading PRP and BMSC cells onto the hydrogels significantly promoted the proliferation of ATDC-5 cells. Figure 7 As shown.

[0046] Test Example 4: Antibacterial Performance Test Test Methods: The antibacterial activity of the AgLNPs-PPA prepared in Example 2 was evaluated using Staphylococcus aureus and Escherichia coli. Briefly, AgLNPs-PPA hydrogel was loaded into 48-well plates. Then, 100 μL of solution containing 1 x 10⁻⁶ Pb / mL was added. 8 Bacterial suspensions with a total colony count of [number] units were cultured for 24 hours, with the control group (without hydrogel) as an example. Then, 1 mL of sterile PBS was added to each well to resuspend the bacterial solution, centrifuged, and fixed with 2.5% glutaraldehyde. Finally, the solution was dehydrated in a gradient of 30%, 50%, 70%, 75%, 80%, 90%, 95% and anhydrous ethanol, respectively. 20 μL of the treated bacterial suspension was dropped onto aluminum foil, allowed to air dry, and then photographed using an electron scanning microscope. The experiment was repeated three times. The test results are as follows: Figure 8 As shown, AgLNPs-PPA hydrogel has good bactericidal effects against both Staphylococcus aureus and Escherichia coli.

[0047] Experimental Example 5: Evaluation of the cartilage differentiation-promoting properties The hydrogel prepared in Example 2 was co-cultured with ATDC-5 cells in a 24-well plate for 14 days. After removing the culture medium, the cells were washed with PBS, fixed with paraformaldehyde for 30 min, washed with PBS to remove excess paraformaldehyde, stained with Alican staining solution (Seville, Wuhan) for 15 min, washed with PBS to remove excess staining solution, and observed and photographed under an optical microscope.

[0048] like Figure 9 As shown, the AgLNPs-PPA / PRP and AgLNPs-PPA / PRP / ACB groups had a larger staining range and a deeper color, suggesting that they had a better effect on promoting cartilage differentiation.

[0049] The above embodiments are merely illustrative examples of preferred embodiments of the present invention and do not encompass all possible implementations. Any modifications and refinements made by those skilled in the art without departing from the spirit and scope of the present invention are considered to fall within the protection scope of the claims.

Claims

1. A method for preparing a biomimetic hydrogel for treating cartilage defects, characterized in that, Includes the following steps: S1: Synthetic lignin-silver nanoparticles (AgLNPs); S2: Preparation of lignin-silver nanoparticles-pectin-polyacrylic acid hydrogel (AgLNPs-PPA). S3: Loading platelet-rich plasma (PRP), including the following steps: The AgLNPs-PPA hydrogel prepared by S3 was repeatedly washed with sterile PBS buffer and equilibrated in PBS at 4℃ for 12–24 h to remove unreacted monomers, initiator residues or rapidly released Ag+ bursts. Place the hydrogel in a sterile well plate, add enough fresh PRP to completely submerge the hydrogel, with a hydrogel to liquid volume ratio of 1:5–1:10, and gently shake at 4°C for 4 hours. S4: Preparation of nucleoshell microcapsules (ACB) of mesenchymal stem cells (BMSCs) using microfluidic technology. S5: Loading of microcapsules (AgLNPs / PPA / PRP / ACB), including the following steps: Under aseptic conditions, the hydrogel prepared by S3 is drawn up with a sterile syringe, inserted and dispersed into the core-shell microcapsules prepared by S4, and thus obtained.

2. The preparation method according to claim 1, characterized in that, S1 includes the following steps: (1) Preparation of lignin solution A: Weigh 0.5 g of lignin and 0.5 g of sodium hydroxide, add 10 mL of water, and dissolve by sonication; (2) Preparation of silver ammonia solution B: Weigh 33-134 mg of silver nitrate and add 10 mL of water to prepare a silver nitrate solution with a concentration of 3.3-13.4 mg / mL; take 9 mL of silver nitrate solution in a beaker and slowly add 6 mL of 5 M ammonia water to prepare a silver ammonia solution with a concentration of 0.25%-0.99%; (3) Synthesis of lignin-silver nanoparticles: 2.5 mL of solution A was added dropwise to 7.5 mL of solution B and reacted at room temperature for 1 h.

3. The preparation method according to claim 1, characterized in that, S2 includes the following steps: 4 mL of lignin-silver nanoparticle solution, 0.135 g of ammonium persulfate, and 6 mL of water were poured into a beaker and stirred until completely dissolved. Then, 0.27 g of pectin, 2.7 mL of acrylic acid, and 50 μL of polyethylene glycol dimethacrylate were added, and the mixture was sonicated until completely dissolved. Finally, the sample was placed in a N2 atmosphere and reacted at room temperature for 20 minutes to obtain AgLNPs-PPA hydrogel.

4. The preparation method according to claim 1, characterized in that, In S3, wash repeatedly with sterile PBS buffer at least 3 times, 10 minutes each time.

5. The preparation method according to claim 1, characterized in that, S4 includes the following steps: the outer phase is a 2% sodium alginate (ALG) solution, and the inner phase is a 1:1 volume ratio of BMSC medium suspension and a solution containing 2% carboxymethyl cellulose (CMC). Both the outer and inner phases are propelled using a syringe pump at a set rate: 30 μl / min for the outer phase and 10 μl / min for the inner phase. At the terminal junction, the inner phase liquid is encapsulated by the outer phase liquid and cleaved by voltage to form microcapsules. The microcapsules are collected using a solution containing 2% calcium chloride, rapidly cross-linked into a hydrogel, washed three times with MEM medium, and then added to the medium and placed in an incubator for further cultivation.

6. A biomimetic hydrogel prepared by the method according to any one of claims 1-6.

7. The use of the biomimetic hydrogel according to claim 6 in the preparation of a drug for treating cartilage defects.

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