Composition for repairing meniscus, nano-particles, hydrogel material and preparation method and application thereof

Abstract

The invention relates to a composition for repairing meniscus, nanoparticles, a hydrogel material and a preparation method and application thereof. Mesoporous silicon dioxide nanoparticles construct a'macrophage simulant 'form, two growth factors are encapsulated in the nucleus, BSA protein outside the nucleus and the mesoporous silicon dioxide nanoparticles are grafted on the surface of the nucleus through reversible disulfide bonds, the growth factors can be slowly released outside the nucleus, and the macrophage simulant is formed. The cationic polymer BPEI is physically adsorbed around the anionic BSA protein and wraps the mesoporous silicon dioxide nanoparticles, adsorbs the inflammatory factors carrying negative charges when the physicochemical properties of the microenvironment change, and dissociates from the nanoparticles together with the BSA protein. The strong cationic polymer BPEI is used for adsorbing and removing inflammatory factors in an early stage and slowly releasing growth factors for promoting ADSCs to be differentiated into cartilage cells in a middle stage, so that regeneration of cartilage at a meniscus defect part is promoted, repair and regeneration of the cartilage are promoted, in-situ repair is performed on a constructed rabbit meniscus cartilage injury inflammation early model, and the survival rate of the rabbit meniscus cartilage injury inflammation early model is increased. And an ideal cartilage repair effect is achieved.

Description

Technical Field

[0001] This invention relates to the field of biomaterials, and in particular to a composition, nanoparticles, hydrogel materials, preparation methods and applications for repairing the meniscus. Background Technology

[0002] The meniscus is a fibrocartilaginous structure in the knee joint, responsible for load transfer, shock absorption, stability maintenance, and lubrication. Meniscus injuries are often caused by acute sports trauma or degenerative changes and are a common type of knee injury, especially when they occur in the poorly blood-supplied white-white zone, making healing difficult. If left untreated, they can lead to altered stress distribution in the knee joint, decreased stability, cartilage degeneration, and ultimately osteoarthritis. Current treatments include partial or total meniscectomy and suture repair, but the former can accelerate cartilage damage, while the latter has a limited success rate.

[0003] Clinically, there is a greater focus on the injury and its subsequent arthritis, while insufficient attention is paid to the inflammation associated with early cartilage damage. Novel minimally invasive repair strategies urgently need to intervene early and regulate the local inflammatory environment. Existing methods, such as exosomes, collagen or extracellular matrix, bone marrow and blood, factors, stem cells, and synthetic hydrogels, are limited by insufficient bioactivity, easy degradation, or the risk of immune reactions.

[0004] Patent CN119564941A discloses a composition for meniscus repair, comprising growth differentiation factor (GDF) and emodin, which improves inflammation and oxidative stress in the microenvironment and promotes chondrogenic differentiation. The provided core-shell co-delivery nanocarrier utilizes mesoporous silica nanoparticles to encapsulate two bioactive compounds: emodin and GDF, the latter promoting fibrocartilage differentiation. This dual-drug nanodelivery system provides sustained release of emodin and GDF, synergistically modulating harsh inflammatory and oxidative environments to promote in vitro and in vivo fibrocartilage regeneration. Furthermore, a cartilage-specific matrix hydrogel was used to integrate a 3D-printed meniscus-shaped PCL framework with the composition to construct an enhanced-stability meniscus replacement model. This model synergistically modulates the cartilage immune microenvironment, achieving successful repair in animal (rabbit) total meniscus replacement surgery.

[0005] Patent CN117717655A discloses a composition containing microspheres, comprising: a first type of microsphere that releases a first type of active substance into the environment to promote chondrogenesis in the early stages of mesenchymal stem cell differentiation; and a second type of microsphere that releases a second type of active substance into the environment to inhibit angiogenesis or cartilage invasion, promote cartilage growth, and form cartilage lacunae and cartilage-specific ECM deposition. This not only achieves regulation of early chondrogenesis and inhibition of late-stage angiogenesis, thus meeting the dynamic requirements of bone marrow mesenchymal stem cell cartilage regeneration, but also achieves stable regeneration of cartilage tissue in an ectopic environment, eliminating the need for pre-induced cartilage tissue implantation in vitro, significantly improving the convenience of tissue repair.

[0006] Studies have found that cell-free DNA released by injured meniscus cells can activate the TLR-9 pathway and promote immune cell inflammatory responses, accelerating the progression of arthritis, suggesting the potential of early intervention targeting inflammatory factors.

[0007] Mesenchymal stem cells (MSCs) possess multi-lineage differentiation and immunomodulatory capabilities. Adipose-derived stem cells (ADSCs) are readily available, can differentiate into various cell types, and exhibit anti-inflammatory, angiogenic, and multi-growth factor secretion properties. They can also regulate macrophage polarization to alleviate inflammation. However, the in situ meniscus repair effect of ADSCs alone is not as good as that of chondrocytes.

[0008] Therefore, it is necessary to construct a repair system that regulates the inflammatory environment in the early stages of meniscus cartilage injury and promotes the repair of cartilage defects. Summary of the Invention

[0009] The purpose of this invention is to regulate the inflammatory environment in the early stage of meniscus cartilage injury and promote the repair of cartilage defects, and to provide a composition, nanoparticles, hydrogel materials, preparation methods and applications for repairing the meniscus.

[0010] The objective of this invention can be achieved through the following technical solutions: The present invention first provides a composition for repairing the meniscus, comprising a cationic polymer and a growth factor, wherein the cationic polymer is used to adsorb and remove inflammatory factors, and the growth factor is used to promote the process of chondrogenic differentiation of cells. The cationic polymer is BPEI, and the growth factor is a combination of TGFβ3 and IGF1.

[0011] Furthermore, in one embodiment of the present invention, the composition for repairing the meniscus further includes mesoporous silica nanoparticles.

[0012] Furthermore, in one embodiment of the invention, the composition for repairing the meniscus also includes BSA protein.

[0013] Furthermore, in one embodiment of the invention, the composition for repairing the meniscus further includes branched polyethyleneimine (BPEI).

[0014] The present invention further provides nanoparticles for repairing the meniscus, the nanoparticles for repairing the meniscus being referred to as MSN. TI -BPEI, or PMMs for short, includes mesoporous silica microspheres, which are surface-thiolized mesoporous silica microspheres. The mesoporous silica microspheres are loaded with growth factors TGFβ3 and IGF1, and the surface of the mesoporous silica microspheres is bound with BSA protein and grafted with branched polyethyleneimine (BPEI).

[0015] In one embodiment of the present invention, the nanoparticles for repairing the meniscus can regulate the level of inflammation during the meniscus injury process and promote the induction of differentiation of adipose stem cells into chondrocytes.

[0016] In one embodiment of the present invention, the nanoparticles for repairing the meniscus have a core-shell structure and are in the form of a "macrophage mimic." The core carries two growth factors, and the shell is a structure formed by BSA protein and a cationic polymer. The BSA protein and mesoporous silica nanoparticles are reversibly grafted onto the core surface via disulfide bonds. The strong cationic polymer BPEI is physically adsorbed around the anionic BSA protein and encapsulates the mesoporous silica nanoparticles, regulating the inflammation level during meniscus injury and promoting the induced differentiation of adipose-derived stem cells into chondrocytes. The TGFβ3 and IGF1 encapsulated within the core can be slowly released outside the nucleus, further promoting the induced differentiation of adipose-derived stem cells into chondrocytes.

[0017] Extranuclear BSA proteins and mesoporous silica nanoparticles are reversibly grafted onto the nuclear surface via disulfide bonds. Upon changes in the physicochemical properties of the microenvironment, these proteins dissociate, slowly releasing growth factors extranuclearly and promoting the differentiation of adipose-derived stem cells into chondrocytes. The strongly cationic polymer BPEI is physically adsorbed around the anionic BSA protein and encapsulates mesoporous silica nanoparticles. Upon changes in the physicochemical properties of the microenvironment, it adsorbs negatively charged inflammatory factors and dissociates from the nanoparticles along with the BSA protein, slowly releasing growth factors extranuclearly. This regulates the inflammatory environment following meniscus injury and promotes the differentiation of adipose-derived stem cells into chondrocytes.

[0018] The present invention further provides a method for preparing nanoparticles for repairing the meniscus, comprising the following steps: First, mesoporous silica microspheres (denoted as MSNs) are prepared. TGFβ3 and IGF1 were infused into MSNs by ultrasound, followed by the addition of 3-mercaptopropyltrimethoxysilane (MPTMs) to prepare surface-thiolized microspheres (denoted as MSNs).TI -SH); Then, it is reversibly grafted with BSA protein to obtain MSN. TI -BSA; Finally, the strong ionic polymer BPEI was physically adsorbed onto the surface of BSA protein to prepare mesoporous silica microspheres loaded with growth differentiation factors and strong cationic polymers. These are the nanoparticles used for meniscus repair, and are classified as MSN. TI -BPEI microspheres or PMMs.

[0019] The morphology, diameter, elemental composition, XPS, ZETA potential, solid-state nuclear magnetic resonance (NMR) and FTIR spectra under transmission electron microscopy (TEM) all showed significant differences, proving that the mesoporous silica nanoparticles of the present invention were successfully grafted with BSA protein and the strong cationic polymer BPEI.

[0020] In one embodiment of the present invention, the method for preparing the nanoparticles for repairing the meniscus includes the following steps: (1) Synthesis of mesoporous silica microspheres (MSNs); (2) Preparation of surface-thiolized mesoporous silica microspheres (MSN-SH microspheres): MSNs microspheres were dispersed in deionized water, and 3-mercaptopropyltrimethoxysilane (3-MPS) was added. The mixture was stirred overnight at room temperature. After stirring, the mixture was centrifuged to remove unreacted substances, and surface-thiolized mesoporous silica microspheres (MSN-SH microspheres) were obtained. (3) Surface-thiated mesoporous silica microspheres (MSN) loaded with growth factors TI Preparation of -SH microspheres: MSN-SH microspheres were dispersed in deionized water, and TGF-β3 and IGF-1 were added. The mixture was shaken in an ice bath to ensure complete dissolution and uniform dispersion of the growth factors. Subsequently, the solution was transferred to room temperature and stirred overnight to obtain surface-thiolized mesoporous silica microspheres (MSN-SH) loaded with growth factors. TI -SH microspheres); (4) MSN TI Preparation of BSA Based on step (3), in the case of MSN TI BSA protein was added to the solution of -SH microspheres, and stirring was continued to ensure that the BSA protein fully bound to the surface of the microspheres. After stirring, the solution was transferred to a low-temperature high-speed centrifuge, centrifuged, and the precipitate was separated. The obtained product was MSN. TI -BSA; (5) MSN TI Preparation of -BPEI (PMMs) microspheres Get MSNTI BSA microspheres were dispersed in deionized water, branched polyethyleneimine (BPEI) was added, and the mixture was stirred overnight at room temperature to ensure the BPEI and MSN were properly mixed. TI After the BSA microspheres were fully combined and stirred, the solution was transferred to a low-temperature high-speed centrifuge and centrifuged to obtain MSN. TI -BPEI (PMMs) microspheres.

[0021] The present invention further provides a hydrogel material for repairing the meniscus, comprising a base GelMA hydrogel, wherein MA-modified decellularized meniscus matrix (mACMMA) and nanoparticles for repairing the meniscus are loaded in the base GelMA hydrogel. The hydrogel material for repairing the meniscus is abbreviated as PMMs@mGC.

[0022] In one embodiment of the present invention, the concentration of the base GelMA hydrogel is 10% w / v, the concentration of the MA-modified meniscus decellularized matrix (mACMMA) is 1% (w / v), and the concentration of the nanoparticles for repairing the meniscus is 1% (w / v).

[0023] This invention incorporates nanoparticles prepared for meniscus repair into a gel composed of methacrylamide hydrogel (GelMA) and methacrylamide-treated porcine meniscus decellularized matrix (mmACMMA) (denoted as PMMs@mGC, in a 10:1 ratio) to form a hydrogel with a meniscus cartilage matrix. This hydrogel exhibits good biocompatibility and mechanical properties, providing a favorable microenvironment for loading adipocyte stem cells (ADSCs). Furthermore, in an early-stage inflammatory model of meniscus cartilage injury constructed, after 8 weeks of in situ repair, PMMs@mGC played a role in regulating inflammation levels and promoting the differentiation of adipose-derived stem cells into chondrocytes, thereby repairing meniscus damage.

[0024] In one embodiment of the present invention, the hydrogel material for repairing the meniscus also contains adipose-derived stem cells (ADSCs).

[0025] The present invention further provides the application of compositions, nanoparticles, and hydrogel materials for meniscus repair in the preparation of biomaterials for meniscus repair.

[0026] In one embodiment of the invention, the application of the hydrogel material for meniscus repair and the 3D-printed PCL scaffold is provided in the preparation of biomaterials for meniscus repair.

[0027] This invention uses a 3D-printed PCL scaffold as a mechanical support, and a hydrogel material for repairing the meniscus wraps around the 3D-printed PCL scaffold to form a "reinforced concrete" structure, wherein the 3D-printed PCL scaffold acts as the "reinforcement" and the hydrogel material for repairing the meniscus acts as the "concrete".

[0028] To complement total meniscus replacement surgery, this invention also constructed a 3D-printed PCL meniscus scaffold containing adipose-derived stem cells in a mesoporous silica hydrogel, denoted as PMMs@mGC-PCL (with PMMs particles at 10 μg / mL). In a total meniscus cartilage injury replacement model, 12 weeks after the replacement surgery, PMMs@mGC-PCL achieved morphological and functional reconstruction of the meniscus while regulating inflammation levels.

[0029] Compared with the prior art, the present invention has the following beneficial effects: 1. By utilizing the modification capabilities of mesoporous silica microspheres, growth factor systems that promote chondrocyte differentiation are encapsulated inside them, and a strong cationic polymer BPEI is adsorbed on the surface of the microspheres. This enables the early adsorption and removal of inflammatory factors, and the mid-term release of growth factors that promote the differentiation of ADSCs into chondrocytes, thereby promoting the regeneration of cartilage in the meniscus defect site.

[0030] 2. By combining with GelMA hydrogel, an extracellular environment and support for ADCSs were provided, while also enabling in-situ fixation at the defect site, further promoting cartilage repair and regeneration. Ultimately, in-situ repair was successfully achieved in a constructed early-stage inflammatory model of rabbit meniscus cartilage injury.

[0031] 3. Mesoporous silica nanoparticles can inhibit the differentiation of macrophages from M0 to M1 types and can effectively regulate the levels of inflammatory factors related to cartilage inflammation.

[0032] 4. Mesoporous silica nanoparticles exhibit good biocompatibility and can support the good spreading morphology of cell microfilaments.

[0033] 5. Under the induction of mesoporous silica nanoparticles, ADSCs expressed high levels of cartilage-related genes. Attached Figure Description

[0034] Figure 1 Preparation and characterization of PMMs. A represents MSN. TI -SH、 MSN TI -BSA and MSN TI Representative transmission electron microscopy (TEM) images of BPEI nanoparticles. B stands for MSN. TITypical energy dispersive spectroscopy (EDS) analysis of BPEI nanoparticles reveals the content of oxygen, nitrogen, and sulfur elements. C represents MSN. TI -BPEI nanoparticles represent the corresponding elemental percentage. D represents MSN. TI -SH、 MSN TI -BSA and MSN TI - Particle size distribution of BPEI nanoparticles. E represents MSN. TI -SH、 MSN TI -BSA and MSN TI - Representative atomic force microscopy image of BPEI nanoparticles. F stands for MSN. TI -SH, MSN TI -BSA and MSN TI -Surface thickness spectrum of BPEI nanoparticles. G represents MSN. TI -SH, MSN TI -BSA and MSN TI Morphological profiles of BPEI nanoparticles. H represents MSNTI-SH and MSN. TI -BSA and MSN TI -Zeta potential change of BPEI nanoparticles; I = 1 H Solid-state nuclear magnetic resonance spectrum; J is Fourier transform infrared spectrum; K is X-ray photoelectron spectrum (including O1s, N1s, and S2p spectra); L is the spectrum loaded on MSN. TI - Release curves of TGFβ3 and IGF1 growth factors in BPEI nanoparticles.

[0035] Figure 2 This section describes the preparation and characterization of meniscus-specific hydrogels. A shows a schematic diagram of the main components of the meniscus-specific hydrogel PMMs@mGC, including GelMA, mmACMMA, and PMMs. B shows the mmACMMA preparation process: i) obtaining porcine meniscus tissue, ii) decellularization, iii) dissolution, and iv) methacrylate modification. C shows the histological and immunohistochemical analyses of natural and decellularized menisci, including hematoxylin-eosin (H&E) staining, Masson's trichrome staining (MT), and type I and type II collagen (COL I) staining. D, E, and F show the quantitative statistics of double-stranded DNA content, collagen content, and glycosaminoglycan (GAG) content before and after decellularization. G shows the mGC (i–iii) and PMMs@mGC (iv–vi) hydrogels after photopolymerization (365nm LED, 20mW / cm²). 2 The sol-gel transition process before and after lyophilization and the corresponding scanning electron microscope (SEM) images of the lyophilized hydrogel scaffold. H represents the PMMs@mGC hydrogel after light irradiation (365nm LED, 20mW / cm²). 2Rheological curves after compression. I represents a statistical comparison of the storage modulus of mGC and PMMs@mGC hydrogels. J is a photograph of the PMMs@mGC hydrogel at approximately 50% compressive strain, showing no fracture. K represents typical compressive stress-strain curves of mGC and PMMs@mGC hydrogels. L and M represent a statistical comparison of elastic modulus and compressive strength. N is a photograph of the water contact angle test of mGC and PMMs@mGC hydrogels, and O represents the corresponding contact angle measurement results. P and Q represent the swelling rate and degradation rate of mGC and PMMs@mGC hydrogels.

[0036] Figure 3 Biological assessment of pro-inflammatory factor self-clearance and fibrocartilage induction. A: Flow cytometry analysis of the self-clearance capacity of pro-inflammatory factors after LPS-induced polarization of M1 macrophages treated with BPEI or MSNTI-BPEI. B: Histogram of the percentage of M1 (CD86+) macrophages in each group. C: Relative mRNA expression levels of pro-inflammatory factors (TNF-α, iNOS, IL-1β, IL-6, and Cox-2). D: Cytotoxicity assay of PMMs@mGC hydrogel and its components after co-culturing with ADSCs for 1, 4, and 7 days. E: Fibrocartilage-related genes (FCR) in ADSCs treated with TGFβ3 / IGF1 complex growth factor and the control group as determined by q-PCR. Acan , Sox9 , COL2 , COL1 and α-SMA The relative expression levels of fibrocartilage-related genes (F) in ADSCs treated with TGFβ3 / IGF1 complex growth factor and in the control group after Western blot analysis. Acan , Sox9 , COL2 , COL1 , α-SMA and β- actin The relative expression level of ).

[0037] Figure 4In vivo assessment of the repair effect of inflammatory meniscus defects. A shows typical macroscopic observation of the meniscus surface 8 weeks postoperatively in different groups (ADSCs / mGC, ACs / mGC, ADSCs / mGC (+PMMs)); C shows typical macroscopic observation of the femoral cartilage surface. B shows the immunohistochemical staining results of hematoxylin-eosin (H&E), safranin (SO), Masson's trichrome (MT), and α-smooth muscle actin (α-SMA) in the repaired meniscus area. C shows the immunohistochemical staining results of H&E, Alsin blue (AB), safranin / fast green (SO / FG), Masson's trichrome (MT), and type II collagen (COL II), TNF-α, IL-6, and IL-1β in the repaired femoral cartilage area. D shows the Mankin score heatmap for each group; E shows the Mankin score histogram for each group. F shows the Osteoarthritis Research Society International (OARSI) score histogram for each group.

[0038] Figure 5 This study assesses the in vivo inflammatory response after total arthroplasty. A shows gross visual observation of the articular cartilage and femoral cartilage surfaces at 12 weeks post-surgery in each group; B shows a comparison image using magnetic resonance imaging (MRI). ADSCs-loaded PMMs@mGC-PCL scaffolds served as the experimental group, while pure PCL scaffolds served as the control group. C shows the results of H&E staining, hematoxylin-hematoxylin (H&E) staining, safranin (SO) staining, Masson's trichrome (MT) staining, and α-SMA immunohistochemical staining in horizontal and sagittal sections for each group. D shows the results of H&E staining, AB staining, safranin / fast green (SO / FG) staining, Masson's trichrome (MT) staining, and immunohistochemical staining for type II collagen (COL II), TNF-α, IL-6, and IL-1β in the repaired femoral cartilage region for each group. Detailed Implementation

[0039] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0040] The specific experimental methods used in the following embodiments of the present invention are described below: 1. MSN TI Synthesis of BPEI microspheres (1) Synthesis and purification of mesoporous silica microspheres (MSNs).

[0041] The specific steps are as follows: First, add 7.85 mL of ammonia and 50 mL of deionized water to 357 mL of anhydrous ethanol, stir magnetically until homogeneous, and continue stirring for about 10 minutes. Then, slowly add 10 mL of tetraethyl orthosilicate (TEOS). Continue stirring the mixture at room temperature for 1 hour to promote the hydrolysis of TEOS and the formation of a silicon-oxygen network, thus obtaining a silica sol.

[0042] Meanwhile, in another beaker, 40 g of hexadecyltrimethylammonium chloride (CTAC) and 4 mL of triethanolamine (TEA) were added to 400 mL of deionized water. The mixture was magnetically stirred at room temperature for 1.5 hours to allow CTAC to fully dissolve and form micelle structures, thus obtaining a CTAC solution containing TEA, which provides a template for the subsequent formation of mesoporous structures.

[0043] After the TEOS hydrolysis reaction was complete, the resulting silica sol was centrifuged at 12,000 rpm and 4°C for 20 minutes in a low-temperature high-speed centrifuge, and the precipitate was collected. The precipitate was then redispersed in anhydrous ethanol, and the centrifugation-dispersion step was repeated three times to remove unreacted precursors and byproducts. During the final wash, the anhydrous ethanol was replaced with deionized water to improve purity. Next, the washed silica sol was slowly added to a CTAC solution containing TEA, and stirring was continued for 1.5 hours to promote the self-assembly of the mesoporous structure. After stirring, the resulting mixture was transferred to an 80°C water bath, and 3 mL of TEOS was slowly added dropwise under constant temperature and stirring conditions, with the reaction continuing for 1 hour to further promote the growth and stabilization of the silica network.

[0044] The system was then slowly cooled to room temperature, allowed to stand for a short while, and then heated back to 50°C. At this point, 12.72 g of sodium carbonate (Na2CO3) was added, and the reaction was continuously stirred for 30 minutes to promote the removal of CTAC, thereby forming a stable mesoporous structure.

[0045] After the reaction was complete, the product was transferred to a cryogenic centrifuge and centrifuged at 12,000 rpm and 4°C for 20 minutes to remove the supernatant. The precipitate was then washed with sodium chloride-methanol solution, and this process was repeated three times to ensure complete removal of CTAC. Finally, the obtained product was freeze-dried to obtain purified mesoporous silica microspheres (MSNs).

[0046] To ensure biosafety, the obtained MSNs microspheres were sterilized by 60 Co irradiation and then sealed and stored in a -20°C refrigerator for later use.

[0047] (2) Preparation of surface-thiolized mesoporous silica microspheres (MSN-SH microspheres) Weigh 1 g of MSNs microspheres and disperse them in deionized water. Add 2 mL of 3-mercaptopropyltrimethoxysilane (3-MPS) and stir overnight at room temperature. After stirring, transfer the solution to a cryogenic centrifuge and centrifuge at 12,000 rpm and 4°C for 20 minutes to remove unreacted substances. Then, freeze-dry the resulting product and store it at -20°C for later use.

[0048] (3) Surface-thiated mesoporous silica microspheres (MSN) loaded with growth factors TI Preparation of -SH microspheres 100 mg of MSN-SH microspheres were weighed and dispersed in deionized water. 10 μg of TGFβ3 and 10 μg of IGF1 were added, and the solution was ultrasonically agitated in an ice bath for 30 minutes to ensure complete dissolution and uniform dispersion of the growth factors. The solution was then transferred to room temperature and stirred overnight to obtain surface-thiolized mesoporous silica microspheres (MSN-SH) loaded with growth factors. TI -SH microspheres).

[0049] (4) MSN TI Preparation of BSA Based on step (3), in the case of MSN TI Add 40 mg of BSA protein to the solution of -SH microspheres and continue stirring for 8 hours to ensure that the BSA protein is fully bound to the surface of the microspheres. After stirring, transfer to a low-temperature high-speed centrifuge and centrifuge at 12,000 rpm and 4 °C for 20 minutes to separate the precipitate. The obtained product is MSN. TI -BSA. The resulting product was freeze-dried and stored at -20°C for later use.

[0050] (5) MSN TI Preparation of -BPEI (PMMs) microspheres Weigh 100 mg of MSN TI BSA microspheres were dispersed in deionized water, and 10 mL of branched polyethyleneimine (BPEI) was added. The mixture was stirred overnight at room temperature to ensure the BPEI and MSN were properly mixed. TI - The BSA microspheres were fully bound. After stirring, the solution was transferred to a cryogenic centrifuge and centrifuged at 12,000 rpm and 4 °C for 20 minutes. The precipitate was collected and freeze-dried to obtain MSN. TI -BPEI (PMMs) microspheres. Finally, the lyophilized product was stored in a -20 °C freezer.

[0051] Finally, MSN was observed using transmission electron microscopy (TEM). TI -SH, MSN TI -BSA and MSNTI The morphology and particle size of BPEI (PMMs) microspheres were determined, and the elemental composition of different microspheres was analyzed using energy dispersive spectroscopy (EDS).

[0052] 2. MSN TI -SH, MSN TI -BSA and MSN TI Characterization of microspheres such as -BPEI (PMMs) Multiple characterization methods were used to analyze MSN. TI -SH, MSN TI -BSA and MSN TI The morphology, particle size, elemental composition, surface properties and sustained-release performance of -BPEI (PMMs) were systematically analyzed.

[0053] (1) Morphology and particle size observation: The morphology and particle size distribution of different microspheres were observed using transmission electron microscopy (TEM). The elemental composition of different microspheres was analyzed by energy dispersive spectroscopy (EDS).

[0054] (2) ZETA potential measurement: The ZETA potential of different microspheres was measured using a ZETA potential analyzer to evaluate their surface charge characteristics and stability.

[0055] (3) Structural and compositional analysis: Fourier transform infrared absorption spectrometry (FTIR) was used to analyze different microspheres and evaluate their composition, structure and corresponding physicochemical properties.

[0056] (4) Elemental surface analysis: X-ray photoelectron spectroscopy (XPS) was used to analyze the elemental composition of different microspheres and to further study the binding energy and chemical shift of the elements.

[0057] (5) Structural change analysis: The structural changes between different microspheres were detected by 1H solid-state nuclear magnetic resonance (NMR) technology, with particular attention paid to the differences in their intermolecular interactions.

[0058] (6) Evaluation of sustained-release performance: The MSN was tested using an ELISA kit. TI -BPEI (PMMs) encapsulated TGF-β3 and IGF-1 in vitro sustained-release performance. MSN TI -BPEI (PMMs) were placed in PBS solution containing 0.5% dithiothreitol (DTT, Yuntian Biotechnology Co., Ltd.) and samples were collected and detected on days 1, 4, 7, 14, 21 and 28. Each experiment was repeated 3 times.

[0059] (7) Plotting standard curve and sustained-release time curve: Plot the standard curve based on the OD value of the standard sample, and plot the sustained-release time curve of growth factor based on the OD value measured at different time points to further evaluate the sustained-release performance of microspheres.

[0060] 3. Preparation of porcine methacrylic anhydride-modified decellularized meniscus matrix (mACMMA) This experiment used commercially purchased fresh porcine meniscus for decellularization to prepare methacrylic anhydride-modified decellularized meniscus matrix (mACMMA). The specific steps are as follows: (1) Decellularization of the meniscus. First, fresh porcine meniscus was frozen at -80°C and then thawed to room temperature. This freeze-thaw process was repeated three times to promote cell lysis and remove cellular components. Subsequently, the frozen-thawed meniscus tissue was cut into uniform fragments of 1 mm × 1 mm and added with deionized water and 0.1 mol / L Tris-HCl buffer. After adjusting the pH to 7.5, the mixture was magnetically stirred at room temperature for 20 hours. After the reaction was completed, the supernatant was discarded after centrifugation (4°C, 3000 rpm, 10 min). Then, 1% (v / v) Triton X-100 was dissolved in PBS buffer and added to the meniscus fragments. The mixture was magnetically stirred at room temperature for 24 hours to further remove cellular components. After stirring, the supernatant was discarded after centrifugation (4°C, 3000 rpm, 10 min), and the mixture was washed three times with PBS to remove residual surfactant.

[0061] (2) Nucleic acid removal. A working solution of nuclease was prepared by adding 157.6 g Tris-HCl, 16.7 mg DNase, and 7.15 mg RNase sequentially to 500 mL of deionized water. After thorough dissolution, the resulting solution was mixed with fragments of meniscus tissue and stirred at room temperature for 4 hours to degrade residual nucleic acids. Subsequently, the supernatant was discarded by centrifugation (4℃, 3000 rpm, 10 min), and the tissue was washed 6 times with PBS buffer to remove residual enzymes. After decellularization, the tissue was freeze-dried to obtain dried decellularized meniscus matrix (mACM).

[0062] (3) Powdering of decellularized meniscus matrix. The lyophilized mACM was ground into powder using a ball mill, and then sieved through a 300-mesh sieve to obtain uniform powder particles. The sieved mACM powder was weighed and dissolved in pepsin digestion solution (2 mg pepsin per 10 mg ACM), and magnetically stirred for 24 hours to improve solubility. After digestion, the sample was lyophilized to obtain water-soluble mACM powder.

[0063] (4) Methacrylic anhydride modification (MA-modification). Dissolve 100 mg of water-soluble mACM powder in XX g or mL of deionized water and place in an ice bath. Then, slowly add 0.1 mL of methacrylic anhydride (MA) dropwise under stirring to allow for complete reaction. Adjust the pH of the solution to 8-9 using 5 M NaOH and continue stirring overnight to ensure complete grafting of methacrylic anhydride. After the reaction is complete, the sample is freeze-dried, and the resulting MA-modified decellularized meniscus matrix (mACMMA) is sealed and stored at -20°C for later use.

[0064] (5) Physicochemical property analysis of mACMMA. To evaluate the decellularization effect and modification status of mACMMA, histological analysis was performed on mACMMA before and after modification, and the contents of glycosaminoglycans (GAG), collagen, and double-stranded DNA (dsDNA) residue were determined. These indicators were used to evaluate the decellularization efficiency, retention of major matrix components, and degree of nucleic acid removal of mACMMA.

[0065] 4. Preparation of PMMs@mGC (1) Preparation of mGC hydrogel First, weigh 10% (w / v) GelMA and mix it thoroughly with a 0.25% (w / v) solution of the photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphate (LAP) pre-dissolved in deionized water. Dissolve and stir at room temperature until completely dissolved to prepare a basic GelMA hydrogel, then store it at -20°C for later use. Next, mix the basic GelMA hydrogel (10% w / v) with 1% (w / v) MA-modified decellularized meniscus matrix (mACMMA) to prepare an mGC hydrogel.

[0066] (2) Preparation of PMMs@mGC Add 1% (w / v) MSN to the mGC hydrogel (10% w / v GelMA, 1% w / v mACMMA). TI -BPEI and after thorough mixing, PMMs@mGC hydrogel was obtained and used for subsequent experimental analysis.

[0067] Next, the different hydrogels were divided into a control group (GelMA), an ACM group (mGC), and a microsphere group (PMMs@mGC). The hydrogels in different groups were photographed in their liquid state and under light irradiation (365 nm; 20 mW / cm²). 2The state of the hydrogel samples at 30 seconds and after photocuring were observed. The morphology, pore structure, and porosity of different hydrogel materials were observed using a scanning electron microscope (SEM, GeminiSEM360) at an accelerating voltage of 10 kV after freeze-drying. After photocuring, the changes in water contact angle were observed and plotted as bar graphs (each group repeated three times). The mechanical properties of the hydrogels after photocuring were tested, and force-displacement curves were plotted (each group repeated three times). The changes in elastic modulus and viscous modulus of the hydrogels before and after photocuring were observed using a rheometer (Mars40), and curves and bar graphs were plotted.

[0068] 5. In vitro swelling rate test (1) Sample preparation. After curing the hydrogels of different groups under ultraviolet light, their initial mass was weighed using an electronic analytical balance and recorded as W0.

[0069] (2) Swelling process determination. The solidified hydrogel samples were immersed in deionized water or PBS solution (selected according to experimental requirements), and the swelling time points were set as 0 h, 2 h, 4 h, 20 h, 22 h, and 24 h. At each time point, the sample was taken out, excess water on the surface was gently absorbed with filter paper, and its wet weight was quickly weighed and recorded as Ws.

[0070] (3) Swelling Ratio Calculation. The swelling ratio (SR) of the hydrogel is calculated as follows: SR(%) = Ws / W0 * 100% where W0 is the initial mass of the hydrogel and Ws is the wet weight of the hydrogel at different time points. Each group of experiments is repeated 3 times to ensure the reproducibility of the data.

[0071] 6. In vitro degradation rate test.

[0072] (1) Sample freeze-drying and weighing. After the swelling experiment, the hydrogel samples of each group were freeze-dried until they were completely dry. The dry weight was then weighed using an electronic analytical balance and recorded as W. d .

[0073] (2) Enzymatic degradation experiment. The lyophilized hydrogel samples were immersed in 0.1% (w / v) collagenase solution and continuously shaken in a constant temperature shaker at 115 rpm, maintaining the temperature at 37℃. Degradation time points were set at 0h, 2h, 4h, 6h, 8h, 10h, and 12h. Hydrogel samples were collected at each time point, and after being lyophilized again, their remaining dry weight was measured and recorded as W. t .

[0074] (3) Degradation rate calculation. Calculate the remaining mass percentage (DegradationRatio, DR) of the hydrogel at different time points: DR(%) = W t / W d * 100%. Among them, W d W represents the initial dry weight after freeze-drying. t The dry weights were measured at different time points. Similarly, each group was tested three times to ensure data reliability.

[0075] 7. Isolation and culture of adipose-derived stem cells This experiment used adipose tissue from 12-week-old New Zealand white rabbits as the source of adipose-derived stem cells (ADSCs). The specific experimental steps are as follows: (1) Animal handling for obtaining adipose tissue. Healthy 12-week-old New Zealand white rabbits were selected and euthanized in accordance with the ethical requirements for laboratory animals, ensuring that the operation complied with the ethical standards for animal experiments. Under aseptic conditions, the skin was longitudinally incised along the groin area to expose and completely dissect the groin adipose tissue. The adipose tissue was collected and immediately transferred to 50 mL sterile centrifuge tubes pre-filled with 10 mL of sterile PBS (Phosphate Buffered Saline) to keep the tissue moist and prevent it from drying out. Subsequently, it was transferred to a laminar flow hood for further processing.

[0076] (2) Isolation of adipose-derived stem cells: The adipose tissue was repeatedly rinsed three times with PBS + triple antibody solution (5 mL triple antibody mixture dissolved in 100 mL PBS) to remove blood and impurities from the tissue surface. Under aseptic conditions, the blood vessels and capsule surrounding the adipose tissue were removed using sterile surgical scissors, and then the adipose tissue was minced to approximately 1-2 mm. 3 Digest the sample into small pieces and transfer them to 50 mL centrifuge tubes. Add 0.25% (w / v) type II collagenase solution (prepared in low-glucose DMEM medium) to the centrifuge tubes and incubate in a 37 °C water bath with shaking for 30-60 min, gently shaking during the process to promote even digestion.

[0077] (3) Primary cell culture: Observe the digestion status of the tissue in the centrifuge tube. When the tissue presents a uniform suspension with few residual fat clumps, stop digestion immediately. Centrifuge at 1500 rpm (4℃, 5 min) to collect the cell pellet and remove the supernatant and free fat liquid. Resuspend the cells in PBS and centrifuge again to remove residual enzyme solution, so as to reduce the influence of collagenase on the cells. Filter the cell suspension through a 100-mesh cell sieve to remove incompletely digested tissue clumps. Centrifuge the filtered cell suspension again (1500 rpm, 4℃, 5 min), discard the supernatant, and add 20 mL of low-glucose adipose-derived stem cell culture medium (50 mL fetal bovine serum + 5 mL triple antibody + 500 mL low-glucose DMEM medium). Gently pipette to mix the cell suspension and count the cell concentration using a hemocytometer at a concentration of 1×10⁻⁶. 6 Seeds were placed into culture dishes at a density of 10 cells / dish. The dishes were gently tapped to distribute the cells evenly, and then transferred to a cell culture incubator (37°C, 5% CO2, 100% humidity) for 48 h. After 48 h, cell morphology was observed under an inverted microscope to ensure cell adhesion. In a clean bench, the culture medium was carefully aspirated, 2 mL of PBS was added for washing, and then the PBS was aspirated again. Fresh adipose-derived stem cell culture medium was replaced, and the cells were cultured until they reached an appropriate density, at which point they were passaged.

[0078] (4) Passaging of adipose-derived stem cells: When the adipose-derived stem cells reach 80-90% confluence in the culture dish, transfer them to a clean bench, aspirate the adipose-derived stem cell culture medium, add 2 ml of PBS to wash the culture dish, and aspirate the liquid. After washing, add 2 ml of trypsin to digest the adherent cells. After 5 minutes, observe the cells under a microscope to see a quicksand-like state, then add culture medium containing fetal bovine serum to stop the digestion. Transfer to a centrifuge and centrifuge (1500 rpm; 4°C; 5 minutes). Aspirate the liquid, add 20 ml of culture medium to resuspend the cells, count them with a hemocytometer, and seed the cells at a rate of 1 million cells per dish. Repeat the above steps until the adipose-derived stem cells have expanded to the second generation before in vitro and in vivo experiments can be performed.

[0079] (5) Cryopreservation of adipose-derived stem cells: After digesting and resuspending the passaged adipose-derived stem cells as described above, count them using a hemocytometer to calculate the number of cells required for in vitro and in vivo experiments. Centrifuge any excess cells, remove the supernatant, and add cell cryopreservation solution. Based on the cell count, distribute the cells into cryovials at a concentration of 5 million cells per milliliter and transfer them to a -80°C freezer for storage.

[0080] 8. Hydrogel Cytotoxicity Detection Experiment To assess the cytotoxicity level of the hydrogel, an extract of the hydrogel needs to be extracted for testing. The specific experimental steps are as follows: (1) Preparation of hydrogel extract. Hydrogel samples from different groups were placed in intact culture medium (500 ml low-glucose medium; 50 ml fetal bovine serum; 5 ml penicillin-streptomycin-amphoteric B mixture) and shaken in a constant temperature shaker at 37℃ for 72 h (120 rpm). After shaking, the samples were centrifuged (the specific speed and time can be adjusted according to experimental requirements), and the supernatant was collected as the extract for subsequent adipose stem cell culture.

[0081] (2) Cell culture and CCK-8 assay. Adipose-derived stem cells were seeded into 24-well culture plates at a density of 1000 cells per well. Five replicates were set up for each time point, and CCK-8 reagent (Beyotime Biotechnology Co., Ltd.) was added to each well on days 1, 4, and 7, at a density of 30 μL. The culture plates were incubated in the dark for 2 hours (cell culture incubator: 37℃, 5% CO2). The absorbance (OD value) of each group was measured at 450 nm using a microplate reader (Thermo, USA), and the data were recorded. The experiment was repeated three times to ensure the reliability of the results.

[0082] 9. Assessment of cell viability and microfilament extension status Second-generation adipose-derived stem cells were mixed with different hydrogel groups (GelMA; mGC; PMMs@mGC) at a ratio of 1:9 (v / v), and the mixture was then dropped into a cylindrical hydrogel mold (9 mm in diameter and 2.5 mm in height). Curing was performed using a curing lamp (365 nm, 20 mW / cm²). 2 After solidification (30 seconds), the adipose-derived stem cell-hydrogel scaffolds of each group were placed in culture dishes, and adipose-derived stem cell culture medium (50 ml fetal bovine serum, 5 ml triple antibody, 500 ml low-glucose medium) was added. The scaffolds were then cultured in a cell culture incubator (37℃, 5% CO2, 100% humidity). The culture medium was changed every 3 days, and the cell viability in the adipose-derived stem cell-hydrogel scaffolds was detected on days 1, 4, and 7 using a live / dead cell survival kit (live cells stained green, dead cells stained red; Beyotime Biotechnology Co., Ltd.). In addition, the extension status of cell microfilaments was detected using a phalloidin and DAPI staining kit (phalloidin stained red, cell nuclei stained blue; Beyotime Biotechnology Co., Ltd.), and the staining results were observed and recorded using a laser confocal fluorescence microscope (Leica, Germany).

[0083] 10. Assessment of the in vitro inflammatory factor clearance capacity of microspheres Raw 264.7 cell line was selected for in vitro anti-inflammatory experiments. At a concentration of 1×10⁻⁶ cells... 6After incubating macrophages at a concentration of 10 cells per dish for 4 days, excess culture medium was aspirated, 3 mL of PBS was added, and the cells were repeatedly pipetted until they were completely detached. The collected liquid was centrifuged (1500 rpm; 37°C; 5 min), the supernatant was aspirated, and the cells were resuspended in high-glucose cell culture medium (50 mL fetal bovine serum, 500 mL high-glucose medium). The cells were then transferred to 6-well plates and incubated at 3 × 10⁶ cells / well. 5 Seed each well with 10 cells and gently tap the 6-well plate to ensure even cell distribution. Observe cell morphology 4 hours after the cells have adhered to the culture plate.

[0084] Except for the control group (cell-only group), 1 μg of lipopolysaccharide (LPS, ThermoFisher, USA) was added to each well for 24 hours for activation. The cells were then divided into four groups: control group, LPS group (LPS-only group), BPEI-only group (1% (w / v) concentration), and MSN group. TI -BPEI (PMMs) group (5% w / v) was added and co-cultured with activated macrophages for 24 hours. After 24 hours of co-culture, macrophage samples were collected after washing with PBS for subsequent analysis. (1) q-PCR analysis: q-PCR was used to detect four groups (blank group, LPS group, BPEI group, MSN group, and blank group). TIChanges in the expression levels of inflammatory factors in macrophages (BPEI group). Total RNA was extracted using an RNA extraction kit (Beyotime Biotechnology Co., Ltd.). After aspirating the liquid from the cell samples, 1 mL of lysis buffer was added for lysis. The supernatant containing total RNA was transferred to a new EP tube. Isopropanol was added at a ratio of 0.5 mL anhydrous isopropanol per 1 mL of lysis buffer, and the mixture was shaken several times and incubated at room temperature for 10 minutes. The mixture was then centrifuged in a low-temperature high-speed centrifuge (12000 g; 4℃; 10 min). After aspirating the supernatant, 1 mL of washing buffer was added, and the mixture was centrifuged again (7500 g; 4℃; 5 min). After aspirating the supernatant, the mixture was centrifuged again (7500 g; 4℃; 1 min). The liquid was carefully aspirated, and after the RNA was slightly dry, 20-50 μL of DEPC water was added to dissolve the RNA. The samples were then stored at -80℃. cDNA synthesis: cDNA was synthesized using a cDNA synthesis kit (containing gDNA EZeraser, Beyotime Biotechnology Co., Ltd.). Thaw the RNA samples and reagents together at room temperature (15-25℃), then heat denature them at 65℃ for 5 minutes, and immediately cool them in ice water. Incubate the reverse transcription mixture in a water bath at room temperature for 2 minutes, then quickly switch to a 55℃ water bath for 5 minutes, and then transfer the mixture to ice. Add 6 μL of water (DEPC-treated water) and 4 μL of cDNA first-strand synthesis premix (5X) to each RNA sample tube, making a total volume of 20 μL. Gently mix using a pipette, then transfer the mixture to a low-temperature high-speed centrifuge (12000 g, 4℃, 10 minutes) to allow sedimentation. Next, incubate the reaction tubes in a 42℃ water bath for 10-60 minutes, then transfer to an 80℃ water bath for 10 minutes to inactivate the reverse transcriptase and terminate the reverse transcription reaction. Finally, use the reaction solution for amplification detection. Using GAPDH as a reference gene, the relative expression level was determined by the 2−ΔΔCt method, with each sample group being repeated three times.

[0085] (2) The culture conditions for flow cytometry analysis were the same as those for q-PCR. First, the treated macrophages were collected, digested with trypsin, and then fixed with 70% ethanol at 4°C. Subsequently, the cells were labeled with the following antibodies: PE-CD86 (Biolegend) and APC-CD68 (Biolegend). After cell labeling, data analysis was performed using flow cytometry.

[0086] 11. In vitro chondrogenic induction experiment of adipose-derived stem cells Adipose-derived stem cells frozen at -80 °C were removed and quickly placed in a 37 °C water bath for rewarming until completely thawed. The cells were then transferred to centrifuge tubes and centrifuged at 1500 rpm (37 °C, 5 min), discarding the supernatant. The cells were resuspended in adipose-derived stem cell culture medium, thoroughly mixed, and then counted. Cells were cultured in 6-well plates at a density of 3 × 10⁶ cells per well. 5 Cells were evenly seeded at a uniform density on a culture plate, and the plate was gently tapped to ensure even distribution. Cells were then cultured in a cell culture incubator (37°C, 5% CO2, 100% humidity). After 3 days of culture, the culture medium was changed to chondrogenic induction medium, and thereafter changed every 3 days for 28 days. At the end of the induction culture (day 28), cells were collected, and PCR and Western blotting were used to detect changes in the expression levels of cartilage-related genes (ACAN, COL1, COL2, SOX9, and α-SMA) before and after adipose-derived stem cell induction to chondrogenesis, to assess the effectiveness of chondrogenesis.

[0087] 12. Experiment on an early inflammatory environment model of meniscus defects in rabbits This experiment used 12-week-old healthy New Zealand white rabbits. 0.5 mL of type II collagenase solution (4 mg / mL, prepared with PBS) was injected into the meniscus joint cavity of the right leg to induce a meniscus injury model. Two weeks later, the experimental animals underwent meniscus defect modeling surgery, followed by simultaneous in-situ repair surgery. All surgeries were performed under sterile conditions, and postoperative care was strictly implemented in accordance with animal ethics requirements to minimize postoperative complications and ensure the reproducibility of the experiment.

[0088] 13. In-situ repair of meniscus defects in rabbits To investigate the ability of PMMs@mGC hydrogel to repair in situ meniscal cartilage defects, meniscal cartilage defect modeling surgery was performed on treated rabbits. After modeling, different groups of hydrogels (ADSCs / mGC, ACs / mGC, and ADSCs+ADSCs / mGC(+PMMs)) were injected into the defect site. Eight weeks post-surgery, the repair status and inflammation level of the meniscal defect site were assessed, and histological analysis and immunohistochemical characterization were performed to explore the mechanism of action of ADSCs / mGC(+PMMs) hydrogel in meniscal repair.

[0089] 14. Histological characteristics Eight weeks later, the samples were fixed in 4% paraformaldehyde solution (Sigma, USA) for 72 hours. Subsequently, the samples were dehydrated and embedded in paraffin using a tissue embedding machine (Falcon, USA). After embedding, tissue sections were prepared and stained as follows: hematoxylin and eosin (H&E), safranin / fast green (SO / FG), and Masson's trichrome (MT).

[0090] 15. Immunohistochemical characterization The collected meniscus cartilage was fixed in a 4% paraformaldehyde solution and then subjected to decalcification for 8 weeks. During decalcification, the decalcification solution was changed every 3 days. After decalcification, subsequent procedures were performed according to standard protocols. All femoral cartilage tissue samples were analyzed using immunohistochemical staining with antibodies including α-SMA, COL1, COL2, TNF-α, IL-6, and IL-1β.

[0091] 16. Statistical Analysis Unless otherwise specified, each experiment was repeated in triplicate. Data were statistically processed and plotted using GrapPad Prism 10 and are presented as mean ± standard deviation. Multiple groups were evaluated using t-tests for continuous variables, and two groups were statistically analyzed using repeated measures ANOVA and unpaired t-tests. *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001 were considered statistically significant.

[0092] Example 1: Structural characterization of MSNs microspheres.

[0093] Mesoporous silica microspheres (MSNs) were prepared using hexadecyltrimethylammonium chloride (CTAC) as a template agent and tetraethyl orthosilicate (TEOS) as a silicon source. Then, fibrocartilage-specific growth factors TGFβ3 and IGF1 were encapsulated into the MSN core using ultrasonic treatment. Subsequently, thiol-modified MSNs were obtained by stirring with mecaptopropyltrimethoxysilane (MPTMS). TI -SH). Next, bovine serum albumin (BSA) containing free thiol groups is reversibly grafted onto disulfide bonds to form disulfide-modified MSN (MSN). TI Finally, the highly cationic polymer BPEI was introduced, and the strongly ionic polymer BPEI was physically adsorbed onto the surface of the BSA protein to prepare a core-shell structured nanocarrier MSN. TI -BPEI (PMMs). This reversible BPEI modification is designed to adsorb and scavenge negatively charged pro-inflammatory factors at an early stage. Once this stage is complete, the encapsulated TGFβ3 / IGF1 growth factors will be released in a timely manner to promote fibrocartilaginous differentiation of ADSCs.

[0094] Furthermore, morphological features were characterized using transmission electron microscopy and atomic force microscopy. For example... Figure 1 As shown in Figure A, the transmission electron microscope image shows MSN. TI-SH nanoparticles are spherical in shape, indicating that they possess a mature porous structure suitable for growth factor encapsulation. Similarly, regardless of the external modification of the mesoporous silica surface, MSN TI -BSA and MSN TI -BPEI nanoparticles all maintain a spherical shape and have uniform dispersion.

[0095] Energy dispersive spectroscopy and elemental intensity distribution maps confirmed that, with MSN TI -SH and MSN TI -BSA nanoparticles are different, MSN TI -BPEI nanoparticles are mainly composed of oxygen, nitrogen, and sulfur elements, which indirectly confirms that the BPEI layer was successfully modified through disulfide bonds. Figure 1 B, C).

[0096] Quantitative analysis shows that MSN TI -BPEI nanoparticles have the largest average diameter, approximately 118 nanometers, while MSN TI -SH and MSN TI - The diameters of the BSA nanoparticles are approximately 86 nm and 89 nm, respectively. Figure 1 D). MSN TI The increased size of BPEI nanoparticles is mainly attributed to the adsorption effect of the outer layer of BPEI.

[0097] Furthermore, atomic force microscopy images verified the typical morphology and particle size characteristics of each group. Figure 1 E, F). Surface thickness measurements further confirmed that, with MSN TI -SH (approximately 3.5 nm) and MSN TI Compared to BSA (approximately 2.6 nm) nanoparticles, MSN after BPEI adsorption... TI - The thickness of the BPEI nanoparticles was significantly increased (approximately 8.1 nm). Figure 1 G).

[0098] In addition to morphological characterization, the surface potential, chemical composition, and growth factor release of the nanoparticles were also evaluated. Zeta potential analysis showed that MSN TI -SH and MSN TI -BSA nanoparticles are negatively charged, while MSN modified with BPEI... TI -BPEI nanoparticles exhibit a strong positive charge ( Figure 1 H). This charge change may help clear negatively charged inflammatory factors. For example... Figure 1As shown in Figure I, the 1H NMR spectrum confirmed the structural features of the three nanoparticles: the peak at approximately 2 ppm corresponds to the grafted BSA and protein amide bonds, while the peak near 6 ppm indicates silane-sulfur bonds formed by thiol-silicon interactions. Fourier transform infrared spectroscopy further characterized the microsphere structure, such as... Figure 1 As shown in J, MSN TI -SH nanoparticles at 1080 cm⁻¹ -1 The appearance of a peak at this point confirms the thiol modification. MSN TI -BSA and MSN TI The weakened thiol peak in BPEI nanoparticles is likely due to the surface coating masking the siloxane signal, thus supporting the successful modification of BPEI. MSN TI -BPEI nanoparticles at 3439 cm⁻¹ -1 The peak enhancement at that location is attributed to the BPEI amino group. X-ray photoelectron spectroscopy further confirmed the surface composition (…). Figure 1 K). MSN TI The displacement of the CO region in the -BPEI nanoparticles is likely due to BPEI adsorption. MSN TI -BSA and MSN TI -BPEI nanoparticles all exhibited strong C-NH2 peaks, while MSN TI This peak was not observed in -SH nanoparticles. TI -SH and MSN TI A bimodal S2p signal corresponding to the -SH group was detected in the -BSA nanoparticles, while MSN... TI The signal in BPEI nanoparticles is significantly weakened, which is consistent with the properties of the surface being coated with BPEI. Figure 1 L showcased MSN TI - 28-day release curves of growth factor (TGFβ3) and insulin-like growth factor 1 (IGF1) from BPEI nanoparticles. Approximately 28% of the growth factors were rapidly released within 0–4 days, likely due to surface adsorption or incomplete encapsulation of molecules. A stable release phase was observed from 5–14 days, attributed to the encapsulation effect of BPEI. Delayed release was observed from 15–22 days, with dissociation of the BSA-BPEI interface, followed by sustained release over 28 days. These results indicate that MSN... TI -BPEI nanoparticles can effectively remove negatively charged inflammatory factors and achieve sustained release of growth factors, providing support for the construction of macrophage mimics to regulate signal cascade reactions and repair fibrocartilage.

[0099] Example 2: Preparation and characterization of meniscus-specific hydrogels.

[0100] Methacrylated cell-free matrix (mACMMA) was prepared using porcine meniscus tissue. mACMMA polymer was combined with methacrylated gelatin (GelMA) to form mGC composite material, and PMMs were added to obtain PMMs@mGC composite material. Figure 2 A).

[0101] The preparation of mACMMA involves three key steps: decellularization, enzymatic hydrolysis, and methacrylation. Figure 2 B). Histological and immunohistochemical analyses, including hematoxylin-eosin (H&E) staining, Masson's trichrome staining, and type I / II collagen (COL I / II) staining, confirmed successful decellularization; no cell nuclei were observed in the cell-free group. Figure 2 C). It is noteworthy that most of the collagen and fibrous components remained stable after decellularization, as evidenced by the positive staining results of COL I / II protein.

[0102] like Figure 2 As shown in the diagram, quantitative analyses were also performed on the content of double-stranded DNA (dsDNA), total collagen, and glycosaminoglycans (GAG) before and after decellularization. The decellularized mACMMA retained most of the glycosaminoglycans (GAG) and collagen components while effectively removing double-stranded DNA (dsDNA), thus creating a favorable microenvironment for meniscal fibrocartilage repair.

[0103] In addition, the rheological, mechanical, and other physicochemical properties of the hybrid photocrosslinked hydrogels were characterized. Figure 2 As shown in G, two hydrogels, mGC and PMMs@mGC, were used under a 365 nm LED light source (20 mW / cm²). 2 Both can rapidly crosslink under irradiation. Scanning electron microscopy images show the lyophilized morphological characteristics of the hydrogel scaffold. Figure 2 H indicates that the storage modulus (G′) rapidly exceeds the loss modulus (G″) after crosslinking, confirming the rapid gelation process and the formation of a stable hydrogel network. Comparative analysis of storage modulus shows that the PMMs@mGC hydrogel reaches 2817±122 Pa, significantly higher than the 2379±41 Pa of the mGC hydrogel. Figure 2 I). This nanoscale increase in shear modulus is mainly attributed to the incorporation of high-modulus nanoparticles into the low-modulus hydrogel matrix. Stress-strain compression tests show that both mGC and PMMs@mGC hydrogels possess elastic properties. Figure 2 JM). Although the compressive modulus and strength of PMMs@mGC hydrogel are slightly lower than those of mGC hydrogel, they still meet the basic mechanical requirements for meniscal fibrocartilage regeneration. Water contact angle measurements showed that the contact angles of both hydrogels were below 90°, indicating moderate hydrophilicity, which is beneficial for cell adhesion. Figure 2 N, O). Furthermore, regardless of the addition of PMMs, mGC and PMMs@mGC hydrogels did not show significant differences in swelling ratio and degradation rate. Figure 2 (P, Q). Notably, the PMMs@mGC hydrogel exhibits a degradation rate of approximately 30% after collagenase digestion, demonstrating ideal biodegradability characteristics for tissue engineering scaffolds. Overall, these results indicate that the hydrogel scaffold of this invention can be rapidly gelled via photopolymerization, while possessing the rheological and compressive properties suitable for meniscal fibrocartilage regeneration.

[0104] Example 3 Evaluation of self-clearance of inflammatory factors and fibrocartilage induction.

[0105] Macrophages, acting as immune defenders, exert immunomodulatory effects by clearing pro-inflammatory cytokines, thereby promoting tissue regeneration. Mesoporous silica nanomaterials (PMMs) can similarly self-clear pro-inflammatory cytokines through electrostatic interactions and, as nanomaterials mimicking macrophages, gradually regulate the cartilage regeneration process. Positively charged BPEI-modified PMMs selectively adsorb negatively charged pro-inflammatory factors (such as IL-1β, IL-6, and TNF-α), subsequently dissociating from the mesoporous silica core and effectively clearing the bound cytokines through metabolic processes. Subsequently, the TGFβ3 / IGF1 complex growth factor encapsulated in the core is released promptly upon the dissociation of the outer shell, inducing ADSCs to differentiate into fibroblast chondrocytes. CCK-8 assays confirmed that the PMMs@mGC hydrogel extract and its components did not exhibit significant cytotoxicity after co-culturing with ADSCs for 1, 4, and 7 days. Figure 3 D).

[0106] Furthermore, the anti-inflammatory effects of PMMs were evaluated by inducing macrophage polarization with lipopolysaccharide (LPS) followed by co-culturing with BPEI or PMM extracts. Flow cytometry results showed that both BPEI and PMMs inhibited M1 macrophage polarization compared to the LPS group, with the PMMs group exhibiting the lowest polarization rate. Figure 3 A). Quantitative analysis further confirmed that PMMs can effectively inhibit the pro-inflammatory M1 phenotype ( Figure 3 B). As previously reported, meniscus injury often triggers the release of key inflammatory cytokines, including TNF-α, IL-1β, IL-6, and COX-2. The adsorption and clearance capabilities of macrophage-mimicking nanomaterials for cytokines were verified by quantitative polymerase chain reaction (qPCR). The LPS-induced group showed the highest mRNA expression levels of TNF-α, iNOS, IL-1β, IL-6, and COX-2, while the groups treated with BPEI and PMMs showed significantly reduced expression levels, attributed to the efficient clearance of pro-inflammatory cytokines. Figure 3 C). Notably, the reduction detected by qPCR was more pronounced than that detected by flow cytometry, which may be due to the physical adsorption of negatively charged cytokines by positively charged BPEI, rather than direct inhibition of the inflammatory response. After confirming the cell compatibility and cytokine clearance capacity of PMMs, their fibroblast-inducing potential was further evaluated by qPCR and Western blotting. In this experiment, ADSCs treated with TGFβ3 / IGF1 complex growth factor were used as the experimental group for inducing fibrochondrocyte formation, while untreated ADSCs served as the control group. After 28 days of culture, both qPCR and Western blotting results showed that, compared with the control group, the expression levels of fibrochondrocyte-related genes and proteins (Acan, Sox9, COL2, COL1, and α-SMA) in the TI group were significantly increased. Figure 3 (E, F). These findings confirm that the combined use of TGFβ3 and IGF1 effectively induces ADSCs to differentiate into fibrochondrocytes, as evidenced by the upregulation of fibrochondrogenesis markers. Overall, this macrophage-mimicking nanomaterial exhibits good cell compatibility, effectively scavenges pro-inflammatory cytokines, and significantly promotes fibrochondrogenesis, supporting its role in the cascade regulation from anti-inflammatory modulation to meniscal fibrochondrogenesis.

[0107] Example 4: In vivo assessment of inflammatory meniscus defect repair.

[0108] To further explore the feasibility of repairing inflammatory meniscus defects, a rabbit osteoarthritis model was first established within the knee joint cavity. Specifically, 1 ml of saline solution containing 8 mg of type II collagenase was injected intra-articularly. Two weeks after inducing early inflammation in the joint cavity, a cylindrical defect with a diameter of 2 mm was created in the meniscus using a drilling instrument. Repair was then achieved by injecting a gel precursor material loaded with seed cells, which was then tested under a 365 nm LED light source (20 mW / cm²). 2 Rapid cross-linking to form a hydrogel scaffold under irradiation. This study evaluated the repair effect of meniscus defects in three groups: i) mGC hydrogel loaded with ear chondrocytes (ACs / mGC; ear chondrocyte loading of 10M / ml), ii) mGC hydrogel loaded with ADSCs (ADSCs / mGC; ADSCs loading of 10M / ml), and iii) PMMs@mGC hydrogel loaded with ADSCs [ADSCs / mGC(+PMMs)]. Eight weeks post-surgery, samples of the repaired meniscus and joint tissue were collected for further analysis. Figure 4As shown in Figure A, macroscopic observation revealed that the ADSCs / mGC (+PMMs) group achieved almost complete macroscopic healing. In contrast, significant cartilage defects were still visible in both the ACs / mGC and ADSCs / mGC groups, likely due to the intense inflammatory environment within the joint cavity. Osteoarthritis-related lesions were observed on the femoral cartilage surface in both the ACs / mGC and ADSCs / mGC groups, while the ADSCs / mGC (+PMMs) group exhibited a nearly normal appearance. This is attributed to the self-clearance of pro-inflammatory factors by PMMs. Figure 4 B). Histological and immunohistochemical analyses were performed to evaluate the meniscus regeneration effect and the inhibitory effect on joint inflammation. Figure 4 As shown in Figure C, the ADSCs / mGC (+PMMs) group exhibited sustained tissue regeneration and fibrocartilage-specific matrix deposition. H&E staining and Safranin (SO) staining showed cartilage-positive staining, while MT and α-SMA staining showed fibrous tissue positivity. In contrast, both the ACs / mGC and ADSCs / mGC groups showed significant tissue defects, consistent with the inflammatory state of the osteoarthritis model. Further evaluation of the articular cartilage surface (including immunohistochemical staining for TNF-α, IL-6, and IL-1β) revealed that the ADSCs / mGC (+PMMs) group had the lowest levels of pro-inflammatory cytokines. Figure 4 D). This indicates that PMMs effectively clear pro-inflammatory factors in vivo. Notably, this anti-inflammatory effect also promotes cartilage repair, as evidenced by positive staining of cartilage-specific matrix markers such as Alcian blue staining, safranin / fast green staining, and type II collagen staining. Figure 4 Figures E and F show the Mankin scores and corresponding histograms for each group. Statistical analysis showed that the ADSCs / mGC group had the highest score, indicating severe cartilage damage; while the ADSCs / mGC(+PMMs) group had the lowest score, reflecting a significant protective effect. Similarly, the Osteoarthritis Research Society International (OARSI) score also confirmed that the ADSCs / mGC(+PMMs) group had a better repair effect. Figure 4 (G). Therefore, PMMs@mGC hydrogel can effectively scavenge pro-inflammatory cytokines and protect articular cartilage from inflammatory damage in vivo. More importantly, these findings demonstrate the success of a cascade regulatory strategy from inflammation elimination to regeneration of meniscal fibrocartilage.

[0109] Example 5: In vivo evaluation of inflammatory total meniscus replacement surgery.

[0110] Besides repairing small-scale meniscus defects, larger injuries usually require total meniscus replacement. To explore the feasibility of total meniscus replacement in inflammatory cases, a rabbit osteoarthritis model was established using previous methods, and a type II collagenase solution (1 ml of physiological saline containing 8 mg of type II collagenase) was injected into the joint. To achieve total meniscus replacement, a "reinforced concrete" tissue engineering strategy was adopted: a 3D-printed crescent-shaped polycaprolactone (PCL) scaffold was used as the "reinforced" mechanical support, and then an mGC (+PMMs) hydrogel loaded with adipose-derived mesenchymal stem cells (ADSCs) was dropped and solidified to form the "reinforced concrete" regenerative component.

[0111] Two weeks after osteoarthritis induction, the experimental group underwent surgical removal of the original meniscus and replaced it with a PMMs@mGC-PCL scaffold loaded with meniscal ADSCs, while the control group received only a PCL scaffold. Macroscopic observation at 12 weeks postoperatively showed that the meniscus in the experimental group was highly similar to the original meniscus in terms of meniscal structure and fibrocartilaginous texture. Figure 5 A). In contrast, the control group showed softer fibrous tissue and residual PCL material. Magnetic resonance imaging confirmed that the meniscus in the experimental group maintained its crescent shape and the signal intensity was comparable to that of the original meniscus, while the control group lacked obvious signs of meniscus regeneration. Figure 5 B).

[0112] To assess the meniscus reconstruction in the horizontal and sagittal planes, histological and immunohistochemical examinations were performed, including H&E staining, AB staining, safranin / fast green (FG) staining, and MT staining. Figure 5 As shown in Figure C, the experimental group (Exp) exhibited characteristics similar to fibrocartilage, with positive staining for GAG and collagen fibers, while the control group (Ctrl) showed disordered fibrous tissue and a lack of cartilage-related structures. Notably, the sagittal section of the regenerated meniscus in the experimental group showed heterogeneous tissue structure and integration, while the control group had a large gap in the central region. These results indicate that the PMMs@mGC-PCL scaffold loaded with ADSCs effectively restored the morphology and function of the original meniscus. In addition to meniscus reconstruction, the condition of articular cartilage and the levels of inflammatory cytokines were also assessed. Figure 5As shown in Figure D, histological and immunohistochemical analyses revealed that the PMMs@mGC-PCL scaffold significantly protected the articular cartilage surface, especially in the lateral region. In contrast, the PCL-only scaffold resulted in more pronounced cartilage wear, likely due to friction generated by the rigid scaffold. Furthermore, the expression levels of TNF-α, IL-6, and IL-1β in the PMMs@mGC-PCL group were lower than those in the PCL-only group, attributed to the effective cytokine clearance effect of PMMs in the OA model. Mankin score heatmaps, histograms, and OARSI scores confirmed that the experimental group achieved the best regenerative effect. Overall, the ADSC-loaded PMMs@mGC-PCL scaffold successfully reconstructed the meniscus, restored joint functional integrity, and provided significant protection for articular cartilage by effectively regulating the inflammatory response. These results fully demonstrate the powerful therapeutic potential of the proposed "reinforced concrete" tissue engineering strategy in meniscus reconstruction and joint protection.

[0113] In summary, this invention has developed a macrophage-mimicking nanomaterial that achieves meniscus treatment through a cascade regulatory mechanism, promoting both the self-clearance of pro-inflammatory cytokines and the regeneration of fibrocartilage. This meticulously designed macrophage-mimicking material (PMMs) effectively removes pro-inflammatory factors through physical adsorption, while simultaneously promoting the timely release of TGFβ3 and IGF1 growth factors, thereby driving the differentiation of adipose-derived stem cells (ADSCs) into fibrocartilage cells within an inflammatory environment. In clinical applications, PMMs@mGC hydrogels loaded with ADSCs have shown good efficacy in meniscus defect repair and fibrocartilage regeneration. Furthermore, total meniscus replacement was successfully achieved using a "reinforced concrete" tissue engineering strategy combined with a crescent-shaped polycaprolactone (PCL) scaffold.

Claims

1. A composition for repairing a meniscus, characterized by, The composition for repairing meniscus comprises a cationic polymer and a growth factor, the cationic polymer is used for adsorbing and removing inflammatory factors, and the growth factor is used for promoting the process of cell chondrogenic differentiation. The cationic polymer is BPEI, and the growth factor is a combination of TGFβ3 and IGF1.

2. A composition for repairing meniscus according to claim 1, wherein The composition for repairing meniscus further comprises mesoporous silicon dioxide nanoparticles and / or BSA protein and / or branched polyethyleneimine.

3. A nanoparticle for repairing a meniscus, characterized by, The nanoparticles for repairing meniscus are called MSNs TI -BPEI or PMMs, including mesoporous silica microspheres, the mesoporous silica microspheres are surface thiolated mesoporous silica microspheres, the mesoporous silica microspheres are internally loaded with growth factors TGFβ3 and IGF1, the mesoporous silica microspheres are surface combined with BSA proteins and grafted with branched polyethyleneimine.

4. A nanoparticle for repairing a meniscus according to claim 3, wherein The nanoparticles for repairing meniscus are a core-shell structure, wherein the core is mesoporous silicon dioxide microspheres, the shell is a structure formed by BSA protein and a cationic polymer, the BSA protein is grafted on the surface of the core through a reversible disulfide bond, and the strong cationic polymer BPEI is physically adsorbed around the anionic BSA protein and wraps the mesoporous silicon dioxide nanoparticles, and TGFβ3 and IGF1 are encapsulated in the core.

5. A method of making nanoparticles for repairing meniscus according to claim 3 or 4, wherein, The method comprises the following steps: (1) synthesis of mesoporous silicon dioxide microspheres; (2) preparation of mesoporous silicon dioxide microspheres with surface thiol groups: Take the MSNs microspheres, disperse them in deionized water, add 3-mercaptopropyl trimethoxysilane, stir at room temperature overnight, centrifuge after stirring is completed, and remove unreacted substances to obtain mesoporous silicon dioxide microspheres with surface thiol groups; (3) Surface thiolated mesoporous silica microspheres loaded with growth factors, i.e. MSN TI Preparation of -SH microspheres: Take the MSN-SH microspheres, disperse them in deionized water, add TGFβ3 and IGF1, and shake under ice bath conditions to ensure sufficient dissolution and uniform dispersion of the growth factors, then transfer the solution to room temperature and continue to stir overnight to obtain mesoporous silicon dioxide microspheres with surface thiol groups loaded with growth factors; (4) MSN TI Preparation of BSA On the basis of step (3), add BSA protein into the solution containing MSN TI -SH microspheres, continue to stir, ensure that the BSA protein is fully combined with the surface of the microspheres, after the stirring is completed, transfer to a low-temperature high-speed centrifuge, centrifuge, separate the precipitate, and the obtained product is MSN TI -BSA; (5) MSN TI Preparation of BPE microspheres Take MSN TI - BSA microspheres were dispersed in deionized water, branched polyethyleneimine (BPEI) was added, and stirred at room temperature overnight to ensure that BPEI was combined with MSN TI - BSA microspheres were fully combined, after completion of stirring, the solution was transferred to a low-temperature high-speed centrifuge, centrifuged, and MSN TI - BPEI microspheres, namely nanoparticles for repairing meniscus.

6. A hydrogel material for repairing a meniscus, characterized by, The composition for repairing meniscus comprises a cationic polymer and a growth factor, the cationic polymer is used for adsorbing and removing inflammatory factors, and the growth factor is used for promoting the process of cell chondrogenic differentiation.

7. The hydrogel material for repairing meniscus according to claim 6, wherein, The concentration of the basic GelMA hydrogel is 10% w / v, the concentration of the MA meniscus acellular matrix is 1% (w / v), and the concentration of the nanoparticles for repairing meniscus is 1% (w / v).

8. The hydrogel material for repairing meniscus according to claim 6, wherein, The hydrogel material for repairing meniscus further comprises adipose-derived stem cells ADSCs.

9. Use of the composition for repairing meniscus of claim 1, the nanoparticles for repairing meniscus of claim 3, and the hydrogel material for repairing meniscus of claim 6 in the preparation of a biomaterial for meniscus repair.

10. Use according to claim 9, characterized in that, Use of the hydrogel material for repairing meniscus and the 3D-printed PCL scaffold in the preparation of a biomaterial for meniscus repair; The 3D-printed PCL scaffold serves as a mechanical support, and the hydrogel material for repairing meniscus wraps the 3D-printed PCL scaffold to form a "rebar-concrete" structure, wherein the 3D-printed PCL scaffold serves as "rebar", and the hydrogel material for repairing meniscus serves as "concrete".

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