Compositions for repairing menisci and their meniscal replacement applications

Abstract

The application discloses a composition for repairing meniscus, which comprises a growth differentiation factor and emodin, improves inflammation and oxidative stress in a microenvironment, and promotes cell chondrogenic differentiation process. It is verified that the "core-shell" co-delivery nano carrier provided by the application uses mesoporous silica nanoparticles to wrap two bioactive compounds, emodin and a growth differentiation factor, and the latter is used for promoting fibrocartilage differentiation. The dual-drug nano delivery system provides sustained release of emodin and GDF, synergistically regulates a harsh inflammatory and oxidative environment, and promotes fibrocartilage regeneration in vitro and in vivo. Benefited from this, the application also combines a three-dimensionally printed meniscus-shaped PCL frame with the composition by using a cartilage-specific matrix hydrogel, constructs a reinforced stability meniscus replacement model, has a synergistic regulation on a cartilage immune microenvironment, and realizes successful repair of a whole meniscus replacement operation of an animal (rabbit).

Description

Technical Field

[0001] This invention relates to a biomaterial, and more particularly to a composition carrying differentiation-promoting active molecules for promoting cartilage differentiation and regeneration, and its application in medical devices. Background Technology

[0002] The meniscus is a crescent-shaped fibrocartilaginous tissue with key biological functions, including shock absorption and providing overall mechanical stability to the knee joint. Meniscus injuries are common in sports, and for severe meniscus injuries requiring total meniscus replacement, current clinical treatment strategies include meniscus allograft (MAT) and meniscus prosthesis repair. However, their long-term efficacy is not significant due to graft size and shape mismatches. Recently, advancements in cartilage tissue engineering have provided a novel strategy for clinical meniscus replacement, enabling three-dimensional cartilage regeneration in vivo using seed cells combined with scaffold materials. For the regeneration of complex cartilage shapes and heterogeneous structures, our research group proposes a 3D-printed reinforced concrete tissue engineering strategy for applications in the ear, nose, trachea, and meniscus.

[0003] Despite significant progress in cartilage tissue regeneration, successful regeneration at defect sites requires the creation of a favorable regenerative microenvironment. Adverse microenvironments caused by chronic inflammation and oxidative stress or acute trauma during clinical surgery severely hinder tissue regeneration, making the development of innovative cartilage tissue engineering strategies a challenging and rare clinical breakthrough. Particularly in meniscectomy, where the cruciate ligament is severed and the joint surface is worn, the meniscal defect site leads to intense oxidative stress and unavoidable inflammatory stimulation. Therefore, it is necessary to develop a novel construction strategy to synergistically regulate the correction of adverse microenvironments and promote cartilage tissue regeneration.

[0004] Previous reports have indicated that growth differentiation factor (GDF) can effectively promote the differentiation of fibroblasts into fibroblasts and chondrocytes. However, the lack of a sustained release system has resulted in unsatisfactory long-term effects.

[0005] Therefore, it is necessary to develop a technique that combines the improvement of the adverse microenvironment caused by inflammation and oxidative stress with satisfactory meniscal functional reconstruction. Summary of the Invention

[0006] One object of the present invention is to provide a composition for use in inflammation and oxidative stress to promote the chondrogenic differentiation process of cells.

[0007] Another object of the present invention is to provide a composition for use in inflammation and oxidative stress, promoting chondrogenic differentiation of cells, which is beneficial for meniscus function reconstruction.

[0008] Another objective of this invention is to provide a hydrogel containing microspheres, wherein the microspheres are loaded within the hydrogel to provide a cell growth environment, which facilitates the release of active substances from the microspheres to regulate the process of cell chondrogenic differentiation, promote cartilage growth and repair, and promote cell chondrogenic differentiation.

[0009] Another objective of this invention is to provide an application for meniscus replacement, in which microspheres loaded with GDF and emodin are applied to a scaffold to improve inflammation and oxidative stress in the microenvironment, promote the process of chondrogenic differentiation of cells, and facilitate the functional reconstruction of the meniscus.

[0010] A composition comprising a growth differentiation factor and emodin that improves inflammation and oxidative stress in the microenvironment and promotes chondrogenic differentiation.

[0011] Another composition is a microsphere containing growth differentiation factors and emodin, which improves inflammation and oxidative stress in the microenvironment and promotes the chondrogenic differentiation process of cells.

[0012] Another composition consists of mesoporous silica nanoparticles forming a "core-shell" structure, in which growth differentiation factors and emodin are loaded, improving inflammation and oxidative stress in the microenvironment and promoting the chondrogenic differentiation process of cells.

[0013] Another composition is a "core-shell" structure constructed from mesoporous silica nanoparticles. The core contains emodin, and growth differentiation factors can be released and bound to the core surface, improving inflammation and oxidative stress in the microenvironment and promoting the chondrogenic differentiation process of cells.

[0014] Another composition is a "core-shell" structure constructed from mesoporous silica nanoparticles. The core contains emodin, and growth differentiation factors are grafted onto the core surface via reversible disulfide bonds, which improves inflammation and oxidative stress in the microenvironment and promotes the chondrogenic differentiation process of cells.

[0015] In vitro experiments showed that sustained-release emodin can effectively regulate macrophage M1 / M2 polarization, maintain a good regenerative microenvironment, and at the same time release GDF to promote fibroblast / chondrocyte seed cell differentiation into fibroblasts.

[0016] In addition, particles made from the above-mentioned compositions are added to a gel composed of methacrylamide hydrogel (GelMA) and chondroitin sulfate methacrylate (CSMA) (denoted as GC, with a weight ratio of 6:1) to form a cartilage-specific matrix hydrogel, which has good cell compatibility, rheological and mechanical properties, and is also conducive to loading cells, such as chondrocytes and fibrochondrocytes.

[0017] To facilitate total meniscus replacement surgery, this invention also provides a PCL scaffold, manufactured by 3D printing, which contains chondrocytes and fibroblasts (a mixture of fibroblasts and chondrocytes in a 7:3 ratio), GC gel, and Em@MSN-GDF, denoted as: Em@MSN-GDF / GC-PCL (wherein the concentration of Em@MSN-GDF is 10 μg / mL). This scaffold synergistically regulates the cartilage immune microenvironment during total meniscus replacement surgery, synergistically regulates inflammation and oxidative stress, as well as the harsh microenvironment and fibrocartilage regeneration, promoting meniscus cartilage differentiation and shaping.

[0018] The microspheres of this invention are prepared by first loading emodin onto dSiO2 silica spheres, then using hexadecyltrimethylammonium chloride (CTAC) as a template agent and tetraethyl orthosilicate (TEOS) as a silicon source to prepare mesoporous silica microspheres (denoted as: Em@MSN). 3-mercaptopropyltrimethoxysilane (MPTMs) is added to obtain surface-thiolized microspheres (denoted as: Em@MSN-SH). These are then mixed with GDF protein to obtain mesoporous silica microspheres loaded with growth differentiation factors and emodin, denoted as: Em@MSN-GDF. The average particle size of the microspheres is 169±15.37 nm, and the Zeta potential is 12.77±1.07 mV.

[0019] The microspheres of the present invention were characterized for sustained release over 45 days, with a GDF release rate of 50% and an emodin release rate of nearly 60%.

[0020] Verification has shown that the "core-shell" co-delivery nanocarrier (Em@MSN-GDF) provided by this invention encapsulates two bioactive compounds: emodin (a natural anti-inflammatory and antioxidant) and growth differentiation factor (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 fibrocartilage regeneration both in vitro and in vivo.

[0021] Furthermore, thanks to the role of Em@MSN-GDF, this invention also utilizes cartilage-specific matrix hydrogel to combine a three-dimensionally printed meniscus-shaped PCL framework with Em@MSN-GDF to construct an enhanced stability meniscus replacement model (scaffold). This model has the ability to synergistically regulate the cartilage immune microenvironment, and has achieved successful repair of total meniscus replacement surgery in animals (rabbits). Attached Figure Description

[0022] Figure 1The prepared Em@MSN-GDF microspheres and their characterization results are shown in Figure A. A schematic diagram of the Em@MSN-GDF microsphere preparation process is shown in Figure B. Transmission electron microscopy (TEM) images (scale bar = 100 nm) of Em@MSN, Em@MSN-SH, and Em@MSN-GDF are shown in Figure C. The average particle size distribution of Em@MSN, Em@MSN-SH, and Em@MSN-GDF is shown in Figure D. The zet density of Em@MSN, Em@MSN-SH, and Em@MSN-GDF is measured by dynamic light scattering (DLS). a) Potential results; E) Energy dispersive spectroscopy (EDS) image of Em@MSN-GDF (scale bar = 200 nm); F) Percentage of elements in the EDS of Em@MSN-GDF; G) Particle size distribution histogram of Em@MSN-GDF; H) Solid-state NMR spectrum of Si in Em@MSN, Em@MSN-SH and Em@MSN-GDF; i) Solid-state NMR spectrum of H in Em@MSN, Em@MSN-SH and Em@MSN-GDF; J) XPS S-2p spectrum of Em@MSN; K) XPS S-2p spectrum of Em@MSN-SH; L) XPS S-2p spectrum of Em@MSN-GDF; M) Statistical graph of emodin encapsulation efficiency; N) GDF sustained-release curve; O) Emodin sustained-release curve.

[0023] Figure 2 Figures show the preparation and performance characterization of GC and Em@MSN-GDF / GC; where A is a bright-field photograph of the gelation process of GelMA / CSMA (GC) and Em@MSN-GDF / GC under 365nm UV irradiation, B is a SEM image of GC and Em@MSN-GDF / GC, C is a graph showing the storage modulus (G') and loss modulus (G"), D is the compressive strain curve, E is a statistical graph of the expansion ratio of each group, F is a statistical graph of the degradation rate of each group, and G is the concentration of AM / PI and F-actin (10 μg / mL). Cell staining images of Em@MSN-GDF / GC at 1, 4, 7 and 14 days, respectively. H is the cell viability statistics of Em@MSN-GDF / GC at 1, 4, 7 and 14 days, I is the quantitative statistics of live cell count at 1, 4, 7 and 14 days, J is the quantitative statistics of f-actin fluorescence at 1, 4, 7 and 14 days, and K is the CCK-8 detection results of Em@MSN-GDF fibroblasts at 48h.

[0024] Figure 3Figure 1 shows the in vitro antioxidant and anti-inflammatory effects of Em@MSN-SH and Em@MSN-GDF. A represents the ROS fluorescence image after co-culturing with Em@MSN or Em@MSN-GDF at 1mM H2O2 for 24 h; B is the fluorescence intensity graph shown in Figure A; C is the relative mRNA ratio of GPX1 / GAD(P)H after co-culturing with Em@MSN or Em@MSN-GDF at 1mM H2O2 for 24 h; D is the relative mRNA ratio of NQO1 / GAD(P)H after co-culturing with Em@MSN or Em@MSN-GDF at 1mM H2O2 for 24 h; E is the flow cytometry result of M1 and M2 polarization in each macrophage group; and F represents the M1 (CD86) polarization in each group. + Macrophage statistical plot, G represents M2 (CD206) in each group. + Macrophage statistics: H is the relative mRNA statistics of TNF-α / GAD(P)H when RAW264.7 cells were co-cultured with Em@MSN or Em@MSN-GDF for 24 h under 100 ng / mL LPS; I is the relative mRNA statistics of IL-6 / GAD(P)H when RAW264.7 cells were co-cultured with Em@MSN or Em@MSN-GDF for 24 h under 100 ng / mL LPS; J is the schematic diagram of the antioxidant and anti-inflammatory mechanism of Em@MSN-GDF.

[0025] Figure 4The graphs show the effects of GDF on fibroblasts in vitro and in vivo. A is a schematic diagram of the Transwell co-culture system; B shows crystal violet staining after 24 and 72 hours of co-culture using an 8 μm Transwell chamber with fibroblasts in the upper chamber and chondrocytes in the lower chamber; C shows alicin blue staining after 7 days of fibroblasts cultured in a 0.4 μm Transwell chamber in the upper chamber, either alone or co-cultured with chondrocytes in the lower chamber; D is a statistical graph of COLII-related mRNA expression levels in fibroblasts analyzed by qRT-PCR; E is a statistical graph of ACAN-related mRNA expression levels in fibroblasts analyzed by qRT-PCR; F is a statistical graph of α-SMA-related mRNA expression levels in fibroblasts analyzed by qRT-PCR; G is a statistical graph of SOX9-related mRNA expression levels in fibroblasts analyzed by qRT-PCR; and H is a Western chromatogram of COLII, ACAN, α-SMA, and SOX9-related proteins in fibroblasts after 7 days of treatment with 100 ng / mL GDF. Blotting electrophoresis images: I is a bright-field photograph of the implant; J is a photograph of the GC hydrogel implantation procedure in nude mice; K is a gross photograph of the mice 6 weeks after implantation; L is a photograph of the approximate morphology of each group of implants 6 weeks after implantation; M is an immunofluorescence staining (blue) image of HE, SO, COL II (green), and α-SMA (red) and cell nuclei in the FBs, FBs7 / CHs3, FBs3 / CHs7, and CHs groups 6 weeks after implantation (scale bar = 100 μm); N is a statistical graph of the fluorescence density of COL II in each group; O is a statistical graph of the fluorescence density of α-SMA in each group (FBs: fibroblasts; FBs7 / CHs3: fibroblast 7 and chondrocyte 3; FBs3 / CHs7: fibroblast 3 and chondrocyte 7; CHs: chondrocytes).

[0026] Figure 5 The in vivo evaluation results of Em@MSN-GDF / GC-PCL in chondrogenesis are shown in the figure. Among them, A is a photographic image of GC-PCL implants carrying cells, B is a bright-field photograph of the gross morphology of GC-PCL, Em@MSN / GC-PCL and Em@MSN-GDF / GC-PCL implants after 6 weeks, C is a staining image of COL II (green), α-SMA (red), HE and SO (cell nuclei are blue, scale bar = 100 μm) of GC-PCL, Em@MSN / GC-PCL and Em@MSN-GDF / GC-PCL groups after 6 weeks, D is a statistical graph of Young's modulus results of GC-PCL group, Em@MSN / GC-PCL group and Em@MSN-GDF / GC-PCL group after 6 weeks, E is a statistical graph of COL II fluorescence density, and F is a statistical graph of α-SMA fluorescence density.

[0027] Figure 6The images show the in vivo evaluation results of meniscus replacement and repair. A is a bright-field photograph of the crescent-shaped Em@MSN-GDF / GC-PCL implant containing FBs7 / CHs3 cells; B is a photograph of the surgical procedure for implanting the crescent-shaped prosthesis in a New Zealand rabbit medial meniscus defect model; C is MRI images of the PCL group and the Em@MSN-GDF / GC-PCL group at 6 and 12 weeks post-operation; D is a bright-field photograph of the gross morphology of the knee joint at 12 weeks post-operation; E is a bright-field photograph of the gross morphology of the crescent-shaped implant at 12 weeks post-operation; F is the staining map of HE, SO, COL II, COL I, α-SMA (red), VEGF (red), and VMF (green) in the Em@MSN-GDF / GC-PCL group; G is a quantitative statistical graph of α-SMA fluorescence intensity; H is a quantitative statistical graph of VEGF fluorescence intensity; I is a quantitative statistical graph of VWF fluorescence intensity; and J is a statistical graph of the ICRS score at 12 weeks post-implantation. Detailed Implementation

[0028] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solution of the invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

[0029] The specific experimental methods used in the following embodiments of the present invention are described below:

[0030] 1) Cell culture

[0031] Skin tissue sections measuring 2 cm × 2 cm were collected from adult New Zealand white rabbits. The tissues were digested overnight at 4°C in 2% neutral protease, followed by washing twice with PBS. Subsequently, the tissues were digested in 2% collagenase for 4 hours to obtain fibroblasts. The cells were then cultured and expanded in a 37°C, 5% CO2 incubator. Second and third generation fibroblasts and chondrocytes were used in this study. Cells were cultured in high-glucose DMEM basal medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

[0032] 2) Preparation and characterization of Em@MSN-GDF

[0033] Synthesis of Em@MSN microspheres: Based on previous research, emodin (S30748, Yuan Ye, Shanghai, China) was accurately weighed and dissolved in 5 ml of water. Then, 35.7 mL of ethanol and 0.785 mL of ammonia solution were added, and the mixture was stirred at room temperature for 10 minutes. Next, 1 mL of TEOS (T819505, Macklin, Shanghai, China) was added, and the mixture was stirred at room temperature for 1 hour. After centrifugation at 10,000 rpm for 20 minutes, the precipitate was washed three times with ethanol and resuspended in 20 mL of deionized water to obtain an aqueous solution of dSiO2.

[0034] 4 g of CTAC (A610332, Sangon Biotech, Shanghai, China) was weighed and dissolved in 40 mL of water. 400 μL of triethanolamine (TEA) (A600970, Sangon Biotech, Shanghai, China) was added, and the mixture was stirred at room temperature for 1 h. Then, 20 mL of the previously prepared dSiO2 aqueous solution was added, and the mixture was stirred at 80 °C for 1 h. 300 μL of TEOS was added dropwise. Once the reaction cooled to room temperature, 1272 mg of Na2CO3 was added to the mixture, and etching was performed for 30 minutes. The product underwent a series of washing steps, starting with water, then a 1% sodium chloride-methanol solution, and finally three washes with anhydrous ethanol. This process yielded Em@MSN-encapsulated emodin microspheres.

[0035] Synthesis of Em@MSN-SH microspheres: 300 mg Em@MSN microspheres were dispersed in 5 ml of deionized water. 200 μL of MPTMs (M812736, Macklin, Shanghai, China) was added, and the mixture was stirred at room temperature for 2 h. After centrifugation at 12,000 rpm for 20 min, the microspheres were washed three times with anhydrous ethanol to obtain Em@MSN-SH microspheres.

[0036] Synthesis of Em@MSN-GDF microspheres: 10 mg of Em@MSN-SH microspheres were dispersed in 1 mL of deionized water. Then, 1 μg of GDF protein (CSB-YP009349HU, CUSABIO, Wuhan, China) was added, and the mixture was stirred at room temperature for 24 h. The resulting precipitate was centrifuged at 15000 rpm for 20 min to obtain Em@MSN-GDF microspheres.

[0037] 3) Encapsulation efficiency of emodin and drug loading of GDF

[0038] The encapsulation efficiency of emodin at 440 nm was determined by ultraviolet spectrophotometry (UV-5100). First, the UV absorbance of the supernatant before stirring and centrifugation was measured, and then the UV absorbance of the supernatant after stirring and centrifugation was measured. The encapsulation efficiency of emodin was calculated using relevant formulas. The drug loading of GDF was determined using an ELISA kit (CSB-EL009349HU, CUSABIO, Wuhan, China). 10 mM DTT was used to dissociate the protein bound to Em@MSN-GDF. After centrifugation at 12000 rpm for 10 minutes, the supernatant was collected for ELISA detection.

[0039] 4) Preparation and characterization of hydrogels

[0040] To prepare a standard hydrogel, 60 mg of GelMA (R201A2, SinoBioPrint, Shanghai, China), 10 mg of CSMA (R203A1, SinoBioPrint, Shanghai, China) and 2 mg of photoinitiator (lithium phenyl-2,4,6-trimethylbenzoylphosphonate, LAP) (G101A2, SinoBioPrint, Shanghai, China) were dissolved in 1 ml of PBS. The solution was heated to 60 °C until completely dissolved, and then irradiated under 365 nm light for 7 s to form a photocrosslinked hydrogel.

[0041] 5) Rheological analysis

[0042] The gel-water solution was tested using a Thermo HAAKE MARS 60 rheometer with a 25°C (25 mm diameter) parallel plate geometry. Dynamic rheological experiments involved exposing the sample to light (365 nm, 20 mW / cm²). 2 The time-scan oscillation test lasted for 120 seconds, with a strain of 10% (CD mode), a frequency of 1 Hz, and a gap of 0.5 mm. It can be observed that the gel point of the storage modulus (G′) will exceed the time of the loss modulus (G′).

[0043] 6) In vitro swelling and degradation test

[0044] Swelling and degradation tests of the hydrogels were conducted using the specific gravity method, as described in previous studies. The initial wet weight of the hydrogel was denoted as W0, and the wet weight of the hydrogel after 24 hours was denoted as Ws. The swelling ratio was defined as Ws / W0 × 100% (n = 4), and the initial wet weight of the hydrogel was denoted as Wd. The remaining wet weight of the hydrogel after soaking in the enzyme solution (1% w / v collagenase) was denoted as Wt. The degradation rate was defined as (Wd - Wt) / Wd × 100% (n = 4).

[0045] 7) Mechanical performance testing

[0046] Samples (n=3) were prepared as cylinders with a diameter of 10 mm and a height of 2 mm. The mechanical properties of the hydrogels were determined using a dynamic mechanical analyzer (Instaston-5542, Canton, USA). The samples were sputtered at 1 mm / min. -1 The compression rate is increased until the compression depth reaches 80% of the initial height. Then, the elastic modulus is calculated from 10% to 20% of the initial strain-stress curve.

[0047] 8) qRT-PCR and Western Blotting Analysis

[0048] Total RNA was extracted using chloroform according to the previously described method, and reverse transcription was performed using a Takara kit. Gene quantification was performed using SYBR Green. The mRNA expression levels of relevant genes, including cartilage-related genes (COLII, ACAM, SOX9, α-SMA), oxidative stress-related genes (NQO1, GPX1), and inflammation-related genes (TNF-α, IL-6), were calculated using the ΔCT method. Total protein was extracted using RIPA lysis buffer (P0013B, Beyotime, Shanghai, China), followed by SDS-PAGE, membrane transfer, and detection of relevant protein bands.

[0049] 9) Cytotoxicity and biocompatibility

[0050] Cells were seeded at a density of 3000 cells per well in 96-well plates and treated with drugs at a specific ratio. 10 μL of CCK-8 reagent (CK04, Dojindo, Shanghai, China) was added to each well, and the cells were incubated in the dark for 2 hours. The absorbance was measured at 450 nm. Fibroblasts and chondrocytes were mixed at a ratio of 7:3 (3 × 10⁻⁶ cells / well). 26 Cells were seeded into 2 ml hydrogels containing Em@MSN-GDF. Cells were cultured in culture dishes for 1, 4, 7, and 14 days. Cell viability was assessed using a Calcein-AM / PI (live / dead) staining kit (C542, Dojindo, Shanghai, China) according to the manufacturer's instructions. F-actin and cell nuclei were stained with Actin-Tracker (C2205S, Beyotime, Shanghai, China) and DAPI (C1006, Beyotime, Shanghai, China), respectively. Fluorescence images were acquired using a Leica Thunder DMI8 fluorescence microscope, and fluorescence intensity was quantified using ImageJ software.

[0051] 10) Antioxidant test

[0052] RAW264.7 (1×10) 5 Cells were seeded in 6-well plates. Oxidative stress was induced by stimulation with 1 mM H₂O₂, followed by treatment with Em@MSN and Em@MSN-GDF for 24 hours. DCFH-DA (1:1000) (S0033S, Beyotime, Shanghai, China) was added, and the plates were incubated at 37°C for 2 hours. Fluorescence intensity was observed and photographed under a Leica Thunder DMI8 microscope, and quantification was performed using ImageJ software.

[0053] 11) Anti-inflammatory experiment

[0054] RAW264.7 cells (density 1×10⁻⁶) 5 Cells were seeded onto 6-well cell slides. Cells were stimulated with 100 ng / mL LPS for 16 h, then treated with Em@MSN and Em@MSN-GDF for 24 h. The supernatant was collected, and the cells were incubated with CD86 antibody and CD206, respectively, and analyzed by flow cytometry.

[0055] 12) Transwell and Alcian staining

[0056] This experiment used Transwell culture chambers with different pore sizes to study the effects of chondrocytes on fibroblasts. First, fibroblasts were implanted into Transwell culture chambers with a pore size of 8 μm and co-cultured with chondrocytes. After 24 and 72 hours of co-culture, crystal violet staining was used to observe the migration of fibroblasts. Crystal violet staining was used to detect the number and migration ability of fibroblasts.

[0057] Simultaneously, fibroblasts and chondrocytes were engraved into separate Transwell culture chambers with 0.4 μm pores and cultured for 7 days. After the culture period, the content of glycosaminoglycans (GAGs) in fibroblasts was detected by Alcian blue staining, which is an important marker of chondrocyte differentiation. Alcian blue staining to detect GAG content can indicate whether fibroblasts are differentiating into chondrocytes.

[0058] 13) In vivo evaluation of cell co-culture ratio

[0059] Cells (FBs, FBs7:CHs3 = 7:3, FBs3:CHs7 = 3:7, CHs) were seeded into hydrogels at a density of 4 x 10⁷ cells / mL to form discs with a diameter of 1 cm. These intervertebral discs were then subcutaneously implanted into nude mice. After 6 weeks, histological staining was performed to determine the optimal cell co-culture ratio for promoting cartilage regeneration.

[0060] 14) Validation of bioprinting in in vivo cartilage regeneration and cartilage formation

[0061] To assess in vivo cartilage regeneration, (REGENOVO) was used. WS developed a 3D bioprinting framework that mixes fibroblasts and chondrocytes in a 7:3 ratio (5 × 10⁻⁶). 7 Cells / mL were loaded with GelMA / CSMA hydrogel. This cell-loaded hydrogel was integrated into a polycaprolactone (PCL) scaffold, which was created using 3D bioprinting technology, measuring 10 × 10 × 2 mm with a 2 mm gap structure. The scaffold assembled from cells and hydrogel was implanted subcutaneously into nude mice. After 6 weeks, chondrogenesis and regeneration were evaluated to validate the potential of different cell ratios to promote in vivo chondrogenesis.

[0062] 15) Establishment and repair of meniscus defect model

[0063] Using one-month-old New Zealand white rabbits, fibroblasts and chondrocytes were collected for autologous implantation. First, a PCL meniscus-shaped scaffold with 2mm pores was 3D printed. Then, hydrogel was mixed with cells and Em@MSN-GDF and injected into the scaffold. During the procedure, either a PCL scaffold or Em@MSN-GDF-PCL was used to replace the medial meniscus. MRI scans were performed at 6 and 12 weeks post-operation to monitor meniscus regeneration. Tissue specimens were collected at 12 weeks for histological staining to assess the degree of meniscus regeneration.

[0064] 16) Histochemical and immunohistochemical analysis

[0065] The tissue was fixed in 4% paraformaldehyde for 2 days, embedded in paraffin, and sectioned into 6 μm thick sections. Following the manufacturer's protocol, the sections were further stained with hematoxylin and eosin (HE), safranin (SO), COL II, and COL I to assess the regenerated tissue structure.

[0066] In addition, after antigen recovery and serum blocking, sections were incubated overnight at 4°C with monoclonal antibodies of COL I (1:300), COL II (1:50), α-SMA (1:500), VEGF (1:200), and VMF (1:100), followed by incubation with fluorescent secondary antibody for 1 hour. The staining results were observed under a fluorescence microscope. Finally, a semi-quantitative histological scoring system was used to further evaluate meniscus regeneration.

[0067] 17) Statistics

[0068] All quantitative data are expressed as mean ± standard deviation (SD). All statistics were analyzed by Student's t-test, and a p-value < 0.05 was considered statistically significant. In the figures and tables shown, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0069] Example 1: Preparation and characterization of Em@MSN-GDF.

[0070] Three types of mesoporous silica microspheres, namely Em@MSN, Em@MSN-SH and Em@MSN-GDF, were prepared using hexadecyltrimethylammonium chloride (CTAC) as a template agent and tetraethyl orthosilicate (TEOS) as a silicon source. Figure 1 A represents the preparation process of Em@MSN-GDF microspheres. The microstructural characteristics of the samples were studied using transmission electron microscopy (TEM), such as... Figure 1 As shown in Figure B, the synthesis of Em@MSN, Em@MSN-SH, and Em@MSN-GDF was observed and analyzed. The image shows a uniformly distributed pore structure on the Em@MSN surface, indicating the successful synthesis of hollow mesoporous silica. Simultaneously, an irregular coating was observed on the surfaces of both the Em@MSN-SH and Em@MSN-GDF samples. This coating masks the surface pores, exhibiting a distinct but uneven coating appearance. The results of this coating layer indicate that surface functionalization and GDF protein loading have a significant impact on the nanoparticles. The uneven distribution of surface functional groups and the differences in the loaded protein lead to uneven coating thickness. Furthermore, Figure 1 C shows the particle size analysis of three mesoporous silica nanoparticle drug delivery systems, with average particle sizes of 151±23.69 nm, 168±18.04 nm, and 169±15.37 nm, respectively. The zeta potentials were -36.76±0.70 mV, -40.07±1.05 mV, and 12.77±1.07 mV, respectively. Figure 1 D). The results showed that with the coupling of thiol-modified Em@MSN and GDF, the particle size of mesoporous silica gradually increased, and the potential of Em@MSN-GDF increased by about 49 mV compared with Em@MSN.

[0071] Through energy dispersive spectroscopy (EDS) images ( Figure 1 Electron microscopy images of Em@MSN-GDF before and after thiol modification and protein loading clearly demonstrate the successful synthesis of the mesoporous silica nanoparticle drug delivery system. The results show that Em@MSN-GDF contains C (52.45%), N (0.81%), O (21.15%), Si (21.49%), and S (4.11%) elements. Figure 1 F), the elements are uniformly distributed within the nanomaterial, and the particle size of Em@MSN-GDF is 170 nm. Figure 1 G). Furthermore, to further verify the success of thiol modification and protein deposition on mesoporous silica surfaces, we performed 29Si NMR ( Figure 1 H) and 1H NMR ( Figure 1 I) Analysis. In Figure 1In the 29Si NMR spectrum, a peak at 3.5 ppm was observed, and the peak value of Em@MSN-GDF decreased, indicating successful thiol modification and protein loading of mesoporous silica. The peak at -65 ppm in the 29Si NMR spectrum further confirmed the success of the thiol modification. Furthermore, X-ray photoelectron spectroscopy (XPS) was used to identify the functional groups in the nanomaterials. The XPS S-2p spectra of Em@MSN, Em@MSN-SH, and Em@MSN-GDF are shown below. Figure 1 J、 Figure 1 K, Figure 1 L. The peaks at 162.5 eV and 164 eV correspond to the SH and SS bonds. After modification with the silane coupling agent, the peak of the new element S is... Figure 1 K and Figure 1 The presence of L clearly indicates the successful synthesis of Em@MSN-SH. Comparing the changes in elemental peaks during the synthesis process, the SS peak area of ​​Em@MSN-GDF was found to have increased significantly, indicating a relative increase in SS content, which may be due to protein loading. Furthermore, the encapsulation efficiency of emodin in the three nanomaterials showed no significant difference, remaining at approximately 14%. Figure 1 Finally, we discussed GDF(M). Figure 1 N) and emodin (N) Figure 1 The sustained-release profile of O was characterized over 45 days, with GDF release reaching 50% and emodin release approaching 60%.

[0072] In summary, these results confirm that emodin and GDF can achieve sustained release over a long period in the Em@MSN-GDF mesoporous silica nanoparticle drug delivery system. This prolonged release is crucial for achieving long-term therapeutic effects in environments with high oxidative stress and inflammation, such as those encountered after meniscectomy. The synergistic release of the two drugs helps modulate the cartilage immune microenvironment.

[0073] Example 2: Physicochemical and biological evaluation of Em@MSN-GDF / GC.

[0074] This embodiment evaluated the physicochemical properties, biocompatibility, and cell proliferation-promoting effect of the Em@MSN-GDF / GC hydrogel. Figure 2 As shown in A, at 365nm and 20mW / cm 2 Under ultraviolet irradiation, the hydrogel rapidly transformed from solution to gel state within 60 seconds. SEM images of GC and Em@MSN-GDF / GC after freeze-drying were observed. Figure 2 B) revealed that both hydrogels possessed microporous structures with minimal pore size variation. Rheological analysis indicated that the hydrogel underwent significant photopolymerization within 60 seconds, forming a gel ( Figure 2C) There was no significant difference in storage modulus (G') and loss modulus (G”) between GC and Em@MSN-GDF / GC (P>0.05).

[0075] Furthermore, there was no significant difference in compressive stress between GC and Em@MSN-GDF / GC. Figure 2 D). Swelling and degradation experiments showed no significant difference in swelling and degradation rates between GC and Em@MSN-GDF / GC. Figure 2 E and Figure 2 F). These results indicate that the introduction of Em@MSN-GDF did not significantly alter the microporous structure, mechanical strength, and compressive strength of the GC hydrogel, demonstrating that the system maintained the same structure and mechanical properties as the original GC hydrogel. This stability is crucial for the application of tissue engineering scaffolds, as the hydrogel must maintain mechanical support during degradation. Furthermore, the rapid photocrosslinking process (below 60 seconds) facilitates rapid manipulation in clinical applications, which is particularly important for reducing intraoperative contamination and minimizing cell damage.

[0076] Furthermore, the biocompatibility of Em@MSN-GDF / GC was assessed through live / dead staining, cell morphology analysis, and cell proliferation studies. Figure 2 As shown in G, AM-PI staining and F-actin staining revealed that FBs and CHs cells were able to proliferate and spread well within the Em@MSN-GDF / GC matrix, indicating that Em@MSN-GDF / GC effectively supports cell growth and proliferation. Figure 2 H showed that the cells exhibited high viability after 14 days of in vitro culture, with cell viability gradually increasing from day 1 to day 14. Figure 2 As shown in Figure I. Simultaneously, F-actin fluorescence statistics showed that cell diffusion increased progressively over the 14-day observation period, with an increasing trend from day 1 to day 14. Figure 2 J).

[0077] To further confirm the promoting effect of Em@MSN-GDF on cell proliferation, cytotoxicity experiments were also conducted. Figure 2 K showed that Em@MSN-GDF had no significant toxicity to cells at concentrations of 10 μg / mL and 20 μg / mL. To protect cells from the potentially harmful effects of drug toxicity, a concentration of 10 μg / mL was used in subsequent cell experiments.

[0078] The results showed that the Em@MSN-GDF / GC hydrogel has good cell compatibility, enabling cells to survive, spread and proliferate in vitro, providing strong support for meniscus regeneration.

[0079] Example 3 Evaluation of in vitro antioxidant and anti-inflammatory effects

[0080] FBs7 / CHs3 cells were treated with 1 mM H2O2 to induce an increase in reactive oxygen species (ROS) levels. Intracellular ROS levels were assessed using the DCFH-DA (1:1000) probe after 24 hours of treatment with 1 mM H2O2 in combination with Em@MSN and Em@MSN-GDF. Results showed that the fluorescence intensity of the Em@MSN and Em@MSN-GDF treatment groups was significantly reduced compared to the H2O2 treatment group alone. Figure 3 A and Figure 3 B), the mRNA expression levels of the related antioxidant genes glutathione peroxidase 1 (GPX1) and NAD(P)H quinone dehydrogenase 1 (NQO1) were also significantly reduced. Figure 3 C and Figure 3 (D) These results indicate that the emodin released by Em@MSN and Em@MSN-GDF can effectively scavenge ROS. This antioxidant effect is particularly important in the meniscus regeneration environment, as continuous ROS accumulation can damage cells and impair their repair capabilities.

[0081] Furthermore, to investigate the roles of Em@MSN and Em@MSN-GDF in macrophage anti-inflammatory phenotypic polarization, we used lipopolysaccharide (LPS) as a pro-inflammatory agent and analyzed macrophage polarization results by flow cytometry. Figure 3 E, Figure 3 F and Figure 3 (G) Compared with the control group, the proportion of M1 (CD86+) macrophages was significantly increased in the LPS treatment group. In contrast, the proportion of M1 macrophages was significantly decreased in the Em@MSN and Em@MSN-GDF treatment groups, while the proportion of M2 (CD206+) macrophages was significantly increased in the Em@MSN and Em@MSN-GDF treatment groups. This indicates that Em@MSN and Em@MSN-GDF promote the transformation of macrophages to an anti-inflammatory phenotype, which helps to suppress excessive inflammation and support tissue repair. To further validate this finding, we used qRT-PCR to measure the relative mRNA expression levels of inflammatory factors in macrophages, including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Figure 3 H and 3I results showed that, compared with the LPS treatment group, the relative expression levels of pro-inflammatory factors in the Em@MSN and Em@MSN-GDF treatment groups were significantly reduced. This anti-inflammatory effect is particularly important for improving the postoperative microenvironment, as persistent inflammation can hinder meniscus regeneration and lead to cartilage degeneration. Figure 3 J provides a brief overview of the antioxidant and anti-inflammatory effects of Em@MSN-GDF.

[0082] The above results indicate that the antioxidant and anti-inflammatory functions of Em@MSN and Em@MSN-GDF are attributed to the release of emodin, which promotes the differentiation of macrophages into an anti-inflammatory phenotype.

[0083] Example 4: Extracellular matrix and GDF promote chondrogenic differentiation of FBs

[0084] The effects of extracellular matrix secreted by chondrocytes on fibroblasts were further investigated using Transwell chambers with different pore sizes for characterization. Figure 4 Figure A shows the experimental setup, in which fibroblasts were cultured in the upper chamber and chondrocytes in the lower chamber. The effect of chondrocytes on the migration ability of fibroblasts was investigated using Transwell chambers with an 8 μm pore size. After co-culturing for 24 and 72 h, fibroblasts were stained with crystal violet, as shown in Figure A. Figure 4 As shown in B, the increased staining intensity indicates that the extracellular matrix of chondrocytes significantly affects fibroblast migration.

[0085] The effect of chondrocytes on chondrogenic differentiation of fibroblasts was investigated using Transwell chambers with a pore size of 0.4 μm. Results are as follows: Figure 4 As shown in Figure C, fibroblasts co-cultured with chondrocytes exhibited higher levels of Alcian blue staining compared to fibroblasts cultured alone. These results suggest that the extracellular matrix derived from chondrocytes has a favorable influence on fibroblast chondrogenic differentiation.

[0086] Similarly, GDF shows great potential in tissue engineering, particularly in promoting stem cell differentiation and chondrogenesis. Studies have shown that GDF can effectively induce fibroblasts to differentiate into fibroblast chondrocytes.

[0087] Chondrocytes were cultured with 100 ng / mL GDF protein to investigate the function of GDF in promoting fibroblast differentiation into fibrochondrocytes. After 7 days of in vitro culture, the expression of cartilage-related genes in FBs (fibroblasts) and the FBs+GDF group was analyzed by Western blotting and qRT-PCR. Figures D, E, F, and G show that compared with the FBs group, the mRNA levels of COL II, ACAN, α-SMA, and SOX9 were increased in the FBs+GDF group. Compared with the FBs group, the protein levels of COL II, ACAN, α-SMA, and SOX9 were also increased in the FBs+GDF group. Figure 4 Both Western blotting and qRT-PCR results indicated that GDF has the ability to promote the differentiation of fibroblasts into fibroblast chondrocytes. These results suggest that both the extracellular matrix of chondrocytes and GDF affect the chondrocyte differentiation of fibroblasts.

[0088] To better promote fibrocartilage regeneration, glycoprotein D (GDF) was combined with the function of the extracellular matrix of chondrocytes. The feasibility of fibrocartilage regeneration with different cell ratios under GDF treatment was investigated by subcutaneously implanting fibroblasts and chondrocytes in nude mice. Figure 4 As shown in Figure I, 30 million cells were mixed with 100 ng / mL GDF in a GC hydrogel to form a 10 mm diameter disc, which was then implanted subcutaneously into nude mice. Figure 4 J shows the surgical procedure of implanting an intervertebral disc under the skin of a nude mouse. Figure 4 L represents the initial formation of cartilage-like samples 6 weeks after implantation of different cell ratios. The tissue transparency of the FBs7 / CHs3 group was similar to that of the FBs3 / CHs7 group. Histological analysis further confirmed that HE and SO staining showed that the regenerated cartilage exhibited similar levels of typical cartilage-specific lacunar structures between the FBs7 / CHs3 and FBs3 / CHs7 groups. Figure 4 M). In the FBs7 / CHs3 cell ratio group, the level of COLII expression in tissues was the same compared to the FBs3 / CHs7 group (M). Figure 4 N). Furthermore, the α-SMA expression level in the FBs7 / CHs3 group was higher than that in the FBs3 / CHs7 group (N). Figure 4 These results indicate that effective cartilage regeneration can be achieved with only 30% chondrocyte ratio during GDF treatment.

[0089] Example 5: Evaluation of Em@MSN-GDF in in vivo chondrogenesis

[0090] To improve the stability and mechanical strength of cartilage regeneration, a rectangular PCL scaffold measuring 1cm long, 1cm wide, and 2mm high was fabricated using 3D printing technology, with 2mm long holes. Figure 5 As shown in Figure A, a GC hydrogel containing 50 million FBs7 / CHs3 cells / mL was implanted into a PCL scaffold to form an implant.

[0091] The grafts were divided into three groups (GC-PCL group, Em@MSN / GC-PCL group, and Em@MSN-GDF / GC-PCL group), and then subcutaneously implanted in nude mice to further investigate the promoting effect of GDF on chondrogenesis. Six weeks after implantation, the morphology of the implants in the three groups remained essentially unchanged. Figure 5 B).

[0092] Next, HE staining, SO staining, COL II and α-SMA immunofluorescence staining were performed. Figure 5 C) Mechanical testing of the subcutaneous implanted tissue was performed 6 weeks later. Figure 5As shown in Figure D, there was no significant difference in Young's modulus between the Em@MSN / GC-PCL group and the Em@MSN-GDF / GC-PCL group, but both were significantly higher than those in the GC-PCL group. Statistical analysis of the fluorescence intensity of COLII and α-SMA showed no significant difference in COLII expression levels between the GC-PCL group and the Em@MSN / GC-PCL group. However, compared with both the GC-PCL group and the Em@MSN / GC-PCL group, the COLII expression level in the Em@MSN-GDF / GC-PCL group was significantly higher. Figure 5 E). Furthermore, compared to the GC-PCL group and the Em@MSN / GC-PCL group, the α-SMA expression level in the Em@MSN-GDF / GC-PCL group was significantly increased (E). Figure 5 F).

[0093] The above results indicate that the cartilage phenotype of the Em@MSN-GDF / GC-PCL group exhibited superior characteristics compared to the GC-PCL and Em@MSN / GC-PCL groups. This is mainly attributed to the sustained release of GDF, which is beneficial for fibrocartilage differentiation and the formation of functional cartilage matrix. Through this method, the Em@MSN-GDF / GC-PCL group not only demonstrated enhanced biocompatibility and mechanical properties but also showed greater potential in promoting chondrocyte differentiation and cartilage matrix formation.

[0094] Example 6: Evaluation of Meniscus Replacement and In Vivo Repair

[0095] This embodiment further verifies the feasibility of Em@MSN-GDF for meniscus regeneration. First, New Zealand rabbits underwent meniscectomy and replacement surgery, and a meniscus-shaped polycaprolactone (PCL) scaffold was fabricated using 3D bioprinting technology. Fibroblasts and chondrocytes were loaded onto the scaffold in a 7:3 ratio and bound to an Em@MSN-GDF / GC hydrogel containing 10 μg / mL Em@MSN-GDF powder. The cell density was set at 60 million cells / mL. The mixture was then cross-linked under ultraviolet light (365 nm) and injected into the PCL scaffold. Figure 6 A).

[0096] These stents were implanted into a New Zealand rabbit meniscus defect model, and the surgical procedure was as follows: Figure 6 As shown in Figure B. Using a PCL stent as a control group, MRI imaging of the knee joint was performed at 6 and 12 weeks post-surgery. Figure 6 C) Observe the cartilage regeneration. Twelve weeks post-surgery, the replaced medial meniscus successfully formed a structure similar to the normal meniscus. Figure 6 D and Figure 6E). Further histological analysis showed that the menisci in the Em@MSN-GDF / GC-PCL group were not only morphologically very similar to normal menisci, but also exhibited regional specificity in cell phenotype and extracellular matrix deposition, consistent with the collagen heterogeneity of normal menisci. Figure 6 F).

[0097] Quantitative fluorescence analysis of α-SMA, VEGF, and VWF in the PCL and Em@MSN-GDF / GC-PCL groups showed that the expression levels of α-SMA, VEGF, and VWF in the Em@MSN-GDF / GC-PCL group were significantly higher than those in the PCL group. Furthermore, the vascular distribution in the lateral and medial regions of the Em@MSN-GDF / GC-PCL group also showed significant differences, consistent with the vascular distribution of a normal meniscus. Figure 6 G, Figure 6 H and Figure 6 I).

[0098] Finally, the articular cartilage was assessed according to the International Cartilage Repair Society (ICRS) classification of cartilage lesions, category 55. The PCL group showed more severe cartilage damage. Figure 6 I). In summary, the meniscus regeneration effect of the Em@MSN-GDF / GC-PCL group was significantly better than that of the PCL group, and was close to the characteristics of a normal meniscus.

[0099] In summary, the Em@MSN-GDF / GC-PCL group showed significantly better meniscus regeneration than the PCL group, exhibiting characteristics similar to those of a normal meniscus. This result demonstrates that combining bioprinting technology with a GDF delivery system can effectively enhance the meniscus regeneration process. Specifically, the release of Em@MSN-GDF promotes cell proliferation and differentiation, leading to favorable characteristics in the regenerated meniscus regarding morphology, cell phenotype, and extracellular matrix deposition.

Claims

1. A composition comprising microspheres, characterized in that... Including growth differentiation factors and emodin, it improves inflammation and oxidative stress in the microenvironment and promotes the process of chondrogenic differentiation. Methods for preparing microspheres of the composition include: First, emodin was loaded onto dSiO2 silica spheres. Then, mesoporous silica microspheres were prepared using hexadecyltrimethylammonium chloride as a template agent and tetraethyl orthosilicate as a silicon source. 3-mercaptopropyltrimethoxysilane was added to obtain surface-thiolized microspheres. After mixing with GDF protein, mesoporous silica microspheres loaded with growth differentiation factors and emodin were obtained.

2. A hydrogel, characterized in that... Microspheres of the composition described in claim 1 are added to a gel GC composed of GelMA and chondroitin sulfate methacrylate (CSMA) to form a cartilage-specific matrix hydrogel.

3. The hydrogel according to claim 2, characterized in that... The weight ratio of GelMA to CSMA is 6:

1.

4. The hydrogel according to claim 2, characterized in that... It also includes chondrocytes and fibroblasts.

5. A PCL scaffold, manufactured by 3D printing, containing chondrocytes and fibroblasts, a gel GC consisting of GelMA and chondroitin sulfate methacrylate (CSMA), and microspheres of the composition of claim 1.

6. The application of the composition according to claim 1, characterized in that... Applications in the manufacture of medical devices for meniscus replacement surgery.

7. The application according to claim 6, characterized in that... The medical device is a stent with PCL as the frame, and also includes at least one of the following components: Microspheres of the composition of claim 1; A gel composed of GelMA and chondroitin sulfate methacrylate CSMA; and Chondrocytes and fibroblasts.

8. A medical device, characterized in that... Microspheres comprising the composition of claim 1.

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