Mitochondria-activated sponge for repairing articular cartilage defects and preparation method thereof

Abstract

This invention belongs to the field of biomaterials and provides a mitochondrial-activated sponge for repairing articular cartilage defects and its preparation method. The sponge is formed by freeze-drying a hydrogel of thiol-modified hyaluronic acid-exfoliated MoS2 nanoparticles, thiol-modified hyaluronic acid, and polyphenol-modified silk fibroin at pH 7-8. The thiol-modified hyaluronic acid-exfoliated MoS2 nanoparticles consist of thiol-modified hyaluronic acid and MoS2 nanoparticles. The thiol-modified hyaluronic acid is located on the surface of the MoS2 nanoparticles and chemically bonded to them via Mo-S bonds. Simultaneously, the thiol-modified hyaluronic acid undergoes oxidative self-crosslinking. This invention improves the dispersibility, stability, and cellular targeting ability of the MoS2 nanoparticles in the hydrogel through the exfoliation modification of thiol-modified hyaluronic acid. The sponge constructed based on this improves the cartilage defect repair performance.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials and relates to mitochondrial-activated sponges for the repair of articular cartilage defects and their preparation methods. Background Technology

[0002] Compared to exogenous stem cells and autologous / allogeneic cartilage transplantation, microfracture surgery relying on endogenous stem cells to promote cartilage defect repair is a safer and more convenient approach. The process by which endogenous stem cells participate in the remodeling of articular cartilage involves cell recruitment, proliferation, and differentiation, accompanied by matrix deposition and structural remodeling; these processes depend on a sufficient energy supply. Mitochondria are the main organelles responsible for energy production within cells. The mitochondrial respiratory chain and adenosine triphosphate (ATP) synthase drive oxidative phosphorylation to continuously produce ATP, providing energy for cellular activities and tissue regeneration. Oxidative phosphorylation involves electron transport processes between multiple complexes (i.e., redox processes). Establishing an electrochemical gradient through electron transport to ensure ATP synthesis plays a decisive role in the rate of oxidative phosphorylation. Regulating electron transport processes is one of the effective means to promote oxidative phosphorylation. Molybdenum (Mo), a transition metal, is one of the trace elements required by all living organisms. It participates in the synthesis of molybdenum cofactors and serves as the catalytic center of molybdenum enzymes, releasing molybdenum ions (Mo) in different valence states. 4+ Mo 5+ Mo 6+ Molybdenum participates in the redox process. Located in the mitochondrial intermembrane space, molybdenum enzymes—sulfite oxidase (SO)—catalyze the oxidation of sulfite to sulfate. The electrons generated in this process can be directly transferred to the physiological electron acceptor cytochrome C, thereby regulating oxidative phosphorylation. Therefore, by introducing molybdenum to regulate electron chain transfer, it is hoped that oxidative phosphorylation can be promoted, leading to the generation of more ATP, which is beneficial for ensuring the energy supply for stem cell chondrogenesis.

[0003] Oxidative stress and the inflammatory microenvironment following articular cartilage injury are key obstacles to the initiation of endogenous stem cell repair processes. Currently, research on molybdenum disulfide (MoS2), with peroxidase-like activity, has made some progress in regulating the oxidative stress microenvironment of damaged tissues. Furthermore, to address the issues of hyaluronic acid hydrogels hindering cell adhesion and spreading, CN115850809A discloses a molybdenum disulfide-enhanced thiolized hyaluronic acid injectable hydrogel, its preparation method, and its applications. By introducing a small amount of MoS2 nanosheets into a thiolized hyaluronic acid solution and forming a hydrogel, the resulting hydrogel contains 70–100 μg / mL of MoS2 nanosheets and 10–30 mg / mL of cross-linked thiolized hyaluronic acid, thus endowing the hydrogel with excellent cell adhesion properties. Therefore, introducing MoS2 into cartilage repair materials may offer hope for improving their repair performance.

[0004] However, MoS2 nanosheets are prone to aggregation in physiological environments, causing localized cytotoxicity, and are easily oxidized, leading to a decrease in their peroxidase-like activity. Furthermore, MoS2 nanosheets are difficult to target into cells and are not effectively taken up by cells to exert their effects. These factors have become technical bottlenecks limiting the effective function of MoS2 in cartilage repair materials. Currently, the solution ultrasonic exfoliation method is mainly used to prepare monolayer or few-layer MoS2 nanosheets from MoS2 crystals. However, after dispersing the MoS2 nanosheets obtained by this method in a thiolized hyaluronic acid aqueous solution, significant aggregation still occurs. After gelling the solution, the MoS2 nanosheets are ultimately unevenly dispersed in the hydrogel as aggregates. Furthermore, current ultrasonic exfoliation processes for preparing monolayer or few-layer MoS2 nanosheets utilize organic solvents such as N-methylpyrrolidone, isopropanol, ethanol, dimethylformamide, and dimethyl sulfoxide, which can lead to limited biocompatibility of the resulting MoS2 nanosheets. When water is used as the exfoliation solvent, stabilizers such as polyvinylpyrrolidone and polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymers are necessary to prevent secondary aggregation and stacking of the exfoliated MoS2 nanosheets; otherwise, stable few-layer MoS2 nanosheets are difficult to obtain. Therefore, if appropriate methods can be used to increase the dispersibility and stability of the exfoliated MoS2 nanosheets, better maintain their catalytic activity and endow them with the ability to target and enter cells, and on this basis, develop articular cartilage defect repair scaffold materials that can regulate the oxidative stress microenvironment and promote the oxidative phosphorylation process, it will, on the one hand, reduce the obstacles of the post-injury oxidative stress microenvironment to the initiation of the repair process by endogenous stem cells, and on the other hand, provide sufficient energy supply for the chondrogenic differentiation of endogenous stem cells, thus having a positive promoting effect on the repair of articular cartilage defects. Summary of the Invention

[0005] To address the issues of insufficient energy supply and limitations imposed by the oxidative stress microenvironment after injury during endogenous stem cell differentiation into chondrocytes, as well as the limitations imposed by the aggregation and uneven distribution of MoS2 in the matrix material in existing MoS2-based cartilage repair materials, this invention provides a mitochondrial-activated sponge for articular cartilage defect repair and its preparation method. The invention improves the dispersibility, stability, and cellular targeting ability of nano-MoS2 in hydrogels through the exfoliation modification of thiol-modified hyaluronic acid, and on this basis, constructs a sponge for cartilage defect repair, thereby enhancing the repair performance of existing cartilage repair materials.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows:

[0007] Mitochondrial-activated sponge for repairing articular cartilage defects is formed by freeze-drying a hydrogel formed by reacting nano-molybdenum disulfide modified with thiol-modified hyaluronic acid, thiol-modified hyaluronic acid, and polyphenol-modified silk fibroin in a mass ratio of (0.002~0.02):(0.5~8):1 at a pH of 7~8.

[0008] The thiol-modified hyaluronic acid-exfoliated molybdenum disulfide nanoparticles are composed of thiol-modified hyaluronic acid and molybdenum disulfide nanoparticles. The thiol-modified hyaluronic acid is located on the surface of the molybdenum disulfide nanoparticles, and the thiol-modified hyaluronic acid and molybdenum disulfide nanoparticles are chemically bonded through Mo-S bonds. At the same time, the thiol-modified hyaluronic acid undergoes oxidative self-crosslinking. The preparation method of the thiol-modified hyaluronic acid-exfoliated molybdenum disulfide nanoparticles is as follows: molybdenum disulfide is added to an aqueous solution of thiol-modified hyaluronic acid, and the mass ratio of thiol-modified hyaluronic acid to molybdenum disulfide is (0.5~5):1. The mixture is subjected to thorough ultrasonic treatment at 1~4 ℃, centrifuged, and the resulting solid phase is washed with water to obtain the final product.

[0009] Furthermore, in the above-mentioned technical solution for mitochondrial-activated sponges, when preparing the thiol-modified hyaluronic acid-exfoliated molybdenum disulfide nanoparticles, the concentration of the aqueous solution of thiol-modified hyaluronic acid is controlled to be 1-10 mg / mL; when preparing the mitochondrial-activated sponge hyaluronic acid-exfoliated molybdenum disulfide nanoparticles, the ultrasonic power is controlled to be 200-500 W, and the ultrasonic time is 2-8 h. Even further, the prepared thiol-modified hyaluronic acid-exfoliated molybdenum disulfide nanoparticles are dispersed in water for storage.

[0010] In the aforementioned mitochondrial-activated sponge technology, during the process of forming a hydrogel by reacting mercapto-modified hyaluronic acid nano-molybdenum disulfide, mercapto-modified hyaluronic acid, and polyphenol-modified silk fibroin, the reactions that occur include oxidative crosslinking between mercapto-modified hyaluronic acid nano-molybdenum disulfide and mercapto-modified hyaluronic acid, oxidative self-crosslinking of mercapto-modified hyaluronic acid, Michael addition reaction between mercapto-modified hyaluronic acid and polyphenol-modified silk fibroin, and Michael addition reaction between mercapto-modified hyaluronic acid nano-molybdenum disulfide and polyphenol-modified silk fibroin.

[0011] In the above-mentioned mitochondrial-activated sponge technical solution, the structure of the thiol-modified hyaluronic acid is shown in formula (I). In the thiol-modified hyaluronic acid, the grafting rate of cysteine ​​is preferably 5% to 65%, and the grafting rate of cysteine ​​is further preferably 10% to 30%. Furthermore, the molecular weight of the hyaluronic acid used as the basis for thiol-modified hyaluronic acid modification does not exceed 1000 kDa. For example, the molecular weight of the hyaluronic acid used as the basis for thiol-modified hyaluronic acid modification can be 300 to 1000 kDa.

[0012] (I)

[0013] In the aforementioned mitochondrial-activated sponge technology, the polyphenol-modified silk fibroin is formed by grafting polyphenols onto silk fibroin, and the polyphenols include dopamine, tannic acid, or caffeic acid. The polyphenol content in the polyphenol-modified silk fibroin is 5-20 μg / mg.

[0014] In the above-mentioned technical solution for mitochondrial-activated sponges, the thiol-modified hyaluronic acid and polyphenol-modified silk fibroin can be prepared using existing methods.

[0015] For example, a feasible method for preparing thiol-modified hyaluronic acid is as follows: hyaluronic acid is dissolved in water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added, the pH of the solution is adjusted to 4.75~5, and the mixture is thoroughly mixed at room temperature. Then cysteine ​​hydrochloride is added and reacted at room temperature for 10~12 h. After dialyzing and freeze drying, thiol-modified hyaluronic acid is obtained.

[0016] For example, a feasible method for preparing polyphenol-modified silk fibroin is as follows: degumming silkworm cocoons to obtain degummed silk fibroin; fully dissolving the degummed silk fibroin in a 9-10 mol / L LiBr solution at 55-65 ℃; then dialyzing with deionized water to obtain a silk fibroin solution; adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and polyphenols to the silk fibroin solution; controlling the pH of the resulting mixture at 4.75-5; reacting at room temperature for 12-24 h under nitrogen protection; dialyzing; and freeze-drying to obtain polyphenol-modified silk fibroin.

[0017] In the aforementioned technical solution for the mitochondrial-activated sponge, the sponge has a pore size of 20–120 μm and a porosity of 60%–75%.

[0018] In the above-mentioned mitochondrial-activated sponge technical solution, the sponge is preferably formed by freeze-drying a hydrogel formed by reacting nano-molybdenum disulfide modified with thiol-modified hyaluronic acid, thiol-modified hyaluronic acid and polyphenol-modified silk fibroin in a mass ratio of (0.01~0.02):(0.5~8):1 under a pH value of 7~8.

[0019] This invention also provides a method for preparing the above-mentioned mitochondrial-activated sponge for repairing articular cartilage defects, comprising the following steps:

[0020] (1) Dissolve thiol-modified hyaluronic acid in water to obtain a thiol-modified hyaluronic acid solution; add a dispersion of thiol-modified hyaluronic acid-exfoliated molybdenum disulfide nanoparticles to the thiol-modified hyaluronic acid solution and mix thoroughly to obtain a mixture; fully disperse polyphenol-modified silk fibroin in water to obtain a polyphenol-modified silk fibroin solution.

[0021] (2) The mixture is thoroughly mixed with the polyphenol-modified silk fibroin solution and the pH value is adjusted to 7-8 to obtain a gel precursor solution. The gel precursor solution is transferred to a mold and allowed to stand until the gel precursor solution is converted into a hydrogel state. The obtained hydrogel is freeze-dried to obtain a mitochondrial activated sponge for the repair of articular cartilage defects.

[0022] In the gel precursor solution, the mass ratio of mercapto-modified hyaluronic acid-exfoliated molybdenum disulfide nanoparticles, mercapto-modified hyaluronic acid, and polyphenol-modified silk fibroin is (0.002~0.02):(0.5~8):1.

[0023] In step (1) of the above preparation method, the dispersion of the mercapto-modified hyaluronic acid exfoliated molybdenum disulfide nanoparticles refers to the nanoparticles of mercapto-modified hyaluronic acid exfoliated molybdenum disulfide that are fully dispersed in water.

[0024] In step (2) of the above preparation method, the concentration of polyphenol-modified silk fibroin in the gel precursor solution is 4~20 mg / mL. Further, the concentration of polyphenol-modified silk fibroin in the gel precursor solution is preferably 4~10 mg / mL.

[0025] In step (2) of the above preparation method, it is preferable to let the gel precursor solution stand at room temperature for 5 to 15 minutes to obtain the hydrogel.

[0026] In the gel precursor solution of step (2) of the above preparation method, the preferred mass ratio of the thiol-modified hyaluronic acid-exfoliated molybdenum disulfide nanoparticles, thiol-modified hyaluronic acid and polyphenol-modified silk fibroin is (0.01~0.02):(0.5~8):1.

[0027] This invention has been experimentally verified to be true that:

[0028] (1) This invention successfully exfoliates nano-MoS2 using thiol-modified hyaluronic acid in one step, reducing the face-to-face stacking of nano-MoS2. The resulting thiol-modified hyaluronic acid-exfoliated molybdenum disulfide nanoparticles (MoS2@HS) exhibit stronger electrostatic repulsion, increasing their stability in aqueous solutions. Compared to nano-MoS2, MoS2@HS demonstrates better dispersibility and the ability to maintain its dispersed state in water. Like nano-MoS2, MoS2@HS also possesses peroxidase-like activity, and it exhibits cell-targeting properties, making it easier to enter cells than nano-MoS2.

[0029] (2) After adding MoS2@HS to a 20 mg / mL thiol-modified hyaluronic acid (HASH) solution, the resulting solution had a uniform color, and no aggregation of MoS2@HS was observed. However, the exfoliated MoS2 obtained by directly ultrasonically exfoliating nano-MoS2 with deionized water showed obvious aggregation after being dispersed in a HASH solution of the same concentration. This indicates that using HASH to exfoliate nano-MoS2 through ultrasonic exfoliation can improve the dispersibility of nano-MoS2 in the matrix material. This means that after gelling the HASH solution, MoS2@HS can be uniformly dispersed in the resulting hydrogel, solving the problem of limited uniformity of MoS2 dispersion in hydrogels in the prior art.

[0030] (3) The mitochondrial activated sponge (sponge M) prepared by the present invention based on the introduction of MoS2@HS, compared with the sponge without the introduction of MoS2@HS (sponge S), significantly improves the reactive oxygen species scavenging ability of the sponge without changing the internal pore structure of the sponge, can better promote the proliferation of bone marrow mesenchymal stem cells, and exhibits good cell compatibility.

[0031] (4) Compared with the sponge without MoS2@HS (sponge S), the mitochondrial-activated sponge (sponge M) prepared by introducing MoS2@HS in this invention can better help cells resist oxidative stress damage and promote mitochondrial oxidative phosphorylation. At the same time, by promoting the production of molybdenum cofactor, sponge M can promote the synthesis of molybdenum enzyme, thereby providing electrons for cytochrome C, enhancing the activity of cytochrome C oxidase, thereby accelerating the electron chain transfer process and promoting ATP production.

[0032] (5) Compared with the sponge without the introduction of MoS2@HS (sponge S), the mitochondrial activated sponge (sponge M) prepared by the present invention with the introduction of MoS2@HS can better promote the expression of cartilage matrix-related genes such as Col II, Sox9, and glycosaminoglycans and the secretion of matrix under oxidative stress chondrogenic induction conditions, and has a better ability to promote bone marrow mesenchymal stem cell chondrogenic differentiation.

[0033] (6) After 20 weeks of repair in a rabbit knee joint cartilage defect model, when sponge M was implanted for repair, the newly formed cartilage was tightly connected with the adjacent cartilage tissue, and the surface of the newly formed cartilage was smoother than when sponge S was implanted. Compared with sponge S, the newly formed cartilage was thicker and closer to the thickness of normal cartilage when sponge M was implanted, and had more glycosaminoglycan matrix secretion. Compared with sponge S, the newly formed fibers were more orderly arranged and closer to natural cartilage when sponge M was implanted. Compared with sponge S, sponge M had more Col II secretion and less Col I and Col X expression. This confirms that the mitochondrial-activated sponge provided by this invention has excellent ability to promote the repair of articular cartilage defects.

[0034] Compared with the prior art, the technical solution provided by the present invention has the following beneficial technical effects:

[0035] 1. This invention involves the one-step ultrasonic exfoliation of MoS2 using an aqueous solution of thiol-modified hyaluronic acid, resulting in thiol-modified hyaluronic acid-exfoliated MoS2 nanoparticles that exhibit better dispersion and biocompatibility in aqueous environments. Simultaneously, the thiol-modified hyaluronic acid encapsulates and covalently binds to the MoS2 nanoparticles, which helps prevent the agglomeration of MoS2 nanoparticles in physiological environments, thus avoiding localized cytotoxicity. Furthermore, the thiol-modified hyaluronic acid encapsulating the MoS2 nanoparticles not only better prevents rapid oxidation of the MoS2 nanoparticles, thus reducing peroxidase-like activity, but also endows the MoS2 nanoparticles with the ability to target and enter cells, thereby facilitating more efficient cellular entry and exertion of their effects. This invention, through a specific method, prepares thiol-modified hyaluronic acid-exfoliated MoS2 nanoparticles, overcoming the problems of poor dispersibility and stability of MoS2 nanosheets prepared by existing technologies in physiological environments, and also solving the problem of the difficulty in targeting and entering cells with MoS2 nanosheets themselves, thus enabling the more effective function of MoS2 nanoparticles in cartilage repair materials.

[0036] 2. This invention provides a mitochondrial-activated sponge for repairing articular cartilage defects. It is formed by freeze-drying a hydrogel created from thiol-modified hyaluronic acid-exfoliated MoS2 nanoparticles, thiol-modified hyaluronic acid, and polyphenol-modified silk fibroin at a mass ratio of (0.002~0.02):(0.5~8):1 under pH 7~8 conditions. On one hand, the thiol-modified hyaluronic acid-exfoliated MoS2 nanoparticles can be more uniformly and stably dispersed in the hydrogel. On the other hand, the thiol-modified hyaluronic acid-exfoliated MoS2 nanoparticles can covalently assemble with thiol-modified hyaluronic acid and polyphenol-modified silk fibroin through chemical bonding, improving the fixation stability of the MoS2 nanoparticles in the sponge and increasing the integration of the components within the sponge. These factors endow the sponge of this invention with good biocompatibility, targeted cell entry, reactive oxygen species scavenging, and the ability to promote mitochondrial oxidative phosphorylation, thus better promoting the repair of articular cartilage defects.

[0037] 3. In the mitochondrial-activated sponge for articular cartilage defect repair provided by this invention, the thiol-modified hyaluronic acid-exfoliated MoS2 nanoparticles effectively maintain the peroxidase-like activity of the MoS2 nanoparticles, endowing the sponge with excellent reactive oxygen species (ROS) scavenging ability. Furthermore, during sponge degradation, the thiol-modified hyaluronic acid-exfoliated MoS2 nanoparticles can target and enter bone marrow mesenchymal stem cells via thiol-modified hyaluronic acid, accelerating mitochondrial oxidative phosphorylation and providing sufficient ATP for chondrogenic differentiation of mesenchymal stem cells. In other words, the sponge of this invention scavenges ROS at the cartilage injury site through the thiol-modified hyaluronic acid-exfoliated MoS2 nanoparticles, creating a favorable microenvironment for the recruitment, proliferation, and differentiation of endogenous stem cells. The targeted entry of the thiol-modified hyaluronic acid-exfoliated MoS2 nanoparticles into cells promotes ATP production, providing sufficient energy for chondrogenic differentiation of stem cells. The synergistic effect of these two aspects can solve the problems of insufficient energy supply and limitation due to the oxidative stress microenvironment after injury during endogenous stem cell chondrogenic differentiation, thereby improving the cartilage repair effect after microfracture surgery.

[0038] 4. The present invention also provides a method for preparing mitochondrial-activated sponges for repairing articular cartilage defects. The method is simple to operate, the process conditions are easy to control, and it is conducive to large-scale production. Attached Figure Description

[0039] Figure 1 The NMR spectra are 1H NMR spectra of hyaluronic acid and the thiol-modified hyaluronic acid prepared in Example 1.

[0040] Figure 2 The images show the 1H NMR spectra of the silk fibroin and polyphenol-modified silk fibroin prepared in Example 2.

[0041] Figure 3Figure (a) shows the Zeta potential test results for nano-MoS2 and MoS2@HS. Figure 3 Figure (b) shows the XRD test results of nano-MoS2, MoS2@HS and HASH. Figure 3 Figure (c) shows the AFM diagram and dimensional measurement results of MoS2@HS.

[0042] Figure 4 Figure (a) shows the AFM test results for MoS2@HS. Figure 4 Figure (b) shows the in-situ elemental analysis results of MoS2@HS, while Figures (b1) to (b4) show the distribution of Mo, S, O and N elements respectively. Figure 4 The scale bars in all of them are 1 μm.

[0043] Figure 5 The figures show the XPS test results for MoS2@HS, MoS2, HASH, and HS-SH. Figures (a) and (b) are the Mo 3d spectra of MoS2 and MoS2@HS, figures (c) and (d) are the S 2p spectra of MoS2 and MoS2@HS, and figures (e) and (f) are the S 2p spectra of HASH and HS-SH.

[0044] Figure 6 Figures (a) and (b) show the dispersion states of nano-MoS2 and MoS2@HS after standing in water for 0 and 24 hours. Figure 6 Figure (c) shows the results of peroxidase activity assays for nano-MoS2 and MoS2@HS. Figure 6 Figure (d) shows the cell entry of nano-MoS2 and MoS2@HS into bone marrow mesenchymal stem cells after interaction. Figure 6 In Figure (d), MoS2 represents control group 1, MoS2@HS represents experimental group, and +HA-MoS2@HS represents control group 2.

[0045] Figure 7 These are optical photographs of the MoS2@HS dispersion and the stripped MoS2 dispersion, formed by dispersing MoS2@HS and stripped MoS2 in a 20 mg / mL HASH solution, respectively.

[0046] Figure 8 The figures show the results of testing the proliferation of BMSCs with different concentrations of MoS2@HS and stripped MoS2. In the figure, Blank represents the Blank group, MoS2@HS50, MoS2@HS100 and MoS2@HS200 represent experimental groups with MoS2@HS concentrations of 50, 100 and 200 μg / mL, respectively, and MoS250, MoS2100 and MoS2200 represent experimental groups with stripped MoS2 concentrations of 50, 100 and 200 μg / mL, respectively.

[0047] Figure 9 Figures (a1) and (a2) provide a general overview of sponge M and S. Figure 9 Figures (b1) and (b2) are SEM images of sponge M and S, with the scale bar in the figures being 100 μm. Figure 9 Figure (c1) is the EDS energy spectrum of sponge M. Figure 9 Figure (c2) is the EDS energy spectrum of Mo in sponge M. Figure 9 The (c3) figure is the EDS energy spectrum of sponge S, and the scale bar in the figure is 50 μm.

[0048] Figure 10 Figures (a) and (b) show the results of enzyme activity in sponge M and sponge S. Figure 10 Figure (c) shows the test results of reactive oxygen species scavenging rates for sponge M and sponge S.

[0049] Figure 11 Image (a) is a staining image showing cell viability and death. Figure 11 Image (b) is a cytoskeleton / nuclear staining image. Figure 11 The scale bar in figures (a) and (b) is 50 μm. Figure 11 Figure (c) shows the cell proliferation results of different sponges as determined using the CCK-8 assay kit. Figure 11 In Figure (c), C represents the control group.

[0050] Figure 12 Image (a) is an image of reactive oxygen species staining in chondrocytes. Figure 12 Figure (b) is a staining image of reactive oxygen species in BMSCs. The scale bars in the figure are all 100 μm.

[0051] Figure 13 Figure (a) shows the items that were upregulated in the GO analysis. Figure 13 Figure (b) shows the chord graph analysis results of differentially expressed genes and entries in the GO upregulated entries. Figure 13 Figure (c) is a heatmap of differentially expressed genes in the upregulated entries. Figure 13 In Figure (c), S_1, S_2, and S_3 represent three parallel samples of sponge M, and M_1, M_2, and M_3 represent three parallel samples of sponge S. Figure 13 Figure (d) shows the q-PCR verification results of differentially expressed genes.

[0052] Figure 14 Figure (a) shows the results of ATP content detection. Figure 14 Figure (b) shows the results of cytochrome C oxidase activity assay, where C represents the control group. Figure 14 Image (c) is an image of intracellular mitochondrial staining. Figure 14Figure (d) shows the expression levels of molybdenum enzyme-sulfite oxidase-related genes (MOCS1, SUOX1).

[0053] Figure 15 Figure (a) is a staining image of Col II, Sox9, and SO. Figure 15 Figure (b) shows the GAG / DNA measurement results. Figure 15 Figure (c) shows the gene expression levels as determined by q-PCR.

[0054] Figure 16 Figure (a) is a gross image of the articular cartilage defect. Figure 16 Image (b) shows the reconstructed cartilage surface as captured by a super depth-of-field microscope. Figure 16 Image (c) shows an HE staining image of the articular cartilage defect. Figure 16 Image (d) shows a picture of safranin-fix-green staining. Figure 16 Image (e) is a picture of Sirius red staining. Figure 16 Figure (f) shows immunohistochemical staining images of Col II, Col I, and Col X. Detailed Implementation

[0055] The following examples further illustrate the mitochondrial-activated sponge for articular cartilage defect repair provided by the present invention and its preparation method. It should be noted that the following examples are only for further illustration of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made to the present invention by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0056] Example 1

[0057] In this embodiment, the preparation of thiol-modified hyaluronic acid (HASH) is carried out through the following steps:

[0058] (1) Hyaluronic acid (HA) with a molecular weight of 1000 kDa was dissolved in deionized water to obtain an HA solution with a concentration of 2.5 mg / mL. N-hydroxysuccinimide (NHS) was added to the HA solution and dissolved completely. Then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) was added and dissolved completely. The pH of the resulting mixture was adjusted to 4.75~5 and reacted at room temperature for 2 h to activate the carboxyl group. Then cysteine ​​hydrochloride (CSA•HCl) was added and reacted at room temperature for 12 h under the condition of pH 4.75~5.

[0059] In this step, the molar ratio of HA, NHS, EDCI and CSA•HCl is controlled to be 1:2:5:5.

[0060] (2) The reaction solution obtained in step (1) was dialyzed in hydrochloric acid aqueous solution with a pH of 3.5~4.5 for 3 days, and then freeze-dried to obtain mercapto-modified hyaluronic acid (abbreviated as HASH), the structure of which is shown in formula (I):

[0061] (I).

[0062] The proton NMR spectra of HA and the HASH prepared in this embodiment are as follows: Figure 1 As shown, the characteristic peak of HASH appears at δ=2.78 ppm, as... Figure 1 As shown in the red box, HA shows no characteristic peak at this location, indicating that thiol groups were successfully grafted onto the HA molecular chain via amidation in this embodiment. The degree of thiol substitution in HASH was calculated based on the integral of the proton NMR spectrum, showing a cysteine ​​grafting rate of 22.5%.

[0063] Example 2

[0064] In this embodiment, the polyphenol-modified silk fibroin (SFD) is prepared by the following steps:

[0065] (1) According to the ratio of 400 mL of 0.212 wt% sodium carbonate aqueous solution for every 1 g of silkworm cocoon, the silkworm cocoon was cut into pieces and peeled and boiled in boiling sodium carbonate aqueous solution twice, each time for 0.5 h. After that, it was washed with deionized water and air-dried at room temperature to obtain degummed silk fibroin. According to the ratio of 10 mL of 9.3 mol / L lithium bromide aqueous solution for every 1 g of degummed silk fibroin, the degummed silk fibroin was immersed in lithium bromide aqueous solution and stirred at 60 ℃ for 4 h. The resulting solution was transferred into a dialysis bag and dialyzed with deionized water for 3 days to obtain a silk fibroin (SF) solution with a concentration of about 10 mg / mL.

[0066] (2) Under nitrogen protection, add NHS, EDCI and dopamine hydrochloride to the SF solution, adjust the pH of the resulting mixture to pH 4.75~5, stir the reaction under nitrogen protection for 24 h, transfer the resulting reaction solution into a dialysis bag, dialyze with deionized water for 3 days to obtain polyphenol modified silk fibroin (SFD) solution, store the SFD solution at 4 ℃, or freeze-dry the SFD solution to obtain SFD.

[0067] In this step, the mass ratio of SF, NHS, EDCI and dopamine hydrochloride is controlled to be 1:23.2:96.5:19.

[0068] The 1H NMR spectra of SF and SFD prepared in this embodiment are as follows: Figure 2 As shown, the characteristic peak of catechins in SFD is located at δ = 7~7.25 ppm, as... Figure 2As shown in the red box, SF shows no characteristic peak at this location, indicating that catechol groups were successfully grafted onto the SF molecular chain via an amidation reaction in this embodiment. The content of catechol groups in SFD was calculated to be 18.375 μg / mg based on UV absorption spectroscopy.

[0069] Example 3

[0070] In this embodiment, HASH-exfoliated MoS2 nanoparticles (MoS2@HS) are prepared by ultrasonic exfoliation of MoS2 nanoparticles using HASH. The steps are as follows:

[0071] The HASH prepared in Example 1 was dissolved in deionized water to obtain a HASH solution with a concentration of 4 mg / mL. Nano MoS2 was added to the HASH solution at a ratio of 1 mg of nano MoS2 per 1 mL of HASH solution. The solution was then sonicated in an ice-water bath for 4 h at a sonication power of 300 W. After centrifugation, the resulting solid phase was washed with deionized water. The washed solid phase was MoS2@HS, which was then dispersed in deionized water for storage.

[0072] MoS2@HS dispersed in deionized water was diluted with deionized water to obtain a MoS2@HS dispersion with a concentration of 1 mg / mL. The MoS2@HS dispersion was added to a potential cell, and the Zeta potential was measured using a Malvern dynamic light scattering particle size analyzer (DLS). At the same time, nano-MoS2 was dispersed in deionized water to obtain a nano-MoS2 dispersion with a concentration of 0.1 mg / mL. The nano-MoS2 dispersion was added to a potential cell, and the Zeta potential was measured. Figure 3 Figure (a) shows the zeta potential test results for nano-MoS2 and MoS2@HS.

[0073] A certain amount of MoS2@HS dispersed in deionized water was freeze-dried and its XRD pattern was measured; at the same time, the XRD patterns of nano MoS2 and HASH prepared in Example 1 were measured. Figure 3 Figure (b) shows the XRD test results of nano MoS2, MoS2@HS and HASH.

[0074] A certain amount of MoS2@HS dispersed in deionized water was dropped onto a mica sheet, air-dried, and its morphology and size were observed using an atomic force microscope (AFM). Figure 3 Figure (c) shows the AFM diagram and dimensional measurement results of MoS2@HS.

[0075] A certain amount of MoS2@HS dispersed in deionized water was diluted with deionized water, and its morphology and elemental distribution were observed using transmission electron microscopy (TEM). The results are as follows: Figure 4 As shown in Figures (a) to (b), Figure 4 Figure (a) shows the AFM test results for MoS2@HS. Figure 4 Figure (b) shows the in-situ elemental analysis results of MoS2@HS, while Figures (b1) to (b4) show the distribution of Mo, S, O and N elements respectively.

[0076] Depend on Figure 3 It can be seen that the Zeta potential of MoS2@HS is approximately -23 mV, which is more negative than that of pure MoS2. This indicates that MoS2@HS can better increase stability through electrostatic repulsion, resulting in better dispersion stability in water. XRD results show that, compared to nano-MoS2, the peaks of MoS2@HS on the (002), (004), and (006) crystal planes are weaker, indicating that MoS2@HS reduces the face-to-face stacking of nano-MoS2, proving successful exfoliation. AFM images show that MoS2@HS exhibits a nanosheet structure with a length of approximately 300 nm and a height of approximately 2 nm. Figure 4 It can be seen that O and N elements are present on the surface of MoS2@HS, indicating that nano MoS2 can be exfoliated into nanosheets by ultrasonic exfoliation in HASH aqueous solution, while HASH is located on the surface of MoS2.

[0077] The HASH self-crosslinked material prepared in Example 1 was designated HS-SH. X-ray photoelectron spectroscopy (XPS) was performed on MoS2@HS, MoS2, HASH, and HS-SH, and the results are as follows: Figure 5 As shown, Figure 5 Figures (a) and (b) are the Mo 3d spectra of MoS2 and MoS2@HS. Figure 5 Figures (c) and (d) are the S 2p spectra of MoS2 and MoS2@HS. Figure 5 Figures (e) and (f) are the S 2p spectra of HASH and HS-SH. Figure 5 It can be seen that a new peak appears at 225.8 eV in the Mo 3d spectrum of MoS2@HS, indicating that a new Mo-S bond has been formed in MoS2@HS. At the same time, a new peak appears at 168.5 eV in the S 2p spectrum of MoS2@HS, indicating that an SS bond has also been formed in MoS2@HS. The above experimental results show that chemical bonds are formed between HASH and MoS2 in MoS2@HS, and HASH has undergone oxidative self-crosslinking.

[0078] Example 4

[0079] In this embodiment, the dispersibility of MoS2@HS prepared in Example 3 in water was tested.

[0080] MoS2@HS was added to deionized water and dispersed by shaking to obtain a MoS2@HS dispersion with a concentration of 0.25 mg / mL. Nano-MoS2 was ultrasonically dispersed in deionized water to obtain a MoS2 dispersion with a concentration of 0.25 mg / mL. The MoS2@HS and MoS2 dispersions were allowed to stand for 24 h, and the changes in their dispersion state before and after standing were observed. The results are as follows: Figure 6 As shown in Figures (a) and (b).

[0081] Depend on Figure 6 As shown in Figures (a) and (b), MoS2@HS exhibits better dispersibility and the ability to maintain its dispersion state in deionized water compared to nano MoS2.

[0082] Example 5

[0083] In this embodiment, the peroxidase-like activity of MoS2@HS prepared in Example 3 was tested.

[0084] Add deionized water to MoS2@HS stored in deionized water, shake to disperse, and obtain a MoS2@HS dispersion with a concentration of 1 mg / mL. Disperse nano-MoS2 in deionized water using ultrasound to obtain a MoS2 dispersion with a concentration of 1 mg / mL.

[0085] 3,3',5,5'-Tetramethylbenzidine (TMB) and H2O2 were added to MoS2@HS dispersion and MoS2 dispersion, respectively, to concentrations of TMB and H2O2 of 0.4 mmol / L and 0.1 mmol / L, respectively. The reactions were carried out at 37 ℃ for 30 min, and the color changes of the liquids were observed by photographing. The absorbance of the solutions in the 300–800 nm wavelength range was measured using a UV spectrophotometer. Simultaneously, the color changes of the TMB solution and the mixed solution of TMB and H2O2 (TMB+H2O2) were observed, and their absorbance in the 300–800 nm wavelength range was measured. The results are as follows: Figure 6 As shown in Figure (c).

[0086] Figure 6 In the inset of Figure (c), the solutions in the test tubes from left to right are TMB solution, a mixed solution of TMB and H2O2 (TMB+H2O2), a MoS2 dispersion with added TMB and H2O2, and a MoS2@HS dispersion with added TMB and H2O2, respectively. The phenomenon that the solution in the test tube turns blue and the absorbance at 650 nm increases indicates that MoS2@HS has peroxidase-like activity similar to MoS2, which shows that using the method of the present invention to exfoliate nano MoS2 with HASH does not affect the peroxidase-like activity of MoS2.

[0087] Example 6

[0088] In this embodiment, the targeting and cell entry ability of MoS2@HS prepared in Example 3 was tested.

[0089] Add ultrapure water to MoS2@HS stored in deionized water, shake to disperse, and obtain a MoS2@HS dispersion with a concentration of 2 mg / mL. Mix the MoS2@HS dispersion with an equal volume of 1 mg / mL Rhodamine B aqueous solution (total volume after mixing is 10 mL), stir overnight, centrifuge at 12000 rpm for 5 min, collect the supernatant, determine the concentration of Rhodamine B, wash the collected precipitate with ultrapure water until no red color is visible to the naked eye, resuspend to 1 mL, and obtain Rhodamine B-labeled MoS2@HS.

[0090] Nano-sized MoS2 was ultrasonically dispersed in ultrapure water to obtain a MoS2 dispersion with a concentration of 2 mg / mL. The MoS2 dispersion was mixed with an equal volume of 1 mg / mL Rhodamine B aqueous solution (total volume after mixing was 10 mL), and the mixture was stirred overnight. After centrifugation at 12000 rpm for 5 min, the supernatant was collected, and the concentration of Rhodamine B was determined. The collected precipitate was washed with ultrapure water until no red color was visible to the naked eye, and the precipitate was resuspended to 1 mL to obtain Rhodamine B-labeled MoS2.

[0091] Rabbit bone marrow mesenchymal stem cells (BMSCs) in the exponential growth phase were collected and seeded in 24-well plates at a density of 20,000 cells / well. After adhesion, the supernatant was discarded. Rhodamine B-labeled MoS2@HS was diluted with α-MEM medium and added to the wells as the experimental group. After 2 h of treatment, Hoechst staining was performed for 3–5 min, and cell infiltration was observed under a laser confocal microscope. Rhodamine B-labeled MoS2 was used instead of Rhodamine B-labeled MoS2@HS as control group 1. Hyaluronic acid (HA, molecular weight 1000 kDa) was first used to treat BMSCs instead of Rhodamine B-labeled MoS2@HS to shield some target binding sites. Then, Rhodamine B-labeled MoS2@HS was diluted with α-MEM medium and added to the wells to treat BMSCs as control group 2. In control groups 1-2 and the experimental group, the concentration of rhodamine B was the same after dilution of rhodamine B-labeled MoS2@HS and rhodamine B-labeled MoS2 in α-MEM medium. The results are as follows: Figure 6 As shown in Figure (d), Figure 6 In Figure (d), MoS2 represents control group 1, MoS2@HS represents experimental group, and +HA-MoS2@HS represents control group 2.

[0092] Depend on Figure 6As shown in Figure (d), Rhodamine B-labeled MoS2@HS is more readily absorbed into cells than Rhodamine B-labeled MoS2. The addition of HA and BMSCs beforehand caused Rhodamine B-labeled MoS2@HS to lose some of its target sites, making it less likely to enter cells. This indicates that MoS2@HS possesses a certain degree of targeting ability and can enter cells via hyaluronic acid.

[0093] Example 7

[0094] In this embodiment, the differences in dispersibility and proliferation-promoting effect of HASH-exfoliated modified nano-MoS2 (MoS2@HS) prepared by HASH ultrasonic exfoliation of nano-MoS2 were examined compared with those of exfoliated MoS2 prepared directly by ultrasonic exfoliation of nano-MoS2 in HASH solution.

[0095] 1. Dispersibility test in HASH solution

[0096] (1) The HASH prepared in Example 1 was dissolved in deionized water to obtain a HASH solution with a concentration of 4 mg / mL. Nano MoS2 was added to the HASH solution at a ratio of 2 mg of nano MoS2 per 1 mL of HASH solution. The solution was sonicated in an ice-water bath for 4 h with the sonication power controlled at 200 W. After centrifugation, the resulting solid phase was washed with deionized water. The solid phase after washing was MoS2@HS. MoS2@HS was dispersed in deionized water and stored.

[0097] (2) Add nano MoS2 to deionized water at a ratio of 2 mg per 1 mL of deionized water, sonicate for 4 h in an ice-water bath, control the sonication power to 200 W, centrifuge, wash the obtained solid phase with deionized water, and the washed solid phase is the stripped MoS2. Disperse the stripped MoS2 in deionized water and store it.

[0098] (3) Add MoS2@HS to a 20 mg / mL HASH solution and mix using a syringe to obtain a MoS2@HS dispersion with a concentration of 0.2 mg / mL. Add the stripped MoS2 to a 20 mg / mL HASH solution and mix using a syringe to obtain a stripped MoS2 dispersion with a concentration of 0.2 mg / mL. Take pictures of the MoS2@HS dispersion and the stripped MoS2 dispersion. The results are as follows. Figure 7 As shown.

[0099] Depend on Figure 7It was found that the exfoliated MoS2 exhibited poor dispersion in the HASH solution, resulting in varying degrees of agglomeration. Ultimately, it was unevenly dispersed in the HASH solution as aggregates, producing a large number of black exfoliated MoS2 aggregates. In contrast, MoS2@HS showed better dispersibility in the HASH solution, resulting in a uniformly black solution. These experimental results demonstrate that using HASH for ultrasonic exfoliation of nano-MoS2 can improve the dispersibility of nano-MoS2 in the matrix material. This means that after gelling the HASH solution, MoS2@HS can be uniformly dispersed in the resulting hydrogel, solving the problem of limited uniformity of MoS2 dispersion in hydrogels in existing technologies.

[0100] 2. Proliferation-promoting effect test

[0101] (1) The HASH prepared in Example 1 was sterilized by soaking in 75% ethanol for 1 h, and then air-dried in a sterile fume hood. The sterilized HASH was dissolved in sterile deionized water to obtain a HASH solution with a concentration of 4 mg / mL. Under sterile conditions, sterilized nano MoS2 was added to the HASH solution at a ratio of 2 mg of nano MoS2 per 1 mL of HASH solution. The solution was sonicated in an ice-water bath for 4 h with the sonication power controlled at 200 W. After centrifugation, the resulting solid phase was washed with deionized water. The solid phase after washing was MoS2@HS. MoS2@HS was dispersed in deionized water to obtain a MoS2@HS dispersion with a concentration of 2 mg / mL.

[0102] (2) Under sterile conditions, add sterile nano-MoS2 to sterile deionized water at a ratio of 2 mg nano-MoS2 per 1 mL of deionized water, sonicate for 4 h in an ice-water bath, control the sonication power to 200 W, centrifuge, wash the obtained solid phase with deionized water, and the washed solid phase is the stripped MoS2. Disperse the stripped MoS2 in deionized water to obtain a stripped MoS2 dispersion with a concentration of 2 mg / mL.

[0103] (3) Rabbit bone marrow mesenchymal stem cells (BMSCs) were seeded in 24-well plates at a density of 30,000 / well, with 1 mL of culture medium per well. After cell adhesion, 100, 50, and 25 μL of MoS2@HS dispersion and MoS2 stripping dispersion were added to the corresponding wells to form experimental groups with MoS2@HS and MoS2 stripping concentrations of 200, 100, and 50 μg / mL, respectively. Three replicates were set up for each concentration, and a Blank group without any nanomaterials was set up as a control. The cells were cultured in a cell culture incubator at 37 ℃ for 48 h. The culture medium was discarded, the cells were washed with PBS buffer, and CCK-8 test solution was added. After incubation for 2 h, the absorbance (OD value) at 450 nm was measured using a microplate reader. Three replicates were set up for each concentration, and three replicates were set up for each replicate. A bar chart of absorbance values ​​was plotted, and the results are shown below. Figure 8 As shown.

[0104] Figure 8 This section presents the results of testing different concentrations of MoS2@HS and exfoliated MoS2 on the proliferation of BMSCs. Blank represents the Blank group; MoS2@HS50, MoS2@HS100, and MoS2@HS200 represent experimental groups with MoS2@HS concentrations of 50, 100, and 200 μg / mL, respectively; and MoS250, MoS2100, and MoS2200 represent experimental groups with exfoliated MoS2 concentrations of 50, 100, and 200 μg / mL, respectively. Figure 8 It was found that, compared with the Blank group, all experimental groups promoted the proliferation of BMSCs to some extent. For the experimental groups with added MoS2@HS, the trend of promoting proliferation became more significant with increasing MoS2@HS concentration. However, for the experimental groups with added MoS2-exfoliated cells, the difference in MoS2 exfoliation concentration had no significant effect on proliferation. Furthermore, at concentrations of 100 μg / mL and 200 μg / mL, MoS2@HS promoted BMSC proliferation more effectively than MoS2, showing a significant difference. This may be because the thiolized hyaluronic acid on the surface of MoS2@HS enhances the dispersibility and stability of the nano-MoS2, while also giving it the possibility of specifically binding to cells via the CD44 receptor, thereby promoting cellular uptake and proliferation.

[0105] Example 8

[0106] In this embodiment, the mitochondrial-activated sponge (M) for repairing articular cartilage defects is prepared by the following steps:

[0107] (1) The HASH prepared in Example 1 was dissolved in ultrapure water to obtain a HASH solution with a concentration of 30 mg / mL. MoS2@HS dispersed in deionized water in Example 3 was added to the HASH solution and mixed thoroughly to obtain a mixture. The concentration of the SFD solution prepared in Example 2 was adjusted to 30 mg / mL with ultrapure water to obtain the adjusted SFD solution.

[0108] (2) Mix the mixture and the SFD solution after adjusting the concentration thoroughly and adjust the pH value to 7-8 to obtain the gel precursor solution. Inject the gel precursor solution into the polydimethylsiloxane (PDMS) mold and let it stand for about 10 min. The gel precursor solution will then be transformed into a hydrogel state. Freeze-dry the obtained hydrogel to obtain a mitochondrial activated sponge for the repair of articular cartilage defects, denoted as M.

[0109] In this step, the mass ratio of MoS2@HS, HASH and SFD in the gel precursor solution is controlled to be 0.01:2:1. More specifically, the concentration of MoS2@HS in the gel precursor solution is 0.1 mg / mL, the concentration of HASH is 20 mg / mL, and the concentration of SFD is 10 mg / mL.

[0110] Comparative Example 1

[0111] In this embodiment, the control sponge (S) is prepared by the following steps:

[0112] (1) The HASH prepared in Example 1 was dissolved in ultrapure water to obtain a HASH solution with a concentration of 30 mg / mL. The concentration of the SFD solution prepared in Example 2 was adjusted to 30 mg / mL with ultrapure water to obtain an SFD solution with adjusted concentration.

[0113] (2) Mix the HASH solution and the SFD solution after adjusting the concentration thoroughly and adjust the pH value to 7~8 to obtain the gel precursor solution. Inject the gel precursor solution into the PDMS mold and let it stand for about 10 min. The gel precursor solution will then be transformed into a hydrogel state. Freeze-dry the obtained hydrogel to obtain the control sponge, denoted as S.

[0114] In this step, the mass ratio of HASH to SFD in the gel precursor solution is controlled to be 2:1. More specifically, the concentration of HASH in the gel precursor solution is 20 mg / mL and the concentration of SFD is 10 mg / mL.

[0115] Macroscopic images of M prepared in Example 8 and Comparative Example 1 were taken, and their microscopic pore structures were observed using scanning electron microscopy (SEM). The elemental distribution of M was identified using energy-dispersive X-ray spectroscopy (EDS). Figure 9Figures (a1) and (a2) show the general appearance of sponges M and S. The introduction of MoS2@HS makes sponge M appear blackish-gray. Figure 9 Figures (b1) and (b2) are SEM images of sponges M and S, with a scale bar of 100 μm. The introduction of MoS2@HS did not significantly alter the internal pore structure of the sponge. Furthermore, the pore size of sponge M is in the range of 20–120 μm, and its porosity is approximately 70%. Figure 9 Figure (c1) is the EDS energy spectrum of sponge M. Figure 9 Figure (c2) is the EDS energy spectrum of Mo in sponge M. Figure 9 Figure (c3) shows the EDS energy dispersive spectroscopy results of sponge S. The scale bar in the figure is 50 μm. Mo element was successfully detected in sponge M, and the Mo element showed a relatively uniform distribution.

[0116] Example 9

[0117] In this embodiment, the peroxidase-like activity and reactive oxygen species scavenging ability of sponge M prepared in Example 8 and sponge S prepared in Comparative Example 1 were examined.

[0118] Sponges M and S prepared in Example 8 and Comparative Example 1 were respectively immersed in a TMB solution with a concentration of 0.4 mmol / L, and then H2O2 was added until the H2O2 concentration was 0.1 mmol / L. The reaction was carried out at room temperature for 24 h, and the color changes of the solution and sponges were observed.

[0119] Sponges M and S, prepared in Example 8 and Comparative Example 1 respectively, were immersed in a 0.1 mmol / L solution of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) for 2 h. The absorbance of the solution was measured using a UV spectrophotometer, and the reactive oxygen species (ROS) scavenging rate was calculated using the following formula. Simultaneously, nano-MoS2 was added to a 0.1 mmol / L DPPH solution and allowed to react for 2 h. The absorbance of the solution was measured using a UV spectrophotometer, and the ROS scavenging rate was calculated using the following formula.

[0120] Reactive oxygen species scavenging rate (%) = (OD0 - OD1) / OD0 × 100%

[0121] Wherein, OD0 represents the absorbance value of the original DPPH solution without sponge action, and OD1 represents the absorbance value of the DPPH solution after sponge action.

[0122] Figure 10Figures (a) and (b) show the results of measuring the activity of sponge-like peroxidases using TMB as a substrate. As can be seen from the figures, sponge M can catalyze the decomposition of H2O2 with TMB as a substrate. During this process, TMB is oxidized to form a blue substance, which is adsorbed on the sponge, making the sponge appear blue. Sponge S, on the other hand, does not undergo a color change. This indicates that sponge M has peroxidase-like activity. Figure 10 Figure (c) shows the test results of reactive oxygen species scavenging rate. As can be seen from the figure, sponge M has a stronger reactive oxygen species scavenging ability compared to sponge S and nano MoS2.

[0123] Example 10

[0124] In this embodiment, the cytotoxicity of sponge M and sponge S prepared in Example 8 and Comparative Example 1 was examined.

[0125] Sponges M and S prepared in Example 8 and Comparative Example 1 were sterilized in a UV chamber for 1 h, and then BMSCs were sterilized at 3 × 10⁻⁶ ppm. 6 Sponges were seeded at densities on sterilized sponges M and S, respectively, and cultured for 2 days in complete culture medium (α-MEM + 10% fetal bovine serum). Live / dead cell staining and cytoskeleton / nuclear staining were then performed. Laser confocal microscopy was used to observe the activity and cytoskeleton morphology of BMSCs on the sponges. BMSCs were also seeded at a density of 5000 / well in 24-well plates. After adhesion, sterilized sponges M and S were added and cultured for 2 days. Cytotoxicity of the sponges was measured using a CCK-8 assay kit. The control group (without sponges) was used.

[0126] Figure 11 Image (a) is a staining image showing cell viability and death. Figure 11 Image (b) is a cytoskeleton / nuclear staining image. Figure 11 The scale bar in figures (a) and (b) is 50 μm. Figure 11 Figure (c) shows the cell proliferation results of different sponges as determined using the CCK-8 assay kit. Figure 11 In figure (c), C represents the control group. Figure 11 As can be seen in Figures (a) to (b), the cells can survive and spread well on the surfaces of both sponge M and sponge S. Figure 11 Figure (c) shows that sponge M can better promote the proliferation of BMSCs compared to sponge S and the control group, demonstrating good cell compatibility.

[0127] Example 11

[0128] In this embodiment, the ability of sponges M and S prepared in Example 8 and Comparative Example 1 to protect chondrocytes and BMSCs under oxidative stress was investigated.

[0129] Sponges M and S prepared in Example 8 and Comparative Example 1 were sterilized in a UV chamber for 1 h, and then BMSCs were sterilized at 2 × 10⁻⁶. 4 BMSCs were seeded at a density of / wells on 24-well plates. After adhesion, sterilized sponges M and S were co-cultured with BMSCs, followed by incubation with 200 μmol / L H2O2 for 24 h. The plates were then removed and stained with ROS fluorescent probes and Hoechst. The production of reactive oxygen species (ROS) was observed under a laser confocal microscope. The cells without sponge and H2O2 incubation were designated as the normal group (NORMAL), and the cells without sponge incubation were designated as the blank group (BLANK).

[0130] Figure 12 Image (a) is an image of reactive oxygen species staining in chondrocytes. Figure 12 Image (b) is a staining image of reactive oxygen species in BMSCs. The scale bars in the image are all 100 μm. Figure 12 It can be seen that both sponge S and sponge M can protect chondrocytes and BMSCs under oxidative stress, causing them to produce less reactive oxygen species. Compared with sponge S, sponge M produces less reactive oxygen species in the cells it affects, indicating that sponge M has a better ability to help cells resist oxidative stress damage.

[0131] Example 12

[0132] In this embodiment, the effects of sponge M and sponge S prepared in Example 8 and Comparative Example 1 on BMSCs mitochondria were investigated.

[0133] Sponges M and S prepared in Example 8 and Comparative Example 1 were sterilized in a UV chamber for 1 h, and then BMSCs were sterilized at 3 × 10⁻⁶ ppm. 6 Sponges were seeded at a density on sterilized sponge M and sponge S, and cultured for 14 days in DMEM high-glucose medium (containing ITS, TGF-β, dexamethasone, ascorbic acid, and L-proline). Sponge-cell cultures were then collected and subjected to reference transcriptome sequencing analysis. The results are as follows: Figure 13 As shown.

[0134] Figure 13 Figure (a) shows the upregulated items in the GO analysis. This figure shows the functional items with significant enrichment of differentially expressed genes from three dimensions: molecular function (MF), cellular components (CC), and biological processes (BP). Each bar represents a GO item, and the length corresponds to the significance of enrichment (-log10 (P value), the larger the value, the more significant). Figure 13 Figure (b) shows the chord graph analysis results of differentially expressed genes and entries in the GO upregulated entries. Figure 13 Figure (c) is a heatmap of differentially expressed genes in the upregulated entries. Figure 13 In Figure (c), S_1, S_2, and S_3 represent three parallel samples of sponge M, and M_1, M_2, and M_3 represent three parallel samples of sponge S. Figure 13 Figure (d) shows the q-PCR validation results of differentially expressed genes. Figure 13 The vertical axis of graph (d) represents the gene expression fold. For example... Figure 13 As shown in Figure (a), among the items upregulated by GO, mitochondrial-related items such as oxidative phosphorylation, ATP synthesis coupled with electron transport, and electron transport were significantly upregulated; Figure 13 As shown in Figure (b), the significantly upregulated genes in the chord graph are all attributed to processes related to oxidative phosphorylation; such as Figure 13 As shown in Figure (c), in the differential gene expression heatmap, compared to sponge S, sponge M significantly upregulated the expression of genes related to ND1, SDHB, COX1, COX2, UQCRQ, ATP6V1B2, and SUOX, indicating that sponge M increased the expression of complex genes involved in mitochondrial oxidative phosphorylation. Further q-PCR results confirmed that sponge M can promote mitochondrial oxidative phosphorylation, such as... Figure 13 As shown in Figure (d).

[0135] Example 13

[0136] In this embodiment, we verify the possible mechanism by which the sponge M prepared in Example 8 promotes the mitochondrial oxidative phosphorylation process of BMSCs.

[0137] Sponges M and S prepared in Example 8 and Comparative Example 1 were sterilized in a UV chamber for 1 h, and then BMSCs were sterilized at 3 × 10⁻⁶ ppm. 6 Sponges were seeded at a density on sterilized sponges M and S and cultured for 7 days in chondrogenesis medium (DMEM high-glucose medium containing ITS, TGF-β, dexamethasone, ascorbic acid, and L-proline). Sponge-cell cultures were collected, and ATP concentration was measured using an ATP assay kit. Cytochrome C oxidase activity was measured using a cytochrome C oxidase activity assay kit. A control group (without any sponges) was used for cytochrome C oxidase activity measurement. Mitochondrial morphology was observed using a deep red fluorescent probe. The culture time was extended to 14 days, and the expression of molybdenum sulfite oxidase-related genes (MOCS1, SUOX1) was measured. Results are as follows: Figure 14 As shown.

[0138] Figure 14 Figure (a) shows the results of ATP content detection. Figure 14 Figure (b) shows the results of cytochrome C oxidase activity assay, where C represents the control group. Figure 14 Image (c) is an image of intracellular mitochondrial staining. Figure 14 Figure (d) shows the expression levels of the molybdenum cofactor synthesis 1 gene (MOCS1) and the sulfite oxidase 1 gene (SUOX1). Figure 12 It is known that sponge M can upregulate the expression of the MOCS1 gene, promote the synthesis of molybdenum cofactor, and thus promote the synthesis of molybdenum enzyme, thereby providing electrons for cytochrome C, enhancing the activity of cytochrome C oxidase, and thus accelerating the electron chain transfer process and promoting ATP production.

[0139] Example 14

[0140] In this embodiment, the regulatory effect of sponge M prepared in Example 8 on the chondrogenic differentiation of BMSCs under oxidative stress is verified.

[0141] Sponges M and S prepared in Example 8 and Comparative Example 1 were sterilized in a UV chamber for 1 h, and then BMSCs were sterilized at 3 × 10⁻⁶ ppm. 6 Sponges were seeded onto sterilized sponges M and S at a density of [missing information]. H2O2 was added every two days to a concentration of 200 μmol / L for 24 h of stimulation. The sponges were then cultured for 14 days in chondrocyte induction medium (DMEM high-glucose medium containing ITS, TGF-β, dexamethasone, ascorbic acid, and L-proline). Sponge-cell cultures were then collected, fixed, embedded, and sectioned. Immunofluorescence staining was used to observe the expression of Col II and Sox 9, safranin staining was used to observe glycosaminoglycan secretion, and GAG / DNA quantification was used to determine glycosaminoglycan secretion. q-PCR was used to determine the expression of chondrocyte differentiation-related genes (SOX9 / ACAN / Col II / Col I). The results are as follows: Figure 15 As shown.

[0142] Figure 15 Figure (a) is a staining image of Col II, Sox9, and SO. Figure 15 Figure (b) shows the GAG / DNA measurement results. Figure 15 Figure (c) shows the gene expression levels as determined by q-PCR. Figure 15 It can be seen that, compared with sponge S, sponge M can better promote the expression of cartilage matrix-related genes such as Col II, Sox9, and glycosaminoglycans and matrix secretion under oxidative stress chondrogenic induction conditions. Sponge M shows a superior ability to promote BMSCs chondrogenic differentiation than sponge S.

[0143] Example 15

[0144] In this embodiment, the effect of sponge M prepared in Example 8 on promoting cartilage repair in a rabbit knee joint cartilage defect model was investigated.

[0145] Sponges M and S, prepared in Example 8 and Comparative Example 1, were sterilized in a UV chamber for 1 h. A full-thickness cartilage defect model was constructed in the rabbit knee joint, and microfracture was performed. Subsequently, the sterilized sponges M and S were implanted into the cartilage defect site of the rabbit knee joint, and the skin and flesh were sutured. Twenty weeks after implantation, the rabbits were euthanized, and the joint site was removed for gross imaging. After fixation, decalcification, embedding, and sectioning, histological staining and immunohistochemical staining were performed to analyze the morphology and function of the reconstructed cartilage defect. The case without a full-thickness cartilage defect in the knee joint was designated as the natural group (N). Results are as follows: Figure 16 As shown.

[0146] Figure 16 Figure (a) shows a gross view of the articular cartilage defect. Figure 16 Image (b) shows a reconstructed cartilage surface captured by a super depth-of-field microscope, combined with... Figure 16 As shown in Figures (a) and (b), 20 weeks after implantation of sponge M, the newly formed cartilage is closely connected with the adjacent cartilage tissue, and the repair is smoother than that after implantation of sponge S. Figure 16 Figure (c) shows an HE-stained image of the articular cartilage defect site, which further confirms that the surface of the newly formed cartilage is smoother 20 weeks after implantation of sponge M. Figure 16 Figure (d) shows the Safranin-Fix-Green (SO / FG) stained image. As can be seen from the figure, when sponge M is implanted for repair, the thickness of the newly formed cartilage is greater than that of normal cartilage, and it has more glycosaminoglycan matrix secretion. Figure 16 Figure (e) shows a Sirius red staining image. As can be seen from the figure, when sponge M is implanted for repair, the arrangement of newly formed fibers is more orderly and closer to natural cartilage compared to when sponge S is implanted for repair. Figure 16 Figure (f) shows the immunohistochemical staining images of Col II, Col I, and Col X. As can be seen from the figure, compared with the implanted sponge S, the implanted sponge M has more Col II secretion and less Col I and Col X expression. This indicates that sponge M can better promote the repair of articular cartilage defects than sponge S.

[0147] Example 16

[0148] In this embodiment, the mitochondrial-activated sponge for repairing articular cartilage defects is prepared through the following steps:

[0149] (1) Following the method of Example 1, HASH with a cysteine ​​grafting rate of approximately 30% was prepared. The HASH was dissolved in ultrapure water to obtain a HASH solution. The prepared HASH was dissolved in deionized water to obtain a HASH solution with a concentration of 1 mg / mL. Nano MoS2 was added to the HASH solution at a ratio of 2 mg of nano MoS2 per 1 mL of HASH solution. The solution was sonicated in an ice-water bath for 5 h at a sonication power of 400 W. After centrifugation, the resulting solid phase was washed with deionized water. The washed solid phase was MoS2@HS. The MoS2@HS was dispersed in deionized water and stored. The MoS2@HS dispersed in deionized water was added to the HASH solution and mixed thoroughly to obtain a mixture. The concentration of the SFD solution prepared in Example 2 was adjusted to 30 mg / mL to obtain the adjusted concentration SFD solution.

[0150] (2) Mix the mixture and the SFD solution after adjusting the concentration thoroughly and adjust the pH value to 7~8 to obtain the gel precursor solution. Inject the gel precursor solution into the PDMS mold and let it stand until the gel precursor solution turns into a hydrogel state. Freeze-dry the obtained hydrogel to obtain the mitochondrial activated sponge for articular cartilage defect repair.

[0151] In this step, the concentration of MoS2@HS in the gel precursor solution is controlled to be 0.2 mg / mL, the concentration of HASH is 5 mg / mL, and the concentration of SFD is 10 mg / mL, that is, the mass ratio of MoS2@HS, HASH and SFD is 0.02:0.5:1.

[0152] Example 17

[0153] In this embodiment, the mitochondrial-activated sponge for repairing articular cartilage defects is prepared through the following steps:

[0154] (1) Following the method of Example 1, HASH with a cysteine ​​grafting rate of approximately 10% was prepared. The HASH was dissolved in ultrapure water to obtain a HASH solution. The prepared HASH was dissolved in deionized water to obtain a HASH solution with a concentration of 10 mg / mL. Nano MoS2 was added to the HASH solution at a ratio of 2 mg of nano MoS2 per 1 mL of HASH solution. The solution was sonicated in an ice-water bath for 2 h with a sonication power of 500 W. After centrifugation, the resulting solid phase was washed with deionized water. The washed solid phase was MoS2@HS. The MoS2@HS was dispersed in deionized water and stored. The MoS2@HS dispersed in deionized water was added to the HASH solution and mixed thoroughly to obtain a mixture. Following the method of Example 2, SFD with a catechol group content of approximately 6 μg / mg was prepared. The concentration of the SFD solution was adjusted to 40 mg / mL to obtain the adjusted concentration SFD solution.

[0155] (2) Mix the mixture and the SFD solution after adjusting the concentration thoroughly, adjust the pH of the mixture to 7-8, and obtain the gel precursor solution. Inject the gel precursor solution into the PDMS mold and let it stand until the gel precursor solution turns into a hydrogel state. Freeze-dry the obtained hydrogel to obtain the mitochondrial activated sponge for the repair of articular cartilage defects.

[0156] In this step, the concentration of MoS2@HS in the gel precursor solution is controlled to be 0.05 mg / mL, the concentration of HASH is 40 mg / mL, and the concentration of SFD is 5 mg / mL, that is, the mass ratio of MoS2@HS, HASH and SFD is 0.01:8:1.

[0157] Example 18

[0158] In this embodiment, the mitochondrial-activated sponge for repairing articular cartilage defects is prepared through the following steps:

[0159] (1) Following the method of Example 1, a cysteine-grafted HASH with a grafting rate of approximately 20% was prepared. The HASH was dissolved in ultrapure water to obtain a HASH solution. The prepared HASH was dissolved in deionized water to obtain a HASH solution with a concentration of 3 mg / mL. Nano MoS2 was added to the HASH solution at a ratio of 1 mg of nano MoS2 per 1 mL of HASH solution. The solution was sonicated in an ice-water bath for 8 h with a sonication power of 200 W. After centrifugation, the resulting solid phase was washed with deionized water. The washed solid phase was MoS2@HS. The MoS2@HS was dispersed in deionized water and stored. The MoS2@HS dispersed in deionized water was added to the HASH solution and mixed thoroughly to obtain a mixture. The concentration of the SFD solution prepared in Example 2 was adjusted to 20 mg / mL to obtain the adjusted concentration SFD solution.

[0160] (2) Mix the mixture and the SFD solution after adjusting the concentration thoroughly and adjust the pH value to 7~8 to obtain the gel precursor solution. Inject the gel precursor solution into the PDMS mold and let it stand until the gel precursor solution turns into a hydrogel state. Freeze-dry the obtained hydrogel to obtain the mitochondrial activated sponge for articular cartilage defect repair.

[0161] In this step, the concentration of MoS2@HS in the gel precursor solution is controlled to be 0.08 mg / mL, the concentration of HASH is 20 mg / mL, and the concentration of SFD is 4 mg / mL, that is, the mass ratio of MoS2@HS, HASH and SFD is 0.02:5:1.

Claims

1. A mitochondrial-activated sponge for repairing articular cartilage defects, characterized in that, The sponge is formed by freeze-drying a hydrogel formed by reacting nano-molybdenum disulfide modified with thiol-modified hyaluronic acid, thiol-modified hyaluronic acid, and polyphenol-modified silk fibroin at a mass ratio of (0.002~0.02):(0.5~8):1 under pH 7~8 conditions. The thiol-modified hyaluronic acid-exfoliated molybdenum disulfide nanoparticles are composed of thiol-modified hyaluronic acid and molybdenum disulfide nanoparticles. The thiol-modified hyaluronic acid is located on the surface of the molybdenum disulfide nanoparticles, and the thiol-modified hyaluronic acid and molybdenum disulfide nanoparticles are chemically bonded through Mo-S bonds. At the same time, the thiol-modified hyaluronic acid undergoes oxidative self-crosslinking. The preparation method of the thiol-modified hyaluronic acid-exfoliated molybdenum disulfide nanoparticles is as follows: molybdenum disulfide is added to an aqueous solution of thiol-modified hyaluronic acid, and the mass ratio of thiol-modified hyaluronic acid to molybdenum disulfide is (0.5~5):

1. The mixture is subjected to thorough ultrasonic treatment at 1~4 ℃, centrifuged, and the resulting solid phase is washed with water to obtain the final product.

2. The mitochondrial-activated sponge for repairing articular cartilage defects according to claim 1, characterized in that, In preparing thiol-modified hyaluronic acid-exfoliated molybdenum disulfide nanoparticles, the concentration of the aqueous solution of thiol-modified hyaluronic acid was controlled to be 1~10 mg / mL.

3. The mitochondrial-activated sponge for repairing articular cartilage defects according to claim 1, characterized in that, In the preparation of thiol-modified hyaluronic acid-exfoliated molybdenum disulfide nanoparticles, the ultrasonic power was controlled at 200~500 W and the ultrasonic time was 2~8 h.

4. The mitochondrial-activated sponge for repairing articular cartilage defects according to any one of claims 1 to 3, characterized in that, The grafting rate of cysteine ​​in thiol-modified hyaluronic acid is 5%~65%.

5. The mitochondrial-activated sponge for repairing articular cartilage defects according to claim 4, characterized in that, The molecular weight of hyaluronic acid used as the basis for thiol-modified hyaluronic acid is 300~1000 kDa.

6. The mitochondrial-activated sponge for repairing articular cartilage defects according to any one of claims 1 to 3, characterized in that, Polyphenol-modified silk fibroin is formed by grafting polyphenols onto silk fibroin, wherein the polyphenols include dopamine, tannic acid or caffeic acid.

7. The mitochondrial-activated sponge for repairing articular cartilage defects according to claim 6, characterized in that, In polyphenol-modified silk fibroin, the polyphenol content is 5~20 μg / mg.

8. The mitochondrial-activated sponge for repairing articular cartilage defects according to any one of claims 1 to 3, characterized in that, The sponge has a pore size of 20~120 μm and a porosity of 60%~75%.

9. The method for preparing the mitochondrial-activated sponge for articular cartilage defect repair according to any one of claims 1 to 8, characterized in that, Includes the following steps: (1) Dissolve thiol-modified hyaluronic acid in water to obtain a thiol-modified hyaluronic acid solution; add a dispersion of thiol-modified hyaluronic acid-exfoliated molybdenum disulfide nanoparticles to the thiol-modified hyaluronic acid solution and mix thoroughly to obtain a mixture; fully disperse polyphenol-modified silk fibroin in water to obtain a polyphenol-modified silk fibroin solution. (2) The mixture is thoroughly mixed with the polyphenol-modified silk fibroin solution and the pH value is adjusted to 7-8 to obtain a gel precursor solution. The gel precursor solution is transferred to a mold and allowed to stand until the gel precursor solution is converted into a hydrogel state. The obtained hydrogel is freeze-dried to obtain a mitochondrial activated sponge for the repair of articular cartilage defects. In the gel precursor solution, the mass ratio of mercapto-modified hyaluronic acid-exfoliated molybdenum disulfide nanoparticles, mercapto-modified hyaluronic acid, and polyphenol-modified silk fibroin is (0.002~0.02):(0.5~8):

1.

10. The method for preparing the mitochondrial-activated sponge for repairing articular cartilage defects according to claim 9, characterized in that, In step (2), the concentration of polyphenol-modified silk fibroin in the gel precursor solution is controlled to be 4~20 mg / mL.

Images