Cartilage organ with function of maintaining mitochondrial homeostasis as well as preparation method and application of cartilage organ

Abstract

The invention relates to a cartilage organ with a mitochondrial homeostasis maintaining function as well as a preparation method and application of the cartilage organ. The preparation method comprises the following steps: preparing a DNA (deoxyribonucleic acid)-silk fibroin hybrid hydrogel solution containing MnO2 nanoparticles and Melatonin; the DNA-silk fibroin hybrid hydrogel solution is mixed with bone marrow mesenchymal stem cells, and a DNA-silk fibroin hybrid hydrogel solution loaded with BMSCs is obtained; a cartilage organ precursor is formed through DLP biological 3D printing; and culturing the cartilage organ precursor in a culture medium to obtain the cartilage organ with the function of maintaining the mitochondrial homeostasis. According to the functionalized cartilage organoid, the culture efficiency of the organoid can be improved, and the stable state of cell mitochondria can be better maintained, so that the activity of cartilage cells is improved, cartilage regeneration and repair are promoted, and alternative materials are provided for preparing medicaments or medical instruments for repairing cartilage defects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and in particular to cartilage organoids having the function of maintaining mitochondrial homeostasis, and preparation methods and applications thereof. Background Art

[0002] Cartilage defects refer to the partial breakdown or complete loss of cartilage tissue within a joint. Cartilage defects pose a serious threat to human health and well-being. In recent years, cartilage defects caused by trauma (traffic accidents, sports injuries, work-related injuries), diseases (orthopedic inflammation, bone tumors, infections), and genetic factors have become increasingly common. Cartilage defects in joints can cause even more severe disability. According to incomplete statistics, osteoarthritis (OA), a leading cause of disability among the elderly, affects over 500 million people worldwide.

[0003] Currently, treatments for articular cartilage damage include autologous chondrocyte transplantation, microfracture, autologous / allogeneic cartilage transplantation, and joint replacement. However, these strategies still have significant limitations. For example, the source of autologous chondrocytes is limited and their survival rate during implantation is low; microfracture can easily lead to cartilage fibrosis; autologous / allogeneic cartilage transplantation can lead to immune rejection; and joint replacement surgery is invasive and faces problems such as poor osseointegration and susceptibility to infection. Therefore, there is an urgent need to explore new approaches to achieve rapid repair of articular cartilage damage.

[0004] In recent years, organoid-based repair strategies have become an important research direction in tissue regeneration. Cartilage organoids are miniature cartilage tissue models cultivated using stem cells or chondrocytes through three-dimensional culture techniques. Cartilage organoids exhibit a 3D structure similar to native cartilage, including an ECM rich in type II collagen and proteoglycans, and embedded chondrocytes. They also exhibit mechanical properties very similar to native cartilage. Furthermore, cartilage organoids can integrate into host tissue, facilitating the repair and regeneration of damaged cartilage. Traditional materials used to repair cartilage tissue typically undergo a complex and time-consuming process involving material implantation, cell recruitment, cell proliferation, and matrix secretion. However, cartilage organoids, when used for cartilage regeneration, can secrete matrix directly at the defect site, enabling rapid cartilage regeneration. Therefore, constructing cartilage organoids as in vitro 3D research models or transplant materials for cartilage regeneration offers a new avenue for cartilage tissue engineering.

[0005] During the construction and culture of cartilage organoids, cells are often subject to oxidative stress, which can compromise mitochondrial homeostasis. This is a significant issue. Oxidative stress is primarily caused by the excessive accumulation of intracellular reactive oxygen species (ROS). ROS production is closely related to cellular metabolic activity, environmental stressors (such as hypoxia and nutrient deficiency), and culture conditions (such as high oxygen concentrations or mechanical stimulation). During organoid culture, cells undergo rapid proliferation, differentiation, and self-organization into three-dimensional structures. These processes inherently increase the metabolic burden on the cells, leading to increased stress on mitochondrial function and the generation of excessive ROS. If these ROS are not promptly cleared, they can cause oxidative damage to intracellular lipids, proteins, and DNA, impairing cell survival, proliferation, and functional expression. Furthermore, excessive ROS can cause a decrease in mitochondrial membrane potential, leading to mitochondrial damage. Damaged mitochondria not only fail to produce efficient energy but also continue to generate more ROS, damaging other healthy mitochondria. Ultimately, this can lead to structural and functional impairment of the organoid, and even culture failure. Therefore, maintaining mitochondrial homeostasis in organoids is crucial for successful organoid culture.

[0006] Chinese patent CN119950814A discloses a cartilage organoid constructed based on a DNA-silk fibroin hybrid hydrogel sustained-release system, as well as its preparation method and application. The DNA-silk fibroin hybrid hydrogel sustained-release system is a hydrogel system obtained by printing a premix of the DNA-silk fibroin hybrid hydrogel sustained-release system using a digital light processing system. The premix contains DNA, silk fibroin, acryloyl-RGD peptide, acryloyl-PEG-NHS ester, glucosamine, TD-198946, and lithium 2,4,6-trimethylbenzoyl phosphate. The glucosamine and TD-198946 introduced into the DNA-silk fibroin hybrid hydrogel sustained-release system are intended to enhance the chondrogenic differentiation efficiency of bone marrow mesenchymal stem cells. However, the solution disclosed in this patent does not address the issue of oxidative stress-induced cell damage.

[0007] The existing technology lacks effective organoid materials that can significantly eliminate intracellular ROS and maintain mitochondrial homeostasis during the construction and culture of cartilage organoids. Summary of the Invention

[0008] Based on the current situation in which there is a lack of effective organoid materials in the prior art that can significantly remove intracellular ROS and maintain mitochondrial homeostasis during the construction and culture of cartilage organoids, the present invention provides a cartilage organoid with the function of maintaining mitochondrial homeostasis, and a preparation method and application thereof.

[0009] The purpose of the present invention can be achieved by the following technical solutions: The present invention provides a method for preparing a cartilage organoid having the function of maintaining mitochondrial homeostasis, comprising the following steps: Step 1: Prepare a DNA-silk fibroin hybrid hydrogel solution containing MnO2 nanoparticles and Melatonin; Step 2: Mixing the DNA-silk fibroin hybrid hydrogel solution containing MnO2 nanoparticles and melatonin with bone marrow mesenchymal stem cells (BMSCs) to obtain a DNA-silk fibroin hybrid hydrogel solution loaded with BMSCs; Step 3: The DNA-silk fibroin hybrid hydrogel solution loaded with BMSCs was 3D-printed into cartilage organoid precursors using DLP bioprinting. Step 4: Cultivate the cartilage organoid precursors in culture medium to obtain cartilage organoids with the function of maintaining mitochondrial homeostasis.

[0010] In one embodiment of the present invention, in step 1, the method for preparing the DNA-silk fibroin hybrid hydrogel solution is: Step 1.1, Construction of DNA supramolecular network: In a buffer solution, a Y-shaped DNA single strand and an L-shaped DNA single strand are mixed to obtain a DNA solution, and a DNA supramolecular network is formed by cross-linking the sticky ends of the Y-shaped DNA single strand and the L-shaped DNA single strand; Step 1.2, preparation of DNA-silk fibroin hybrid hydrogel solution: Mix the methacryloylated silk fibroin and photoinitiator with the DNA solution prepared in step 1.1 to obtain a DNA-silk fibroin hybrid hydrogel solution.

[0011] In one embodiment of the present invention, the Y-shaped DNA single strand comprises DNA single strand Y1, DNA single strand Y2, and DNA single strand Y3, wherein the nucleotide sequence of DNA single strand Y1 is shown in SEQ ID NO.1, the nucleotide sequence of DNA single strand Y2 is shown in SEQ ID NO.2, and the nucleotide sequence of DNA single strand Y3 is shown in SEQ ID NO.3; The L-shaped DNA single strand includes DNA single strand L1 and DNA single strand L2, the nucleotide sequence of DNA single strand L1 is shown in SEQ ID NO.4, and the nucleotide sequence of DNA single strand L2 is shown in SEQ ID NO.5; The sticky ends of the Y-shaped DNA single strand are complementary to the sticky ends of the L-shaped DNA single strand.

[0012] The three DNA single strands Y1, Y2 and Y3 each contain 39 nucleotides, and each single strand has three 13-nucleotide sticky ends.

[0013] Both DNA single strands L1 and L2 contain 44 nucleotides, and each single strand has a 13-nucleotide sticky end at both ends, which is completely complementary to the sticky end of the Y-shaped DNA single strand.

[0014] In one embodiment of the present invention, the molar ratio of DNA single strand Y1, DNA single strand Y2 and DNA single strand Y3 is 1:1:1; the molar ratio of DNA single strand L1 and DNA single strand L2 is 1:1; and the total molar ratio of Y-shaped DNA single strand and L-shaped DNA single strand is 1:1 to 1:2.

[0015] Furthermore, the nucleotide sequences of the Y-shaped and L-shaped DNA single strands are as follows: In one embodiment of the present invention, in step 1.2, the concentration of the methacrylylated silk fibroin in the hydrogel solution is 5-15 wt %, preferably 10 wt %.

[0016] In one embodiment of the present invention, in step 1.2, the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, and the concentration of 2,4,6-trimethylbenzoyl lithium phosphate in the hydrogel solution is 0.1-0.5 wt%, preferably 0.25 wt%.

[0017] In one embodiment of the present invention, in step 1, MnO2 nanoparticles and Melatonin are added to the DNA-silk fibroin hybrid hydrogel solution to obtain a DNA-silk fibroin hybrid hydrogel solution containing MnO2 nanoparticles and Melatonin.

[0018] In one embodiment of the present invention, in step 1, the method for preparing MnO2 nanoparticles is as follows: potassium permanganate (KMnO4) is dissolved in deionized water to prepare a solution (the concentration of the solution can be selected to be 0.1 M), and a reducing agent sodium citrate solution is slowly added dropwise to the KMnO2 solution under stirring conditions, and the reaction temperature is maintained at 60-80°C. During the reaction, the color of the solution gradually changes from purple to brown, indicating that MnO2 nanoparticles have been formed.

[0019] In one embodiment of the present invention, in step 1, in the DNA-silk fibroin hybrid hydrogel solution containing MnO2 nanoparticles and Melatonin, the concentration of MnO2 nanoparticles is 0.5-2 wt%, preferably 1 wt%.

[0020] In one embodiment of the present invention, in step 1, the concentration of melatonin in the DNA-silk fibroin hybrid hydrogel solution containing MnO2 nanoparticles and melatonin is 0.5-2 wt%, preferably 1 wt%.

[0021] In one embodiment of the present invention, in step 2, the bone marrow mesenchymal stem cells (BMSCs) are (1-10)×10 6 The concentration of 100 cells / mL was added to a DNA-silk fibroin hybrid hydrogel solution containing MnO2 nanoparticles and Melatonin to obtain a DNA-silk fibroin hybrid hydrogel solution loaded with BMSCs.

[0022] In one embodiment of the present invention, in step 3, the conditions for using DLP bio-3D printing to form cartilage organoid precursors are: DLP printing parameters are light intensity 17mW / cm 2 , exposure time is 15 s, the number of base layers is 5, and the exposure time of the base layer is 10 s.

[0023] In one embodiment of the present invention, in step 4, the cartilage organoid precursors are cultured in a cartilage culture medium for up to 6 weeks to obtain cartilage organoids having the function of maintaining mitochondrial homeostasis.

[0024] In one embodiment of the present invention, the cartilage culture medium is: DMEM high glucose medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, 1 mM sodium pyruvate, 10 ng / ml transforming growth factor-β3 (TGF-β3), 0.1 μM dexamethasone, 1% ITS premix and 1 μM ascorbic acid-2-phosphate.

[0025] In one embodiment of the present invention, in step 4, the culture conditions are: the culture environment is 37° C., 5% CO 2 , and the cartilage culture medium is replaced every 2 days.

[0026] The present invention further provides cartilage organoids prepared based on the above method and having the function of maintaining mitochondrial homeostasis.

[0027] The cartilage organoids having the function of maintaining mitochondrial homeostasis can maintain the mitochondrial homeostasis of BMSCs and promote the chondrogenic differentiation of BMSCs.

[0028] The present invention further provides the use of cartilage organoids with the function of maintaining mitochondrial homeostasis prepared based on the above method in the preparation of cartilage repair materials. When used in cartilage repair materials, the cartilage organoids can promote cartilage regeneration.

[0029] DNA hydrogels are materials primarily composed of DNA, forming a hydrophilic three-dimensional network structure. They retain the excellent biocompatibility, biodegradability, and programmability of DNA, and seamlessly integrate the mechanical properties of its backbone, making them an ideal material for constructing cartilage organoids. Compared to pure DNA hydrogels, composite DNA hydrogels exhibit superior mechanical properties. Current DNA hydrogels synthesized in research are generally measured in microliters, resulting in high synthesis costs. The development of hybrid hydrogels could, to some extent, alleviate the high cost associated with pure DNA hydrogels. Furthermore, compared to the generally weaker mechanical properties of pure DNA hydrogels, composite DNA hydrogels exhibit enhanced mechanical properties. However, the use of traditional monolithic hydrogels remains challenging due to their low solute diffusion efficiency and limitations in submicron applications. Current DNA hydrogels synthesized in research are generally measured in microliters, resulting in high synthesis costs. The development of hybrid hydrogels could, to some extent, alleviate the high cost associated with pure DNA hydrogels. Therefore, the introduction of a silk fibroin network in this application could improve the mechanical properties of the DNA hydrogel network and reduce costs.

[0030] Clearing excess ROS in cells and promoting autophagy of damaged mitochondria can relieve the stress of the cell mitochondrial system to a certain extent and maintain the homeostasis of the mitochondrial system. Therefore, in the present application scheme, MnO2 nanoparticles and Melatonin are loaded into the hydrogel. As a biomaterial, MnO2 nanoparticles are widely used for ROS clearance in vivo due to their excellent antioxidant capacity and biocompatibility. Melatonin is a natural hormone secreted by the pineal gland, which is famous for its circadian rhythm regulation and antioxidant properties. Melatonin can activate the PINK1 and Parkin pathways, which is an important regulatory mechanism of mitochondrial autophagy. PINK1 accumulates stably when the mitochondrial membrane potential decreases, and recruits Parkin to the mitochondrial surface, promoting the ubiquitination of mitochondrial proteins, thereby triggering the formation of autophagosomes.

[0031] This application scheme loads MnO2 nanoparticles and Melatonin into DNA-silk fibroin hybrid hydrogel to construct a cartilage organoid system that can maintain mitochondrial homeostasis, breaking through the limitations of traditional cartilage organoids in oxidative stress microenvironments and providing a multi-dimensional solution for cartilage construction.

[0032] The solution provided by the present invention is of great significance for promoting the development of new cartilage regeneration therapies and improving the quality of life of osteoarthritis patients.

[0033] The introduction of the composite hydrogel silk fibroin network described in the present invention can provide mechanical properties to the DNA hydrogel network and reduce production costs. MnO2 nanoparticles can remove excess ROS produced by cells under oxidative stress, and melatonin can promote autophagy of damaged mitochondria, thereby maintaining mitochondrial homeostasis and enhancing cell viability. The functionalized cartilage organoids can improve organoid culture efficiency and better maintain cell mitochondrial homeostasis, thereby enhancing chondrocyte viability and promoting cartilage regeneration and repair, providing an alternative material for the preparation of pharmaceutical agents or medical devices for repairing cartilage defects.

[0034] Compared with the prior art, the present invention has the following advantages and beneficial effects: This invention constructs Mn-Me@DSH by loading MnO2 nanoparticles and melatonin onto a DNA-SF hydrogel (a DNA-silk fibroin hybrid hydrogel). This hydrogel solution is then mixed with cells, and then bone marrow mesenchymal stem cells (BMSCs) and Mn-Me@DSH are combined using a DLP system. Ultimately, functionalized cartilage organoids (MMCOs) that maintain mitochondrial homeostasis are cultivated for cartilage repair. In vivo experiments have demonstrated that MMCOs significantly improve mitochondrial function in cells. In vitro experiments have also demonstrated that MMCOs enhance cartilage regeneration. This study innovatively utilizes a dual-functional strategy of "ROS scavenging and autophagy activation" to overcome the limitations of traditional cartilage organoids in oxidative stress microenvironments, providing a multi-dimensional solution for cartilage organoid construction and cartilage regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 . Actual image of functional cartilage organoids.

[0036] Figure 2 Immunofluorescence staining of SOX9, Col II, and ACAN proteins in functionalized cartilage organoids from 2 to 8 weeks.

[0037] Figure 3 . ROS fluorescence staining of functionalized cartilage organoids from 2 to 8 weeks.

[0038] Figure 4 . JC-1 membrane potential staining of functionalized cartilage organoids from 2 to 8 weeks.

[0039] Figure 5 Mitochondrial and lysosomal staining of functionalized cartilage organoids from 2 to 8 weeks.

[0040] Figure 6 Macroscopic observation of cartilage repair in functionalized cartilage organoids at 3 and 6 weeks.

[0041] Figure 7. Histological staining of new cartilage in functionalized cartilage organoids after 3 and 6 weeks of cartilage repair.

[0042] Figure 8 Immunohistochemical staining of newly formed cartilage after 3 and 6 weeks of cartilage repair in functionalized cartilage organoids. DETAILED DESCRIPTION

[0043] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0044] To complete the contents of the present invention, the experimental reagents, materials or instruments used in the examples were purchased from commercial products unless otherwise specified.

[0045] Example 1

[0046] This embodiment provides a method for preparing cartilage organoids capable of maintaining mitochondrial homeostasis, comprising the following steps: Step 1: Preparation of DNA-silk fibroin hybrid hydrogel solution containing MnO2 nanoparticles and Melatonin: The specific method is as follows: Step 1.1, Construction of DNA supramolecular network: Y-shaped and L-shaped DNA single strands were mixed at a total concentration of 500 nmol / mL in a phosphate buffered saline solution at pH 7.4 at 37°C to obtain a DNA solution, and a DNA supramolecular network was formed by cross-linking the sticky ends of the Y-shaped DNA single strands and the L-shaped DNA single strands; Wherein, the Y-shaped DNA single strand includes DNA single strand Y1, DNA single strand Y2 and DNA single strand Y3, wherein the nucleotide sequence of DNA single strand Y1 is shown in SEQ ID NO.1, the nucleotide sequence of DNA single strand Y2 is shown in SEQ ID NO.2, and the nucleotide sequence of DNA single strand Y3 is shown in SEQ ID NO.3; the L-shaped DNA single strand includes DNA single strand L1 and DNA single strand L2, the nucleotide sequence of DNA single strand L1 is shown in SEQ ID NO.4, and the nucleotide sequence of DNA single strand L2 is shown in SEQ ID NO.5; the molar ratio of DNA single strand Y1, DNA single strand Y2 and DNA single strand Y3 is 1:1:1; the molar ratio of DNA single strand L1 and DNA single strand L2 is 1:1; in this embodiment, the total molar ratio of Y-shaped DNA single strand and L-shaped DNA single strand is 1:1.

[0047] Step 1.2, preparation of DNA-silk fibroin hybrid hydrogel solution: The methacrylated silk fibroin and the photoinitiator were mixed with the DNA solution in step 1.1 to obtain a DNA-silk fibroin hybrid hydrogel solution, wherein the concentration of the methacrylated silk fibroin was 10 wt %, the photoinitiator was phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, and the concentration of 2,4,6-trimethylbenzoyl lithium phosphate was 0.25 wt %.

[0048] Step 1.3, adding MnO2 nanoparticles and Melatonin to the DNA-silk fibroin hybrid hydrogel solution, the concentration of MnO2 nanoparticles is 1wt%, and the concentration of melatonin is 1wt%, thereby obtaining a DNA-silk fibroin hybrid hydrogel solution containing MnO2 nanoparticles and Melatonin.

[0049] Step 2: 2 ml of DNA-silk fibroin hybrid hydrogel solution containing MnO2 nanoparticles and Melatonin was mixed with bone marrow mesenchymal stem cells (BMSCs) at a concentration of 1×10 6 The concentration of cells / mL was added to obtain a DNA-silk fibroin hybrid hydrogel solution loaded with BMSCs. The DNA-silk fibroin hybrid hydrogel solution loaded with BMSCs can also be called cell-loaded 3D printing bio-ink.

[0050] Step 3: The DNA-silk fibroin hybrid hydrogel solution loaded with BMSCs was 3D-printed into cartilage organoid precursors using DLP bioprinting. The specific conditions were: DLP printing parameters were a light intensity of 17 mW / cm 2 , exposure time 15 s, number of base layers is 5, base exposure time 10 s; Step 4: Cultivate the cartilage organoid precursors in culture medium to obtain cartilage organoids that maintain mitochondrial homeostasis. The culture medium consists of DMEM high-glucose medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 1 mM sodium pyruvate, 10 ng / ml transforming growth factor-β3 (TGF-β3), 0.1 μM dexamethasone, 1% ITS premix, and 1 μM ascorbic acid 2-phosphate. Culture conditions are: 37°C, 5% CO2, with the culture medium replaced every 2 days for 8 weeks.

[0051] The functional cartilage organoids prepared in this example are shown in the figure below. Figure 1 As shown in the figure, we can see that the diameter of the cartilage organoids is about 3 mm, and they are consistent in size and complete in shape.

[0052] Example 2

[0053] This example mainly investigates the growth and development of functional cartilage organoids. Functionalized cartilage organoids cultured at 2, 4, 6, and 8 weeks were collected. Organoid samples were fixed in 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 for 10 minutes, blocked with 10% goat serum for 2 hours, and incubated overnight with primary antibodies (Col II, ACAN, and COL X). Finally, the samples were incubated with secondary antibodies (1:5000) and TRITC-phalloidin for 1 hour at room temperature. After staining with DAPI, images were acquired using an inverted fluorescence microscope.

[0054] from Figure 2 It can be seen that as the functional cartilage organoids cultured for 6 weeks grow, the expression of representative proteins of cartilage development in the organoids increases, such as SOX9, Col II and ACAN. This shows that the functional cartilage organoids promote the cartilage differentiation of BMSCs and the secretion of cartilage matrix. However, at 8 weeks, these representative proteins of cartilage development in the organoids decrease, which means that the functional cartilage organoids may gradually lose their homeostasis during long-term culture, simulating the early stage of cartilage degeneration in vivo. In addition, from Figure 2 It can be seen that the cartilage hypertrophy protein COL X continues to increase with time, which indicates that as the functional cartilage organoids grow over time, their cells are prone to hypertrophy, resulting in a decrease in cartilage matrix secretion.

[0055] Example 3

[0056] As described in Example 1 above, a cartilage organoid constructed by DNA-SF hydrogel loaded with MnO2 and Melatonin and BMSCs is provided, referred to as MMCO.

[0057] For comparison, unlike Example 1, on the basis of Example 1, only Melatonin-loaded DNA-SF hydrogel and BMSCs-constructed cartilage organoids were prepared, referred to as MeCO.

[0058] For comparison, different from Example 1, based on Example 1, only MnO2 was loaded into the DNA-SF hydrogel and cartilage organoids constructed by BMSCs without loading Melatonin, which is referred to as MnCO.

[0059] For comparison, unlike Example 1, on the basis of Example 1, without loading MnO2 and Melatonin, a cartilage organoid was constructed using only DNA-SF hydrogel and BMSCs, referred to as CO.

[0060] In order to investigate the ability of functional cartilage organoids to clear ROS during growth and development, ROS fluorescence staining was performed on different cartilage organoids cultured for 2, 4, 6, and 8 weeks. Figure 3 As shown, from Figure 3 As can be seen in the figure, cartilage organoids without MnO2 and melatonin loading showed elevated ROS levels throughout the culture period, while MnO2-loaded cartilage organoids showed lower ROS levels compared to standard organoids. This suggests that functionalized cartilage organoids can reduce elevated ROS levels caused by oxidative stress during cartilage organoid culture.

[0061] Example 4

[0062] Based on Example 3, in order to explore the ability of functional cartilage organoids to maintain mitochondrial homeostasis during growth and development, JC-1 membrane potential and lysosome staining were performed on different cartilage organoids cultured for 2 weeks, 4 weeks, 6 weeks, and 8 weeks. Figure 4 and Figure 5 As shown, first from Figure 4 It can be seen from the figure that the JC-1 membrane potential of the functional cartilage organoids increased throughout the time period, while the JC-1 membrane potential of the ordinary cartilage organoids decreased. Figure 5 As can be seen in the figure, functional cartilage organoids exhibit high levels of mitochondrial autophagy throughout the entire time period. These two phenomena indicate that functional cartilage organoids can maintain mitochondrial homeostasis by increasing membrane potential and promoting mitochondrial autophagy.

[0063] Example 5

[0064] This example is verified by animal experiments. The specific method is as follows: (1) Construction of rat cartilage defect model: SD male rats aged 7-8 weeks (weighing about 250 g) were selected. After general anesthesia, the joint capsule was cut to expose the femoral trochlea, and a cartilage defect with a diameter of 2 mm and a depth of 1.5 mm was constructed in the middle of the femoral trochlea using a punch.

[0065] (2) Application of functionalized cartilage organoids to repair cartilage defects: The organoids (CO and MMCO) prepared above were transplanted in situ into the cartilage defects and sutured layer by layer. Rats were maintained normally after surgery and sacrificed 6 weeks later to analyze the repair effect.

[0066] This example mainly investigates the repair effect of functional cartilage organoids on cartilage defects. Figure 6These are the macroscopic observation pictures of the femurs of each group of animals at the 6th week of the experiment. The Sham group is a group without modeling or materials, that is, the normal animal group. Compared with the control group (the Control group, that is, the group without any repair materials) and the ordinary cartilage organoids (the group with repaired organoid materials without MnO2 and Melatonin, that is, the CO group), the functionalized cartilage organoid group (MMCO group) has almost no visible cartilage defects, which is closest to the sham operation group. The results show that functionalized cartilage organoid precursors can promote cartilage regeneration and repair.

[0067] Example 6

[0068] Based on Example 5, the new cartilage obtained after repair using the functionalized cartilage organoid group (MMCO group) was fixed in 4% paraformaldehyde for 48 h. The cartilage specimens were decalcified, embedded, and sectioned, and stained with HE and safranin fast green for histological analysis. Figure 7 As shown, HE and Safranin Fast Green staining demonstrated that functionalized cartilage organoids promoted cartilage regeneration and exhibited highly ordered chondrocyte arrangement and intact tissue structure similar to native cartilage. Chondrocytes were evenly distributed in the extracellular matrix, and there was no obvious inflammatory cell infiltration. In contrast, the control group (the group without any repair material) and the conventional cartilage organoids (the group repaired with organoid material without MnO2 and Melatonin loading, the CO group) showed fibrotic repair characteristics (disorganized cell arrangement and loose matrix) at 3 weeks, and immature tissue areas and matrix heterogeneity were still visible at 6 weeks.

[0069] Example 7

[0070] Based on Example 6, the new cartilage tissue sections obtained after repair using the functionalized cartilage organoid group (MMCO group) were incubated with anti-ACAN or anti-Col II antibodies at 4°C overnight. After incubation with secondary antibodies at room temperature, the sections were stained with 3,3′-diaminobenzidine. All tissue sections were photographed under a microscope, as shown in Figure 6. Figure 8 As shown, further ACAN and Col II immunohistochemical staining also showed the significant effect of functionalized cartilage organoids on cartilage formation, and a high-density Col II positive signal (brown) was present at 3 weeks. As the repair time increased (to 6 weeks), its distribution uniformity and staining intensity were significantly close to those of the natural cartilage group, indicating that the collagen network of the extracellular matrix was reconstructed in an orderly manner.

[0071] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A method for preparing cartilage organoids capable of maintaining mitochondrial homeostasis, characterized in that: The following steps are involved: Step 1: Prepare a DNA-silk fibroin hybrid hydrogel solution containing MnO2 nanoparticles and Melatonin; Step 2: Mixing the DNA-silk fibroin hybrid hydrogel solution containing MnO2 nanoparticles and melatonin with bone marrow mesenchymal stem cells to obtain a DNA-silk fibroin hybrid hydrogel solution loaded with BMSCs; Step 3: The DNA-silk fibroin hybrid hydrogel solution loaded with BMSCs was 3D-printed into cartilage organoid precursors using DLP bioprinting. Step 4: Cultivate the cartilage organoid precursors in culture medium to obtain cartilage organoids with the function of maintaining mitochondrial homeostasis.

2. The method for preparing a cartilage organoid having the function of maintaining mitochondrial homeostasis according to claim 1, characterized in that: In step 1, the method for preparing the DNA-silk fibroin hybrid hydrogel solution is: Step 1.1, Construction of DNA supramolecular network: In a buffer solution, a Y-shaped DNA single strand and an L-shaped DNA single strand are mixed to obtain a DNA solution, and a DNA supramolecular network is formed by cross-linking the sticky ends of the Y-shaped DNA single strand and the L-shaped DNA single strand; Step 1.2, preparation of DNA-silk fibroin hybrid hydrogel solution: Mix the methacryloylated silk fibroin and photoinitiator with the DNA solution prepared in step 1.1 to obtain a DNA-silk fibroin hybrid hydrogel solution.

3. The method for preparing a cartilage organoid having the function of maintaining mitochondrial homeostasis according to claim 2, characterized in that: The Y-shaped DNA single strand includes DNA single strand Y1, DNA single strand Y2, and DNA single strand Y3, wherein the nucleotide sequence of DNA single strand Y1 is shown in SEQ ID NO.1, the nucleotide sequence of DNA single strand Y2 is shown in SEQ ID NO.2, and the nucleotide sequence of DNA single strand Y3 is shown in SEQ ID NO.3; The L-shaped DNA single strand includes DNA single strand L1 and DNA single strand L2, the nucleotide sequence of DNA single strand L1 is shown in SEQ ID NO.4, and the nucleotide sequence of DNA single strand L2 is shown in SEQ ID NO.5; The sticky ends of the Y-shaped DNA single strand are complementary to the sticky ends of the L-shaped DNA single strand; The molar ratio of DNA single strand Y1, DNA single strand Y2 and DNA single strand Y3 is 1:1:1; the molar ratio of DNA single strand L1 and DNA single strand L2 is 1:1; and the total molar ratio of Y-type DNA single strand and L-type DNA single strand is 1:1~1:

2.

4. The method for preparing a cartilage organoid having the function of maintaining mitochondrial homeostasis according to claim 2, characterized in that: In step 1.2, the concentration of the methacrylylated silk fibroin in the hydrogel solution is 5-15 wt %; In step 1.2, the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, and the concentration of 2,4,6-trimethylbenzoyl lithium phosphate in the hydrogel solution is 0.1-0.5 wt %.

5. The method for preparing a cartilage organoid having the function of maintaining mitochondrial homeostasis according to claim 1, characterized in that: MnO2 nanoparticles and Melatonin are added to the DNA-silk fibroin hybrid hydrogel solution to obtain a DNA-silk fibroin hybrid hydrogel solution containing MnO2 nanoparticles and Melatonin. In the DNA-silk fibroin hybrid hydrogel solution containing MnO2 nanoparticles and Melatonin, the concentration of MnO2 nanoparticles is 0.5-2wt%, and the concentration of Melatonin is 0.5-2wt%.

6. The method for preparing a cartilage organoid having the function of maintaining mitochondrial homeostasis according to claim 1, characterized in that: In step 2, the bone marrow mesenchymal stem cells are (1-10)×10 6 The concentration of 100 cells / mL was added to a DNA-silk fibroin hybrid hydrogel solution containing MnO2 nanoparticles and Melatonin to obtain a DNA-silk fibroin hybrid hydrogel solution loaded with BMSCs.

7. The method for preparing a cartilage organoid having the function of maintaining mitochondrial homeostasis according to claim 1, characterized in that: In step 3, the conditions for DLP bio-3D printing into cartilage organoid precursors are: DLP printing parameters are light intensity 17mW / cm 2 , exposure time is 15 s, the number of base layers is 5, and the exposure time of the base layer is 10 s.

8. The method for preparing a cartilage organoid having the function of maintaining mitochondrial homeostasis according to claim 1, characterized in that: In step 4, the cartilage organoid precursors are cultured in chondrogenic medium containing DMEM high glucose medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 1 mM sodium pyruvate, 10 ng / ml transforming growth factor-β3 (TGF-β3), 0.1 μM dexamethasone, 1% ITS premix, and 1 μM ascorbic acid 2-phosphate. In step 4, the culture conditions are as follows: the culture environment is 37°C, 5% CO2, and the cartilage culture medium is replaced every 2 days.

9. A cartilage organoid capable of maintaining mitochondrial homeostasis, characterized in that: Prepared based on the method according to any one of claims 1 to 8.

10. Use of the cartilage organoid having the function of maintaining mitochondrial homeostasis as claimed in claim 9 in the preparation of cartilage repair materials.

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