An engineered mesenchymal stem cell spheroid exosome and a preparation method and application thereof

Abstract

The application relates to the technical field of biological medicine, in particular to an engineered mesenchymal stem cell sphere exosome and a preparation method and application thereof. The method comprises the following steps: O-GlcNAc glycosylation modification is performed on mesenchymal stem cells to obtain O-GlcNAc glycosylation modified mesenchymal stem cells; the mesenchymal stem cells are subjected to sphere formation culture to form mesenchymal stem cell spheres; and three-dimensional culture is performed to make the mesenchymal stem cell spheres secrete exosomes containing hsa-miR-423-3p. The exosomes can efficiently deliver hsa-miR-423-3p to target cells, activate a mitochondrial autophagy pathway, selectively remove damaged mitochondria, thereby block ROS excessive release, maintain the level of GSH, SOD and other antioxidant substances, reduce oxidative stress damage, and finally promote chondrocyte survival and cartilage matrix synthesis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an engineered mesenchymal stem cell spheroid exosome, its preparation method, and its application. Background Technology

[0002] External ear reconstruction in patients with congenital microtia presents a significant challenge in the fields of plastic surgery and tissue engineering. Current cartilage tissue engineering methods often suffer from limited seed cell sources, functional decline after in vitro expansion, and post-implantation oxidative stress leading to apoptosis and cartilage matrix degradation. Mitochondrial dysfunction is a core component of oxidative stress damage; the accumulation of damaged mitochondria releases excessive reactive oxygen species (ROS), consuming antioxidants such as glutathione (GSH) and superoxide dismutase (SOD), thereby inhibiting chondrocyte survival and differentiation. In recent years, exosomes (Exo) derived from mesenchymal stem cells have been considered promising therapeutic vectors due to their good biocompatibility and paracrine regulatory capabilities. However, natural exosomes suffer from low loading and limited production of specific functional molecules (such as microRNAs regulating mitochondrial autophagy), restricting their therapeutic efficiency and application scale. Therefore, developing an engineered exosome preparation technology capable of efficiently delivering functional molecules, actively clearing damaged mitochondria, and large-scale production is of great significance for promoting the generation of tissue-engineered cartilage in the auricle. In view of this, the present invention provides an engineered mesenchymal stem cell spheroid exosome, its preparation method and application. Summary of the Invention

[0003] The technical problem to be solved by this invention is to provide an engineered mesenchymal stem cell exosome, its preparation method, and its application. The aim is to provide an engineered mesenchymal stem cell exosome that can efficiently promote mitophagy, reduce oxidative stress damage, and produce significant yields, as well as its preparation method.

[0004] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, a method for preparing engineered mesenchymal stem cell exosomes includes the following steps: Mesenchymal stem cells were modified with O-GlcNAc glycosylation to obtain O-GlcNAc glycosylated mesenchymal stem cells; the O-GlcNAc glycosylated mesenchymal stem cells were cultured into spheres to form mesenchymal stem cell spheres; the mesenchymal stem cell spheres were cultured in three dimensions to secrete exosomes containing hsa-miR-423-3p, which were then isolated and purified to obtain engineered mesenchymal stem cell sphere exosomes.

[0005] Based on the above technical solution, the present invention can be further improved as follows.

[0006] Furthermore, the method for modifying mesenchymal stem cells with O-GlcNAc glycosylation was lentiviral transfection; The mesenchymal stem cells include any one of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, and adipose-derived mesenchymal stem cells.

[0007] Furthermore, the nucleotide sequence of hsa-miR-423-3p is as shown in SEQ ID NO: 1. The nucleotide sequence of hsa-miR-423-3p is: AGCUCGGUCUGAGGCCCCUCAGU.

[0008] Furthermore, the culture medium used for the spheroidization culture is mesenchymal stem cell culture medium, such as MSCM. The culture conditions are as follows: culture at 37°C and 100% saturated humidity in a 5% CO2 incubator. Specifically, mesenchymal stem cell suspension (200 μL / well, 10,000 cells / well) is added to the pre-coated cell spheroids in a 96-well U-shaped plate, and the plate is placed in a 37°C, 100% saturated humidity in a 5% CO2 incubator. Within 24 hours, spheroids gradually form in the U-shaped wells. The medium is gently changed using a 200 μL pipette tip to ensure the integrity of the cell spheroids. The culture medium is replaced after 48 hours. The three-dimensional culture uses a mesenchymal stem cell culture medium, such as MSCM. Specifically, the three-dimensional culture involves seeding the mesenchymal stem cell spheres in methacryloyl hyaluronic acid hydrogel, then placing them in a dynamic culture reactor and culturing them at 37°C and 100% saturated humidity in a 5% CO2 incubator. More specifically, the mesenchymal stem cell spheres are seeded in methacryloyl hyaluronic acid hydrogel (5% (w / v), gelled by 405 nm light source irradiation for 15 seconds), then placed in a dynamic culture reactor and cultured at 37°C and 100% saturated humidity in a 5% CO2 incubator to produce exosomes.

[0009] Secondly, an engineered mesenchymal stem cell exosome is prepared by the aforementioned preparation method.

[0010] Thirdly, an application of engineered mesenchymal stem cell exosomes, wherein the engineered mesenchymal stem cell exosomes are used in the preparation of products that promote the generation of engineered cartilage in auricular tissue.

[0011] Furthermore, the engineered mesenchymal stem cell exocytosis selectively clears damaged mitochondria by delivering the functional nucleic acid molecule hsa-miR-423-3p, blocks ROS release, and reduces the consumption of GSH and SOD antioxidants to alleviate oxidative stress damage, thereby promoting mitophagy for the generation of auricular tissue-engineered cartilage.

[0012] Fourthly, a product that promotes the generation of engineered cartilage in the auricle, the product comprising the engineered mesenchymal stem cell exosomes described above.

[0013] Furthermore, it also includes pharmaceutical excipients and / or carriers.

[0014] Furthermore, the product includes at least one of a reagent kit and a pharmaceutical product.

[0015] The engineered mesenchymal stem cell exosomes are used to prepare bioactive materials that promote cartilage formation. Specifically, the exosomes can be combined with biological scaffold materials (such as collagen, chitosan, PLGA, etc.) to construct an active scaffold for building auricular tissue-engineered cartilage; or they can be directly injected locally into the auricular cartilage damage area to promote mitochondrial autophagy and chondrogenic differentiation of endogenous cartilage precursor cells, thereby achieving functional auricular cartilage reconstruction.

[0016] This invention utilizes lentiviral transfection technology to upregulate O-GlcNAc transferase (OGT) in engineered mesenchymal stem cells. OGT stabilizes and upregulates TSPAN6 expression by upregulating O-GlcNAc glycosylation modification of TSPAN6, thereby increasing the binding of TSPAN6 to Syntenin-1, i.e., activating the OGT / TSPAN6 / Syntenin-1 axis, thus inducing changes in its secretory properties and increasing exosome production. After O-GlcNAc glycosylation modification, engineered mesenchymal stem cell spheres secrete engineered mesenchymal stem cell sphere exosomes, which specifically enrich the functional nucleic acid molecule hsa-miR-423-3p. These exosomes can efficiently deliver hsa-miR-423-3p to target cells, activate the mitophagy pathway, selectively clear damaged mitochondria, thereby blocking excessive ROS release, maintaining the levels of antioxidants such as GSH and SOD, reducing oxidative stress damage, and ultimately promoting chondrocyte survival and cartilage matrix synthesis.

[0017] The beneficial effects of this invention are: (1) Functional enhancement: By modifying the maternal cells of mesenchymal stem cells with O-GlcNAc glycosylation, the selective and efficient enrichment of hsa-miR-423-3p by exosomes was achieved. The content of hsa-miR-423-3p in exosomes was increased by more than 4 times, which significantly improved its delivery efficiency. It targeted and activated the mitochondrial autophagy pathway, effectively cleared damaged mitochondria, and blocked the vicious cycle of oxidative stress.

[0018] (2) Antioxidant protection: The exosomes of the present invention can significantly reduce the intracellular ROS level, protect antioxidants such as GSH and SOD from being over-consumed, create a favorable microenvironment for chondrocytes, and promote their survival and functional expression.

[0019] (3) Significantly increased yield: The O-GlcNAc glycosylation modification process can stimulate the exosome secretion ability of mesenchymal stem cell spheres, increasing the exosome yield by more than 10 times, thus solving the bottleneck problem of low natural exosome yield and difficulty in large-scale preparation.

[0020] (4) The preparation process is simple and stable: the method is engineered during the cell culture stage, without the need for complicated exosome loading or chemical modification steps. The process is simple, reproducible, and avoids potential damage to the integrity of the exosome membrane.

[0021] (5) High biocompatibility and safety: It uses autologous mesenchymal stem cells, which have low immunogenicity. The exosomes are natural vesicles, which have good biocompatibility and low toxicity.

[0022] (6) Clear application potential: In the auricular tissue-engineered cartilage model, the engineered exosomes can significantly promote the formation of cartilage-like tissue and increase the deposition of cartilage-specific matrix such as type II collagen and proteoglycans, showing excellent cartilage regeneration potential and providing a new solution for clinical external ear reconstruction. Attached Figure Description

[0023] Figure 1 The image shows the results of detecting the delivery of hsa-miR-423-3p to chondrocytes by ADSCs-Exo and its promotion of auricular tissue-engineered cartilage formation. (A) qRT-PCR results showing that ADSCs-Exo significantly delivers hsa-miR-423-3p to chondrocytes; (B) Western Blot results showing that ADSCs-Exo significantly inhibits chondrocyte dedifferentiation and promotes cartilage formation; (C) qRT-PCR results showing the expression of ACAN, COL2A1, and SOX9 cartilage formation-related genes; (D) H&E staining showing the lacunar structure of the two groups; (E) Alcian Blue staining showing that ADSCs-Exo significantly increases extrachondral matrix secretion; (F) Immunohistochemical staining. Figure 2 The diagram shows how ADSCs-Exo promotes mitophagy and alleviates oxidative stress damage in chondrocytes. (A) ADSCs-Exo significantly promotes mitophagy in chondrocytes and reduces oxidative stress damage; (B) ROS decreases; (C) GSH / GSSH increases; (D) SOD increases; (E) MDA decreases. Figure 3 The engineered mesenchymal stem cell exosomes (Exo) of this invention OE-OGTThe results of the verification of the specific enrichment of hsa-miR-423-3p and the significant delivery of hsa-miR-423-3p to chondrocytes, which enhanced their ability to promote mitophagy and chondrogenesis, are shown in the figure. Among them, (A) qRT-PCR results show that compared with the control group; (B) ROS is reduced; (C) GSH / GSSH and SOD are increased; (D) MDA is reduced; (E) Collagen II and SOX9 protein expression is increased; Collagen I protein expression is decreased; (F) glycosaminoglycan s-GAG production is increased; (G) ACAN, COL2A1 and SOX9 gene expression is increased, while COL1A1, MMP9 and MMP13 gene expression is decreased. Figure 4 The diagram shows the verification results of the mechanism of action of the O-GlcNAc glycosylation modification process of the present invention; (A) Western Blot results showing that OGT overexpression can increase the O-GlcNAc glycosylation modification of TSPAN6 and increase the expression of TSPAN6 and Syntenin-1; (B) Cycloheximide (CHX) tracking experiment results; (C) Nanoparticle tracking analysis; (D) Transmission electron microscopy showing that the particle size and morphology of different groups of exosomes meet the exosome identification criteria; (E) OGT overexpression can significantly increase the secretion of ADSCs-Exo; (F) Western blot semi-quantitative analysis to identify the surface markers CD81, TSG101, and CD63 of exosomes. Figure 5 A schematic diagram of an in vivo and in vitro culture model of auricular tissue-engineered cartilage for ADSCs-Exo treatment; Figure 6 This is a schematic diagram illustrating the working principle of the O-GlcNAc glycosylation modification process. Detailed Implementation

[0024] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0025] Example 1. Preparation of engineered mesenchymal stem cell exosomes.

[0026] A method for preparing engineered ADSCs exosomes includes the following steps: (1) Glycosylation modification of O-GlcNAc glycosyltransferase (OGT) Figure 6Subcutaneous adipose tissue was surgically harvested from the chest of microtia patients undergoing ear reconstruction surgery. The tissue was placed in centrifuge tubes containing PBS and immediately transferred to a sterile operating table in the cell culture room for isolation and culture of adipose-derived mesenchymal stem cells (ADSCs). The ADSCs were then subjected to O-GlcNAc glycosylation modification via lentiviral transfection to obtain O-GlcNAc glycosylated ADSCs. OGT-overexpressing lentivirus and corresponding negative control lentivirus were purchased from Beijing Hesheng Gene Technology Co., Ltd. (China). The specific transfection process was as follows: 24 hours before lentiviral transfection, ADSCs were transfected at a concentration of 1×10⁻⁶... 6 The virus was seeded into 6-well plates, and the original medium was replaced with 2 mL of fresh medium containing 5 μg / mL polybrene. An appropriate amount of virus suspension was added, and the plates were cultured for 24 hours. The medium containing the virus was replaced with fresh medium, and the plates were cultured for 48 to 72 hours. Western blotting was used to detect the expression level of OGT protein in the cells to assess the overall transfection effect. (2) Obtaining engineered mesenchymal stem cell spheroid exosomes: The O-GlcNAc glycosylated modified ADSCs were cultured into spheroids to form ADSC spheres; the mesenchymal stem cell spheres were then cultured in three dimensions to induce the secretion of exosomes containing hsa-miR-423-3p; these exosomes were then isolated and purified to obtain engineered mesenchymal stem cell spheroid exosomes. Specifically, the following steps were included: ① ADSCs spheroid formation and three-dimensional culture: The O-GlcNAc glycosylated ADSCs were aggregated into ADSCs spheroids using ultra-low adhesion culture technology and then seeded in hyaluronic acid hydrogel for three-dimensional culture. The spheroid formation culture used was mesenchymal stem cell culture medium MSCM (ScienCell, catalog number 7501). The culture conditions were as follows: mesenchymal stem cell suspension (200 μL / well, 10,000 cells / well) was added to the coated cell spheroids in a 96-well U-shaped plate, and the plate was incubated at 37°C, 100% saturated humidity, and 5% CO2. Spheroids gradually formed in the U-wells within 24 hours. The medium was gently changed using a 200 μL pipette tip to ensure the integrity of the cell spheroids. The medium was replaced after 48 hours. The three-dimensional culture used was also mesenchymal stem cell culture medium MSCM. The culture conditions were as follows: mesenchymal stem cell spheroids were seeded in methacryloyl hyaluronic acid hydrogel (200 μL, 5% (w / v), 405... Exosomes were gelled by irradiation with a nm light source for 15 seconds, then placed in a dynamic culture reactor and cultured and produced in an incubator containing 5% CO2 at 37°C and 100% saturated humidity.

[0027] ② Induction and secretion of engineered mesenchymal stem cell spheres exosomes: Modified ADSCs spheres were cultured under specific conditions to stimulate the secretion of a large number of exosomes rich in hsa-miR-423-3p. Specifically, 200 modified ADSCs spheres were seeded in 200 μL of methacrylamide hyaluronic acid hydrogel for three-dimensional culture. The methacrylamide hyaluronic acid hydrogel containing ADSCs spheres was placed in a dynamic culture reactor and cultured in a 37°C, 100% saturated humidity incubator containing 5% CO2 to stimulate the secretion of a large number of exosomes rich in hsa-miR-423-3p in the culture supernatant. The culture supernatant was collected every 48 hours for 14 consecutive days.

[0028] ③ Exosome isolation and purification: The culture supernatant was collected and purified using differential ultracentrifugation to obtain high-purity engineered mesenchymal stem cell spheroids exosomes (ADSCs-Exo). The specific procedure is as follows: The culture supernatant was centrifuged at 4°C at gradients of 300g (10 min), 2000g (10 min), and 10000g (30 min). After each centrifugation, the supernatant was collected, and the white precipitate at the bottom of the centrifuge tube was discarded to remove cells, dead cells, cell debris, and apoptotic bodies. Then, the supernatant was transferred to an ultracentrifugation tube and centrifuged at 4°C at 100000g for 70 min to obtain crude exosomes containing impurity proteins. The exosomes were resuspended in PBS and centrifuged again at 4°C at 100000g for 70 min to obtain higher-purity exosomes. Finally, the exosomes were resuspended in 100 to 200 μL of PBS.

[0029] (3) Identification of engineered mesenchymal stem cell exosomes. Marker proteins (such as TSG101, CD63, and CD81) were detected by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. The enrichment level of hsa-miR-423-3p was verified by qRT-PCR. The reverse transcription primers for qRT-PCR were GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTG. GATACGACACTGAG (SEQ ID NO: 2), positive primer AGCTCGGTCTGAGGCCC for qRT-PCR (SEQ ID NO: 3), and reverse primer CAGTGCGTGTCGTGGAGT (SEQ ID NO: 4).

[0030] 2. This ADSCs exosome (ADSCs-Exo) can selectively deliver the nucleic acid molecule hsa-miR-423-3p with cartilage regeneration potential, clear damaged mitochondria, reduce ROS release and antioxidant consumption, thereby promoting the formation of auricular tissue-engineered cartilage. ADSCs-Exo effect on in vivo and in vitro culture models of auricular tissue-engineered cartilage, such as Figure 5 The specific steps for in vitro and in vivo culture of auricular tissue-engineered cartilage treated with ADSCs-Exo are as follows: Second-generation residual ear chondrocytes (Pronoseudo) were resuspended in 10% methacrylamide gelatin (GelMA) solution to prepare bio-ink loaded with residual ear chondrocytes. The ADSCs-Exo treatment group had ADSCs-Exo (working concentration 0.5 μg / μL) added to the GelMA solution, while the blank control group had an equal volume of PBS added. A three-dimensional digital human ear model was designed using 3D laser scanning technology and a computer-aided design system. Polycaprolactone (PCL) was selected as the support material to improve the shape fidelity of the auricle. Alternating printing was performed using a 3D-Bioplotter bioprinter to construct auricular tissue-engineered cartilage. In vitro, auricular tissue-engineered cartilage samples were completely immersed in high-glucose DMEM medium for culture, with fresh medium replaced every 2 days. Samples were harvested and analyzed after 2 weeks. In vivo, 6-8 week old female immunodeficient BALB / c Nude nude mice were used for in vivo auricular tissue-engineered cartilage transplantation. After anesthesia and disinfection, a longitudinal incision was made in the midline of the back, a subcutaneous tissue bag was freed, and the auricular tissue-engineered cartilage was implanted subcutaneously. The incision was then sutured. Samples were collected for analysis 6 weeks later.

[0031] 2.1 Verification of the effect of ADSCs-Exo on chondrocyte delivery of hsa-miR-423-3p and promotion of auricular tissue-engineered cartilage formation: The experimental steps are as follows: ① Western Blot: Western blotting was performed using standard protocols. Samples were washed three times with PBS at 4°C, the PBS was discarded, and the samples were placed on ice. RIPA lysis buffer, PMSF, and a protein phosphatase inhibitor were added for complete lysis. Protein quantification was performed using BCA protein assay reagent. After protein denaturation, the gel was run at 200V constant voltage using 4%–12% polyacrylamide gel. After electrophoresis, the gel was cut, and an appropriate size NC membrane was cut. Transfer was then performed using a Bio-rad semi-dry transfer tank. The membrane was incubated overnight at 4°C with primary antibodies corresponding to Collagen II, SOX9, Collagen I, and GAPDH. The membrane was then washed three times with 1 x TBST solution on a shaker for 10 min each time. It was then incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibody. The membrane was again washed three times with 1 x TBST solution on a shaker for 10 min each time. After adding chromogenic buffer, the protein bands were imaged using a chemiluminescence analyzer.

[0032] ② qRT-PCR: TRIzol was used in a biosafety cabinet. TMTotal RNA was extracted from samples in each group. After treatment with TRIzol, the cells were incubated at room temperature for 10 min. Cell lysis was accelerated by pipetting, and the RNA was transferred to 1.5 mL EP tubes. 200 μL of chloroform was added, and the mixture was thoroughly mixed and incubated for 15 min. After centrifugation at 12000g for 15 min at 4°C, the solution separated into three layers. The colorless supernatant was collected, and the white intermediate layer and the pink organic lower layer were discarded. 600 μL of isopropanol was added, and the mixture was thoroughly mixed and incubated at room temperature for 15 min. After centrifugation at 12000g for 15 min at 4°C, a white precipitate was visible at the bottom of the tube. The supernatant was carefully aspirated, and 1 mL of 75% ethanol was added. The tube was centrifuged at 12000g for 10 min at 4°C. This process was repeated twice. The tubes were dried at room temperature for about 5 min until the white precipitate became translucent. Then, 30 to 50 μL of DEPC water was added to dissolve the precipitate, and the tubes were placed on ice. RNA purity and concentration were measured using a Nanodrop micro-spectrophotometer. An OD260 / 280 ratio between 1.85 and 2.1 indicated good RNA purity. This was performed according to PrimeScript. TM Reverse transcription was performed using the RT kit protocol, with the following conditions: 37°C for 15 min, followed by inactivation at 85°C for 5 sec, and temporary storage at 4°C. qRT-PCR was performed in a 20 μL reaction volume according to the SYBR Green I Master (Roche) kit protocol. GAPDH and U6 were used as internal control genes for mRNA and miRNA, respectively. Gene expression levels were measured using a 2-1... −ΔΔCT The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. The sequence information of the Forward primer (F) and Reverse primer (R) is shown in Table 1 below: Table 1 ③ Hematoxylin and Eosin (H&E) Staining: Dewax paraffin sections thoroughly by immersing them in xylene, then pass them sequentially through a gradient of ethanol (100%, 95%, 80%, 70%) for 2 minutes each, finally immersing them in distilled water. Stain with hematoxylin for 5 minutes, then rinse briefly with running water. Differentiate in differentiation medium for a few seconds, then immediately rinse with running water to remove excess hematoxylin. Rinse with running water for 15 minutes until the nuclei are a bright blue. Stain with eosin for 1 minute, then rinse briefly with running water to remove any excess stain. Quickly dehydrate by passing them sequentially through a gradient of ethanol (70%, 80%, 95%, 100% I, 100% II), clear with xylene, add neutral resin, and seal with a coverslip.

[0033] ④ Alcian Blue Staining: Paraffin sections are routinely dewaxed with xylene, then diluted with a gradient of ethanol (100%, 95%, 70%) to distilled water. The sections are briefly rinsed with acidic water (3% acetic acid solution) to provide a suitable staining environment. Sufficient 1% Alcian Blue staining solution is added, ensuring complete coverage of the tissue, and stained at room temperature for 30 minutes. The staining solution is discarded, and the sections are gently rinsed with running water to remove excess stain. Nucleotide red staining solution is added to counterstain the cell nuclei for 3 minutes, followed by rinsing with running water for 2 minutes. The sections are then rapidly dehydrated with a routine gradient of ethanol (80%, 95%, anhydrous ethanol), cleared with xylene, coated with neutral resin, and mounted with a coverslip.

[0034] ⑤ Immunohistochemical staining: Paraffin sections are routinely dewaxed with xylene, then diluted with a gradient of ethanol (100%, 95%, 70%) to distilled water. Antigen retrieval: Immerse sections in preheated EDTA or citrate retrieval solution (pH 6.0 or 9.0), heat to above 95°C in an autoclave and maintain for 15-20 minutes, then cool naturally to room temperature. Blocking endogenous enzymes: After rinsing with PBS, add 3% hydrogen peroxide solution and incubate at room temperature in the dark for 10 minutes to block endogenous peroxidase activity, then wash thoroughly with PBS. Blocking: Add normal goat serum blocking solution and incubate at room temperature for 30 minutes to reduce non-specific binding. Primary antibody incubation: Discard the blocking solution, add an appropriate amount of diluted primary antibody (Collagen II, Aggrecan, SOX9) working solution directly, place the sections in a humidified chamber, and incubate overnight at 4°C, then wash thoroughly three times with PBS. Secondary antibody incubation: Add horseradish peroxidase-labeled secondary antibody working solution matching the primary antibody species, incubate at room temperature for 60 minutes, and wash thoroughly three times with PBS. Staining: Add freshly prepared DAB chromogenic solution, control the staining reaction under a microscope, and stop staining immediately by immersing the slide in tap water when the specific brownish-yellow staining is clear and the background remains unstained. Counterstaining: Lightly stain the cell nuclei with hematoxylin for about 1 minute, differentiate with hydrochloric acid and ethanol, and then bluing with running water. Mounting: Dehydrate with a routine gradient of ethanol, clear with xylene, and mount with neutral resin.

[0035] The results are as follows Figure 1 As shown in Figures A to F. Among them, by... Figure 1 As shown in section A, qRT-PCR results indicate that ADSCs-Exo can significantly deliver hsa-miR-423-3p to chondrocytes; Figure 1 As shown in the results of Western Blot analysis, ADSCs-Exo significantly inhibited chondrocyte dedifferentiation and promoted chondrogenesis, manifested by increased expression of Collagen II and SOX9 proteins and decreased expression of Collagen I protein; Figure 1 As shown in the qRT-PCR results, the expression of chondrogenesis-related genes ACAN, COL2A1, and SOX9 was increased, while the expression of chondrogenesis-related genes COL1A1, MMP9, and MMP13 was decreased. Figure 1 As can be seen from D, H&E staining revealed the lacunar infarct structure in both groups; Figure 1 As can be seen from E, Alcian Blue staining showed that ADSCs-Exo significantly increased extrachondral matrix secretion; Figure 1 As shown in the results, immunohistochemical staining revealed that ADSCs-Exo significantly increased the expression of Aggrecan, Collagen II, and SOX9 proteins. In summary, ADSCs-Exo can deliver hsa-miR-423-3p to chondrocytes and promote the formation of auricular tissue-engineered cartilage by increasing the expression of Collagen II, SOX9, Aggrecan and the deposition of extrachondral matrix, and decreasing the expression of Collagen I, MMP9, and MMP13.

[0036] 2.2 Verification of the effects of ADSCs-Exo on promoting mitophagy and alleviating oxidative stress damage: The experimental steps are as follows: ① Western Blot: Western blotting was performed using standard protocols. After removing the cell culture medium, the cells were washed three times with PBS at 4°C. The PBS was discarded, and the cells were placed on ice. RIPA lysis buffer, PMSF, and a protein phosphatase inhibitor were added for complete lysis. Protein quantification was performed using BCA protein assay reagent. After protein denaturation, the cells were run on a 4%–12% polyacrylamide gel at a constant voltage of 200V. After electrophoresis, the gel was cut, and an appropriately sized NC membrane was prepared and transferred using a Bio-Rad semi-dry transfer tank. Primary antibodies corresponding to PINK1, p-Parkin, LC3-II, Beclin1, BNIP3, BNIP3L, and GAPDH were incubated overnight at 4°C. The membranes were then washed three times with 1 x TBST solution for 10 min each time on a shaker. Next, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 hour. The membranes were then washed three times again with 1 x TBST solution for 10 min each time on a shaker. After adding chromogenic buffer, the protein bands were imaged on a chemiluminescence analyzer.

[0037] ② ROS detection: ROS levels were detected using a fluorescence microplate reader (Ex: 490 nm, Em: 500-550 nm) according to the operating procedures of the high-sensitivity ROS detection kit (DOJINDO, R252).

[0038] ③ MDA detection: Following the operating procedures of the MDA detection kit (DOJINDO, M496), use a fluorescence microplate reader to detect the fluorescence intensity (Ex: 540 nm, Em: 590 nm). Calculate the MDA concentration in the sample based on the standard curve.

[0039] ④ SOD detection: Following the operating steps of the SOD detection kit (DOJINDO, S311), read the value at 450 nm using an ELISA reader and calculate the SOD concentration in the sample based on the standard curve.

[0040] ⑤ GSH / GSSH Detection: Following the operating procedures of the oxidized / reduced glutathione quantitative kit (DOJINDO, S311), the absorbance was measured at 405 nm using a microplate reader. The concentration of GSSG in the obtained GSSG sample solution was determined using a GSSG standard curve. The concentration of total glutathione (GSH + GSSG) in the obtained GSH sample solution was determined using a GSH standard curve. The GSH / GSSH ratio was then calculated.

[0041] The results are as follows Figure 2 From A to E. Figure 1 As shown in Figure A, ADSCs-Exo can significantly promote chondrocyte mitophagy and reduce oxidative stress damage, as evidenced by increased protein expression of PINK1, p-Parkin, LC3-II, Beclin1, BNIP3, and BNIP3L; Figure 1 From B, we can see that ROS decreases, from Figure 1 From C, we can know GSH / GSSH, by Figure 1 From D, we can see that SOD increases, from Figure 1 The results showed a decrease in MDA. In summary, ADSCs-Exo can significantly promote chondrocyte mitochondrial autophagy and reduce oxidative stress damage, as evidenced by increased protein expression of PINK1, p-Parkin, LC3-II, Beclin1, BNIP3, and BNIP3L, decreased ROS, increased GSH / GSSH and SOD, and decreased MDA.

[0042] 3. By modifying ADSCs spheres with O-GlcNAc glycosylation, the resulting engineered mesenchymal stem cell sphere exosomes (Exo OE-OGT It specifically enriches hsa-miR-423-3p, enhancing its ability to promote mitophagy and chondrogenesis.

[0043] The experimental steps are as follows: ① The experimental procedures for Western Blot, qRT-PCR, and ROS, MDA, SOD, and SOD detection are the same as described above.

[0044] ② s-GAG / DNA Detection: Tissue-engineered cartilage hydrogel samples were weighed and ground, then digested with proteinase K digestion buffer at 65°C for 5 hours. s-GAG was detected using the Biocolor Blyscan glycosaminoglycan assay kit. The sample was mixed with 1000 μL of Blyscan dye reagent on a shaker for 30 minutes, then centrifuged at 13000g for 10 minutes to collect the precipitate, which was then dissolved in a dissociation reagent. The absorbance at 656 nm was measured using a microplate reader for both samples and standards, and the s-GAG content was calculated based on the standard curve. The Quant-iT assay was performed according to the manufacturer's instructions. TM The Picogreens DNA assay kit is used to quantify sample DNA. Finally, the s-GAG is normalized based on the DNA content of each sample, and the s-GAG / DNA ratio is calculated.

[0045] The results are as follows Figure 3 From A to G. Figure 3 As shown in section A, qRT-PCR results indicate that, compared to the control group, OGT overexpression resulted in increased secretion of exosomes (Exo OE-OGT Enriching hsa-miR-423-3p significantly delivers it to chondrocytes, significantly inhibits chondrocyte dedifferentiation, reduces oxidative stress damage, and promotes the formation of engineered cartilage in auricle tissue; Figure 3 As can be seen from B, the ROS level decreases. Figure 3 From C, we can see that GSH / GSSH and SOD increase; from Figure 3 From D, we can see that MDA decreases, from Figure 3 As shown in the E diagram, the protein expression of Collagen II and SOX9 increased, while the protein expression of Collagen I decreased. Figure 3 From F, it can be seen that the formation of glycosaminoglycans s-GAG increases, from Figure 3 The study revealed increased expression of ACAN, COL2A1, and SOX9 genes, while decreased expression of COL1A1, MMP9, and MMP13 genes. In summary, engineered mesenchymal stem cell exosomes (Exo) showed increased expression. OE-OGT It specifically enriches hsa-miR-423-3p and significantly delivers hsa-miR-423-3p to chondrocytes, enhancing their ability to promote mitophagy and chondrogenesis.

[0046] 4. Verification of the working principle of O-GlcNAc glycosylation modification process.

[0047] The experimental steps are as follows: ① The steps for Western Blot detection of OGT, TSPAN6, Syntenin-1, CD81, TSG101, and CD63 are the same as described above.

[0048] ② Cycloheximide (CHX) tracking assay to assess TSPAN6 protein stability: The effect of OGT on TSPAN6 protein stability in ADSCs was evaluated by inhibiting protein synthesis with CHX. Cells were collected at 0, 6, 12, and 24 hours, and the protein was extracted and Western blot was performed to detect TSPAN6 expression levels and calculate its half-life changes.

[0049] ③ The morphology of exosomes was examined using transmission electron microscopy (TEM). The method was as follows: 10 μL of exosome sample was taken and added to a copper grid for precipitation for 1 min. The surface liquid was then absorbed with filter paper. 10 μL of phosphotungstic acid was added and added to the copper grid for precipitation for 1 min. The surface liquid was absorbed again with filter paper. The sample was dried at room temperature for several minutes. Then, electron microscopy was performed at 100 kV for imaging.

[0050] ④ Use a nanoparticle tracking and analysis instrument to measure the exosome size distribution and particle concentration. The method is as follows: Place the exosome sample on ice, dilute it with 1×PBS, and directly test the particle size distribution and particle concentration on the instrument.

[0051] The results are as follows Figure 4 From A to F. Figure 4 As shown in section A, Western blotting results indicate that OGT overexpression increases O-GlcNAc glycosylation modification of TSPAN6 and increases the expression of TSPAN6 and Syntenin-1; Figure 4 As shown in section B, the cyclohexylimide (CHX) tracking experiment results indicate that OGT can increase the stability of TSPAN6 protein; from Figure 4 As shown in C and D, nanoparticle tracking analysis and transmission electron microscopy revealed that the particle size and morphology of different groups of exosomes met the criteria for exosome identification; Figure 4 As shown in the E study, OGT overexpression significantly increases the secretion of ADSCs-Exo, with a production increase of at least 10-fold; Figure 4 As shown in Figure F, Western blot semi-quantitative analysis identified the surface markers CD81, TSG101, and CD63 of exosomes. In summary, OGT overexpression can increase the O-GlcNAc glycosylation modification of TSPAN6, increase the expression of TSPAN6 and Syntenin-1, and improve the stability of TSPAN6 protein, ultimately increasing the exosome yield by at least 10-fold.

[0052] In summary, this invention discloses a method for preparing and applying engineered mesenchymal stem cell spheroid exosomes that promote mitophagy. This invention engineered the ADSCs spheroid exosomes by performing O-GlcNAc glycosylation on the parent ADSCs spheroids, resulting in the selective enrichment of the functional nucleic acid molecule hsa-miR-423-3p in the secreted engineered mesenchymal stem cell spheroid exosomes, thereby enhancing their role in promoting mitophagy and the formation of auricular tissue-engineered cartilage. The O-GlcNAc glycosylation modification also increases the exosome yield by at least 10 times, meeting the requirements for large-scale clinical applications.

[0053] The ADSCs exosomes can selectively clear damaged mitochondria by delivering the functional nucleic acid molecule hsa-miR-423-3p, blocking ROS release, reducing the consumption of antioxidants such as GSH and SOD, alleviating oxidative stress damage, and thus promoting mitophagy for the generation of auricular tissue-engineered cartilage. The engineered mesenchymal stem cell exosomes in this invention have the advantages of simple preparation method, good biocompatibility, high nucleic acid delivery efficiency, and high exosome yield, and have good application potential in the reconstruction of the outer ear with auricular tissue-engineered cartilage.

[0054] These ADSCs exosomes can serve as active materials that promote cartilage regeneration and are used in the construction of auricular tissue-engineered cartilage to achieve efficient and safe repair and reconstruction of the outer ear.

[0055] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing engineered mesenchymal stem cell spheroids exosomes, characterized in that, Includes the following steps: Mesenchymal stem cells were modified with O-GlcNAc glycosylation to obtain O-GlcNAc glycosylated mesenchymal stem cells; the O-GlcNAc glycosylated mesenchymal stem cells were cultured into spheres to form mesenchymal stem cell spheres; the mesenchymal stem cell spheres were cultured in three dimensions to secrete exosomes containing hsa-miR-423-3p, which were then isolated and purified to obtain engineered mesenchymal stem cell sphere exosomes.

2. The method for preparing engineered mesenchymal stem cell exosomes according to claim 1, characterized in that, The method for modifying mesenchymal stem cells with O-GlcNAc glycosylation is lentiviral transfection; The mesenchymal stem cells include any one of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, and adipose-derived mesenchymal stem cells.

3. The method for preparing engineered mesenchymal stem cell exosomes according to claim 1, characterized in that, The nucleotide sequence of hsa-miR-423-3p is shown in SEQ ID NO:

1.

4. The method for preparing engineered mesenchymal stem cell exosomes according to claim 1, characterized in that, The culture medium used for the spheroidization culture is mesenchymal stem cell culture medium; the spheroidization culture specifically refers to the culture in an incubator containing 5% CO2 at 37°C and 100% saturated humidity. The culture medium used in the three-dimensional culture is mesenchymal stem cell culture medium; the three-dimensional culture specifically involves: seeding the mesenchymal stem cell spheres in methacryloyl hyaluronic acid hydrogel, then placing them in a dynamic culture reactor, and culturing them in an incubator containing 5% CO2 at 37°C and 100% saturated humidity.

5. An engineered mesenchymal stem cell exosome, characterized in that, The engineered mesenchymal stem cell exosomes are prepared by the preparation method according to any one of claims 1 to 4.

6. An application of engineered mesenchymal stem cell exosomes, characterized in that, The engineered mesenchymal stem cell exosomes according to any one of claims 1 to 4 are used in the preparation of products that promote the generation of auricular tissue-engineered cartilage.

7. The application of the engineered mesenchymal stem cell spheroid exosome according to claim 6, characterized in that, The engineered mesenchymal stem cell exocytosis delivers the functional nucleic acid molecule hsa-miR-423-3p to selectively clear damaged mitochondria, block ROS release, and reduce the consumption of GSH and SOD antioxidants to alleviate oxidative stress damage, thereby promoting mitophagy for the generation of auricular tissue-engineered cartilage.

8. A product that promotes the formation of engineered cartilage in the auricle, characterized in that, The product includes engineered mesenchymal stem cell exosomes as described in any one of claims 1 to 4.

9. The product for promoting the formation of engineered cartilage in the auricle according to claim 8, characterized in that, It also includes pharmaceutical excipients and / or carriers.

10. The product for promoting the formation of auricular tissue-engineered cartilage according to claim 8, characterized in that, The product includes at least one of a reagent kit and a pharmaceutical product.

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