A HA-Orn 3D hydrogel for obtaining exosomes from cultured cells and its preparation method and application

Abstract

The present invention provides an HA-Orn 3D hydrogel for obtaining exosomes by culturing cells, as well as a preparation method and application thereof, belonging to the field of cell transplantation technology. A cross-linking strategy is developed at room temperature and in an aqueous solvent using HA and Orn as substrates to form HA-Orn hydrogel. This strategy has the characteristics of mild conditions, low cost, and scalable industrialization. The HA-Orn hydrogel has an ideal microstructure, mechanical properties, biosafety, and biodegradability. Its low adhesion property promotes the natural formation of 3D spherical structures of cells during culture and increases the yield of 3D exosomes. Further, 3D Exo can be used for wound healing, and can exert its effects through mechanisms such as collagen remodeling, promoting cell proliferation, and promoting angiogenesis. The HA-Orn-based 3D hydrogel system has unique advantages in reducing the cost of 3D cell culture, increasing the yield of exosomes, and enhancing the biological activity of exosomes.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell transplantation, and specifically relates to a HA-Orn 3D hydrogel for obtaining exosomes by culturing cells, and a preparation method and application thereof. Background Art

[0002] Advances in regenerative medicine are transforming the treatment of tissue damage and organ failure. This field combines stem cell therapy, tissue engineering, gene editing, and exosomes to effectively repair or replace damaged cells, tissues, and organs. Stem cell therapy, leveraging the stem cells' inherent renewal and multipotential differentiation capabilities, has demonstrated excellent regenerative effects in a variety of applications. However, its application remains limited by complex procedures, high costs, potential cellular heterogeneity, and the risk of tumor formation. In contrast, cell-free therapies, particularly those based on stem cell-derived exosomes, demonstrate enhanced safety and stability. These exosomes carry a variety of active molecules, including growth factors, cytokines, mRNA, and miRNA, which synergistically target damaged tissues to promote repair and regeneration. Thus, exosomes retain the biomedical activity of stem cells while avoiding the potential risks associated with direct cell transplantation.

[0003] Given the advantages of exosomes in cell-free therapies, developing high-yield production methods that maintain the original biological properties of exosomes is crucial to ensure the effectiveness and economic feasibility of exosome therapies and to promote the research and application of exosomes. Currently, the most common method for producing exosomes is to obtain them through traditional two-dimensional (2D) culture systems and collect them by ultracentrifugation.

[0004] Exosomes derived from mesenchymal stem cells have shown great potential in cell-free therapies, particularly in wound care. They exhibit unique regulatory effects at multiple stages of wound repair, such as promoting angiogenesis, accelerating skin cell migration and proliferation, regulating extracellular matrix remodeling, and inhibiting apoptosis. However, traditional two-dimensional culture methods suffer from low yields, and the morphology and behavior of cells in 2D culture are constrained, resulting in the loss of certain exosome activities. Three-dimensional (3D) cell culture techniques have shown promise in improving exosome yield and function. By mimicking the natural three-dimensional growth environment of cells in vivo, they can more realistically recreate the physiological state of cells. This approach promotes the natural expression of cell behavior and optimizes cell-cell interactions and communication with the extracellular matrix, thereby enhancing the quality and bioactivity of exosomes. Studies have shown that exosomes cultured in 3D systems have superior functions to those in 2D systems, including promoting tissue repair and modulating immune responses. However, due to the high operating costs and lack of standardization of 3D technology, variations in culture conditions can lead to inconsistent exosome quality, limiting its widespread clinical application. Therefore, although 3D cell culture technology provides a new way for the production and research of exosomes, solving the complexity and high cost of the operation of this technology is the key to promoting its widespread application. Summary of the Invention

[0005] To address the aforementioned technical issues, the present invention provides a 3D HA-Orn hydrogel for obtaining exosomes from cultured cells, as well as a preparation method and application thereof. The HA-Orn hydrogel is formed using a cross-linking strategy in an aqueous solvent at room temperature. This strategy is characterized by mild conditions, low cost, and scalable industrialization. The HA-Orn hydrogel exhibits ideal microstructure, mechanical properties, biosafety, and biodegradability. Its low adhesion promotes the natural formation of 3D spherical structures in cell culture and increases the yield of 3D exosomes.

[0006] To achieve the above objectives, this scheme first provides a HA-Orn 3D hydrogel for obtaining exosomes from cultured cells, wherein the HA-Orn 3D hydrogel comprises hyaluronic acid (HA), L-ornithine methyl ester dihydrochloride (Orn) and DMTMM.

[0007] Preferably, the molar ratio of the hyaluronic acid, DMTMM and L-ornithine methyl ester dihydrochloride is 1:1:2.

[0008] Preferably, the HA-Orn hydrogel has a pore size of 50-300 μm and a porosity of 69.37 ± 3.94%.

[0009] Based on a general inventive concept, this solution also provides a method for preparing HA-Orn 3D hydrogel, comprising the following steps:

[0010] S1. Dissolve sodium hyaluronate in sterile PBS and stir to dissolve it completely by magnetic stirring. At the same time, dissolve L-ornithine methyl ester dihydrochloride in sterile PBS and stir to dissolve. Mix the two evenly and adjust the pH to 6.5.

[0011] S2. Add DMTMM and stir the reaction at room temperature for 3 days to prepare the primary HA-Orn hydrogel product. The primary hydrogel product is placed in a dialysis bag with a relative molecular mass of 14,000 D and dialyzed in PBS with stirring to obtain the purified HA-Orn hydrogel.

[0012] Preferably, the relative molecular mass of the sodium hyaluronate in step S1 is 400,000-800,000.

[0013] Preferably, the pH of PBS in step S2 is 7.4.

[0014] Based on a general inventive concept, this solution also provides an application of HA-Orn 3D hydrogel in culturing mesenchymal stem cells.

[0015] Based on a general inventive concept, the present invention also provides exosomes obtained by culturing mesenchymal stem cells using HA-Orn 3D hydrogel, and the exosomes can be applied externally to promote wound healing.

[0016] Preferably, the method for culturing mesenchymal stem cells to obtain exosomes is: constructing a 1 mm thick HA-Orn 3D hydrogel coating on a culture plate, inoculating a mesenchymal stem cell suspension thereon for culturing, and separating the exosomes by ultracentrifugation.

[0017] Preferably, the ultracentrifugation speed is 10,000-20,000 g, the time is 90 minutes, and the culture time is 3 days.

[0018] The mechanism of this 3D culture system based on HA-Orn hydrogel is as follows:

[0019] Hyaluronic acid, as one of the main components of the extracellular matrix, provides a framework that simulates the natural cellular environment, promotes interaction between cells and supports the three-dimensional growth of cells; ornithine, as an important cellular nutrient, helps maintain cell growth and physiological function. DMTMM acts as a coupling agent to activate the carboxyl group of HA, promoting its reaction with the amine group on L-ornithine methyl ester dihydrochloride (L-Orn) to form an amide bond to form a hydrogel. The HA / L-Orn hydrogel can provide a low-adhesion surface, promoting cells to form tight three-dimensional aggregates through intercellular adhesion molecules (such as cadherins and integrins) rather than adhering to the surface of the culture plate for growth.

[0020] HA-Orn hydrogels possess a loose and highly interconnected porous network structure with pore sizes primarily ranging from 50 to 300 μm. This structure facilitates the efficient diffusion of culture medium and cellular metabolites. When co-cultured with human umbilical cord mesenchymal stem cells (hUC-MSCs), HA / L-Orn hydrogels form 3D spheroids. The formation of these 3D spheroids is likely driven primarily by two factors: the self-assembly ability of stem cells, a biological property that enables them to aggregate in three dimensions; and the low adhesion of HA-Orn hydrogels, which reduces direct contact between cells and the matrix and promotes the formation of more stable multidimensional structures through intercellular adhesion molecules. The abundant hydroxyl and carboxyl groups in HA impart a highly hydrophilic structure, preventing protein adsorption and reducing direct cell adhesion to the matrix, hindering cell colonization.

[0021] When 3D mesenchymal stem cells are cultured using HA-Orn as a hydrogel matrix, their phenotype and function are largely dependent on complex interactions with neighboring cells, proteins, and the extracellular matrix (ECM). The 3D environment formed by HA-Orn promotes the secretion of exosomes. 3D-Exo have advantages in promoting skin cell proliferation, with a stronger ability to promote collagen deposition, tending to promote early stages of healing. This tendency may contribute to the rapid establishment of soft granulation tissue, improve the elasticity and cell migration capacity of the wound area, and thus accelerate the healing process. Activation of the VEGF / Ang2 axis may be one of the key mechanisms by which 3D-Exo promote angiogenesis.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The newly developed HA-Orn hydrogel in this scheme has ideal microstructure, mechanical properties, biosafety and biodegradability. Its low adhesion properties promote the natural formation of 3D spherical structures of cells during culture and increase the yield of 3D exosomes (3D Exo).

[0024] (2) 3D Exo showed better activity than 2D-Exo in promoting skin cell proliferation, migration, angiogenesis and extracellular matrix remodeling, suggesting that it has better medical potential in wound repair. Based on this finding, we further used 3D Exo for wound healing and confirmed that it has better wound repair ability than 2D-Exo. It can play a role through mechanisms such as collagen remodeling, cell proliferation promotion, and angiogenesis promotion. In addition, 3D-Exo obtained by HA-Orn hydrogel culture has high biosafety when applied locally on the wound surface.

[0025] (3) The HA-Orn-based 3D hydrogel system has unique advantages in reducing the cost of 3D cell culture, increasing the yield of exosomes, and enhancing the biological activity of exosomes, providing a new therapeutic strategy for regenerative medicine applications. The cross-linking strategy at room temperature and in aqueous solvents forms HA-Orn hydrogels, which has the characteristics of mild conditions, low cost, and scalable industrialization.

[0026] (4) Compared with traditional cross-linking agents Divinyl sulfone and 1,4-Butanedioldiglycidyl ether, the DMTMM used in this scheme is more suitable for aqueous reaction conditions, has higher reaction efficiency and lower toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 Synthesis of HA-Orn hydrogel in Example 1, (A) Chemical reaction synthesis process of HA-Orn, (B) Morphology of hydrogels formed at different molar ratios of HA / DMTMM / L-Orn with HA as the control, (C) Biocompatibility evaluation of hydrogels with different molar ratios of HA / DMTMM / L-Orn on hUC-MSCs, (D) Effect of hydrogels with different molar ratios of HA / DMTMM / L-Orn on the formation of hUC-MSCs 3D spheroids, scale bar = 100 μm;

[0029] Figure 2 Characterization of the properties of the HA-Orn hydrogel in Example 2: (A) Scanning electron microscopy (SEM) image of the HA-Orn hydrogel; (B) FTIR spectra of HA and HA-Orn hydrogel; (C) Storage modulus (G'), loss modulus (G''), and tan δ value of the HA-Orn hydrogel under frequency sweep; (D) Changes in the complex viscosity of the HA-Orn hydrogel with frequency; (E) Swelling behavior of the HA-Orn hydrogel in PBS; (F) Degradation behavior of the HA-Orn hydrogel in PBS buffer with and without hyaluronidase; (G) Microstructure of 3D mesenchymal stem cells cultured in the HA-Orn hydrogel, and live / dead cell staining;

[0030] Figure 3Extraction and characterization of 3D stem cell spheroid exosomes based on HA-Orn hydrogel in Example 3. (A) Extraction process of 3D stem cell spheroid exosomes based on HA-Orn hydrogel. (B) Comparison of 2D cell culture and 3D stem cell spheroid culture. (C) Western blotting analysis of the expression of typical exosome marker proteins Alix and Hsp70. TEM morphology (D) and DLS particle size (E) characterization of 3D-Exo and 2D-Exo. (F) BCA protein quantification of exosome protein concentration and comparison of exosome yield.

[0031] Figure 4 To characterize the cell biological properties of 3D-Exo in Example 4, (AD) MTT assays were used to determine the effects of 2D-Exo and 3D-Exo on HaCaT and HSF cell viability at different incubation times and concentrations. (E) Plate colonies of HaCaT and HSF cells treated with 2D-Exo and 3D-Exo. (FI) Quantification of HaCaT and HSF cell colony formation by crystal violet staining. (JK) Wound-wound assay results of HaCaT and HSF cells treated with 2D-Exo and 3D-Exo. Scale bar = 100 µm. (LM) Quantitative analysis of the wound-wound assay of HaCaT and HSF cells. (N) Angiogenesis of HUVECs after 2D-Exo and 3D-Exo treatment. Scale bar = 100 µm. (OP) Quantitative comparison of tube length after 2D-Exo and 3D-Exo treatment. (Q) HYP expression levels after 2D-Exo and 3D-Exo treatment.

[0032] Figure 5In vivo wound healing activity study of 3D-Exo in Example 5. (A) Healing process of a full-thickness skin wound model on the back of a mouse under different treatment groups (PBS, 2D-Exo, 3D-Exo), with wound images from day 0 to day 14, (B) Changes in wound area at different time points, (C) Microscopic images of wound tissues after H&E and Masson staining in different treatment groups on day 14, and (D) Comparison of wound scar width, (E) Double immunofluorescence staining images of collagen I and III in skin sections from different treatment groups, (F) Quantitative percentage of collagen deposition, and (G) Quantitative analysis of collagen I and III immunofluorescence staining, (H) Ki67 immunofluorescence showing cell proliferation, (I) Quantitative analysis of cell proliferation rate, (JK) Double immunofluorescence staining of CD31 and α-SMA to mark new blood vessels, (L) Quantitative analysis of the number of blood vessels labeled by CD31 and α-SMA, (M) VEGF immunofluorescence labeling showed the VEGF expression levels in each group, (N) Quantitative analysis of VEGF expression, (O) Ang immunofluorescence labeling showed Ang expression, and Ang expression was significantly increased in the 3D-Exo group;

[0033] Figure 6 Safety evaluation of 3D-Exo in Example 6: (A) ALT, AST, BUN, and CREA blood biochemical index levels in each treatment group; (B) HE staining of tissue sections of the heart, liver, lung, spleen, and kidney of mice in each group;

[0034] Figure 7 H in Example 2 1 -NMR spectra confirmed the cross-linking of HA and Orn. DETAILED DESCRIPTION

[0035] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0036] The following examples are provided to illustrate the present invention but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0037] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the examples are commercially available.

[0038] Example 1 Synthesis and ratio optimization of HA-Orn hydrogel.

[0039] DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholiniumchloride), a triazine derivative, was used as a coupling agent to activate the carboxyl group of HA and promote its reaction with the amino group on L-Orn to form an amide bond. The ratio of the reactants (HA / DMTMM / L-Orn) was optimized based on the gel-forming properties.

[0040] First, the ratio of HA and L-Orn was fixed at 1:1, and the effect of crosslinker concentration on gel formation was evaluated by changing the amount of DMTMM. HA / DMTMM / L-Orn with a molar ratio of 1:1:1 was first prepared. The specific steps are as follows:

[0041] S1. Dissolve 0.0108 g of sodium hyaluronate (molecular weight 400,000) in 0.45 mL of sterile PBS and stir magnetically until completely dissolved. Simultaneously, dissolve 0.0059 g of L-ornithine methyl ester dihydrochloride (Orn) in 0.45 mL of sterile PBS and stir to dissolve. Mix thoroughly and adjust the pH to 6.5 with 0.1 M NaOH or HCl.

[0042] S2. Add 0.0037 g of DMTMM and stir at room temperature for 3 days to produce the HA-Orn hydrogel. Place the hydrogel in a dialysis bag with a relative molecular mass of 14,000 D and dialyze in PBS (pH 7.4) with stirring to obtain a purified hydrogel.

[0043] Similarly, HA / DMTMM / L-Orn with a molar ratio of 1:2:1 was prepared.

[0044] The results are as follows Figure 1 As shown in the figure, the gelation state is good under the conditions of molar ratio of 1:1:1 and 1:2:1, but further addition of DMTMM (1:4:1) will make the hydrogel texture too stiff ( Figure 1 B) Based on the principle of minimizing crosslinker usage, the HA / DMTMM ratio was fixed at 1:1 to explore the effect of L-Orn dosage on hydrogel formation. When the L-Orn dosage was insufficient (1:1:0.5), the reaction was incomplete and no hydrogel formed. Overall, these results indicate that HA / DMTMM / L-Orn molar ratios of 1:1:1, 1:1:2, and 1:2:1 all formed hydrogels with ideal morphology.

[0045] The biocompatibility of the hydrogel was then investigated. The hydrogel extract was co-cultured with human umbilical cord mesenchymal stem cells (hUC-MSCs) for 48 hours, and cell viability was assessed using an MTT assay. Among the three formulations with good gel formation, HA / DMTMM / L-Orn (1:1:2) had the best biocompatibility, with a cell survival rate exceeding 80% ( Figure 1 C).

[0046] To further evaluate the function of the hydrogel as a cell culture matrix, a 1 mm thick hydrogel coating was constructed on a culture plate and hUC-MSCs suspension was seeded on it for culture. After 24 hours of culture, the cell aggregation degree of the HA / DMTMM / L-Orn (1:1:2) group was the highest ( Figure 1 D). After the third day of culture, uniformly shaped hUC-MSC 3D spheroids began to form, while the other groups exhibited irregular elliptical structures. After seven days of culture, the differences between the groups further widened. The HA / DMTMM / L-Orn (1:1:2) group consistently formed uniform, intact spheroid structures, while the other groups showed significant cell debris at the periphery, suggesting increased cell mortality in the outer layer. Therefore, the HA / DMTMM / L-Orn (1:1:2) hydrogel provides a low-adhesion surface, promoting cell growth through the formation of tight three-dimensional aggregates via intercellular adhesion molecules (such as cadherins and integrins) rather than adherence to the culture plate surface. In comparison, the hydrogels in the other groups performed poorly and exhibited significant cytotoxicity. Taking into account the gel morphology, biocompatibility, and support for 3D cell culture, the HA / DMTMM / L-Orn hydrogel at a molar ratio of 1:1:2 performed optimally and was used for subsequent cell culture exploration.

[0047] Example 2 Performance characterization of HA-Orn hydrogel.

[0048] The HA-Orn hydrogel prepared in Example 1 was observed by SEM. The results showed that the HA-Orn hydrogel had a loose and highly interconnected porous network structure ( Figure 2 A), the pore size is mainly in the range of 50-300 μm, which is conducive to the effective diffusion of culture medium and cell metabolites; the porosity of the hydrogel measured by ethanol displacement volume method is 69.37 ± 3.94%, further confirming its excellent water absorption.

[0049] The chemical structure of HA-Orn was characterized by infrared spectroscopy ( Figure 2 B), HA-Orn and HA at 3397.43 cm -1 The stretching vibrations of -OH and -NH groups are shown at 1610 cm -1The new peak at 1730 cm-1 is due to the C=O stretching vibration in the newly formed amide bond between HA and Orn, which indicates the successful formation of the amide bond. -1 The carbonyl peak was also observed at , which further verified the covalent grafting of HA and Orn. 1 -NMR also confirmed the cross-linking of HA and Orn ( Figure 7 HA showed sugar ring proton signals at 2.82-4.04 ppm, while the HA-Orn polymer showed new resonance peaks at 4.52 ppm (α-H), 1.92 ppm (β-H, 2H), 2.95 ppm (δ-H, -CH2-), and 1.70 ppm (γ-H, 2H), which were attributed to the grafted ornithine structure.

[0050] The mechanical properties of HA-Orn hydrogel were evaluated by rheological tests. Dynamic frequency sweep tests showed that the storage modulus (G') was always significantly higher than the loss modulus (G'') in the frequency range of 0.1-100 rad / s. Figure 2 C), indicating the stability of the hydrogel network structure. The low and stable tanδ value further confirms the excellent elasticity of the hydrogel. As the frequency increases, the composite viscosity (η*) shows a power law downward trend with increasing frequency ( Figure 2 D), showing typical shear thinning behavior, which is conducive to the adaptive deformation of hydrogels under dynamic physiological conditions. The swelling test of hydrogels showed that the hydrogels rapidly absorbed water in PBS within 1 hour ( Figure 2 E), swelling equilibrium was reached within 24 hours, and the equilibrium swelling ratio was 1450 ± 85%. This high water absorption property provides an ideal three-dimensional microenvironment for cell growth.

[0051] Hyaluronidase (100 U / mL) was used as a degradation agent to simulate physiological conditions to investigate the degradation performance of the gel. In the absence of hyaluronidase, the mass loss of HA-Orn hydrogel was less than 10% within 96 hours, showing good stability ( Figure 2 F). In the presence of enzymes, the hydrogel completely degraded within 84 hours, demonstrating controllable enzymatic degradation properties.

[0052] In summary, the HA-Orn hydrogel prepared in this scheme exhibits ideal microstructure, mechanical properties, moderate degradation characteristics and excellent biological properties, which is conducive to its application in 3D cell culture.

[0053] Subsequently, the effect of HA-Orn hydrogel on the support culture of 3D cell spheroids was evaluated. After 3 days of culture of hUC-MSCs, the formation of 3D spheroids was observed ( Figure 2G), and the spheroids increase in size as the culture time increases. The formation of this 3D cell spheroid may be mainly driven by two factors: on the one hand, the self-assembly ability of stem cells, which is a biological characteristic that enables them to aggregate in three-dimensional space; at the same time, the low adhesion of HA-Orn hydrogel reduces the direct contact between cells and the matrix, and promotes the formation of more stable multidimensional structures by cells through intercellular adhesion molecules. The abundant hydroxyl and carboxyl groups in HA give the structure high hydrophilicity, which prevents the adsorption of proteins, reduces the direct adhesion of cells to the matrix through proteins, and is not conducive to cell colonization. Using calcein live cell staining, it was found that most stem cells in the spheres showed strong green fluorescence, indicating that they grew well. At the same time, PI staining found that there was no obvious cell death in the three-dimensional culture system of HA-Orn hydrogel, showing low cytotoxicity.

[0054] Example 3 Extraction and characterization of 3D spheroid stem cell exosomes based on HA-Orn hydrogel.

[0055] A 1 mm thick HA-Orn 3D hydrogel coating was constructed on a culture plate, and a mesenchymal stem cell suspension was inoculated on it and cultured for 3 days. Exosomes were obtained by ultracentrifugation at 20,000 g for 90 minutes. Figure 3 A) Cell phenotype and function depend heavily on complex interactions with neighboring cells, proteins, and the extracellular matrix (ECM). Compared to traditional 2D culture systems, 3D cell culture better mimics the in vivo microenvironment, providing richer cell-cell and cell-ECM interactions. Based on this, we studied 3D spheroid stem cells cultured in HA-Orn hydrogels, using traditional 2D cultured hUC-MSCs as a control, and compared the production and function of exosomes secreted by the two.

[0056] Under 2D conditions, hUC-MSCs showed typical adherent growth and fibroblast-like morphology ( Figure 3 B); in the HA-Orn hydrogel 3D culture system, cells formed spheroids of uniform size. Next, exosomes were isolated and characterized using ultracentrifugation. The results showed that there was no significant difference in the microscopic morphology and particle size of the two exosomes (3D-Exo and 2D-Exo). Figure 3 C, D), and the surface marker proteins Alix and Hsp70 confirmed the successful extraction of exosomes ( Figure 3 E). The BCA protein quantification method was then used to compare the exosome production under the two culture conditions, and it was found that the production of 3D-Exo was significantly higher than that of 2D-Exo ( Figure 3 F) shows that the 3D environment formed by HA-Orn promotes the secretion of exosomes.

[0057] Example 4 Characterization of cell biological properties of 3D-Exo.

[0058] Taking wound healing as the application scenario and 2D-Exo as the control, the biological activity of 3D-Exo was studied at the cellular level. Human epidermal keratinocytes (HaCaT) and human skin fibroblasts (HSF) were used as models to evaluate the cell proliferation-promoting effect of exosomes.

[0059] The results are as follows Figure 4 As shown in Figure 2, both 3D-Exo and 2D-Exo significantly promoted the proliferation of HaCaT and HSF ( Figure 4 AD), and the effect was time- and concentration-dependent. Notably, 3D-Exo exhibited a superior proliferation-promoting effect compared to 2D-Exo, with the difference becoming more pronounced with increasing concentration and prolonged treatment time.

[0060] To further verify the long-term effect of exosomes on cell proliferation, crystal violet staining was used to evaluate their clone formation ability. The results showed that after 3D-Exo treatment, the purple cell colonies formed were larger and more numerous ( Figure 4 E). Quantitative analysis results showed that the clone formation rate of the 3D-Exo treated group was significantly higher than that of the 2D-Exo group and the untreated control group ( Figure 4 FI), further confirming the advantages of 3D-Exo in promoting skin cell proliferation.

[0061] The effects of the two exosomes on cell migration were then compared. The scratch test showed that the wound healing speed of the 3D-Exo treatment group was significantly faster than that of the 2D-Exo group and the control group ( Figure 4 J, K). Specifically, in HSF cells, the 3D-Exo-treated group achieved complete wound closure within 48 hours, while only a few cells in the 2D-Exo-treated group migrated at the same time point. Quantitative characterization of wound closure revealed that 3D-Exo significantly enhanced the migration-promoting effect compared to 2D-Exo (Figure 4L, M).

[0062] To evaluate the potential of 3D exosomes in tissue repair, we investigated their effects on angiogenesis and collagen synthesis. Using a human umbilical vein endothelial cell (HUVEC) in vitro tube formation model, all exosome-treated groups showed a tube formation trend after culturing on Matrigel matrix for 4 hours ( Figure 4 N); After 8 hours of culture, the 3D-Exo-treated group showed a significant advantage, and the cell tubular connections induced by it were significantly longer than those of the 2D-Exo-treated group and the control group. Quantitative analysis of the length of the tube formation further confirmed that the promoting effect of 3D-Exo on angiogenesis was significantly better than that of 2D-Exo ( Figure 4 O, P).

[0063] In terms of extracellular matrix remodeling, the effect of exosomes on collagen synthesis was indirectly assessed by measuring the hydroxyproline (HYP) content. HYP, as a characteristic amino acid of collagen, is a key indicator for measuring collagen levels in tissues. The experimental results showed that the HYP content in cells treated with both exosomes was significantly higher than that in the control group ( Figure 4 Q), indicating that both exosomes can effectively promote the synthesis of HYP. Notably, the 3D-Exo-treated group showed slightly higher HYP levels, suggesting that it has a stronger ability to promote collagen deposition.

[0064] The above results confirm that 3D-Exo exhibits better activity than 2D-Exo in promoting skin cell proliferation, migration, angiogenesis and extracellular matrix remodeling, suggesting that it has better medical potential in wound repair.

[0065] Example 5 Study on the wound healing promoting activity of 3D-Exo in vivo.

[0066] After confirming the biological activity of 3D-Exo at the cellular level, the efficacy of 3D-Exo on wound repair was further evaluated at the animal level. A circular full-thickness skin wound defect model with a diameter of 1 cm was established on the back of mice, and PBS, 2D-Exo, or 3D-Exo were topically administered every three days for treatment. During the entire experiment, all mice did not show obvious adverse reactions, and their diet and mental state remained normal. Starting from the 7th day, the healing rate of the 3D-Exo and 2D-Exo treatment groups was significantly faster than that of the PBS control group ( Figure 5 A). By day 14, the healing rate of the 3D-Exo-treated group was as high as 97% ( Figure 5 B), the healing speed was significantly higher than that of the other two groups. This result shows that 3D-Exo has a stronger ability to promote wound healing, which is consistent with the results of cellular level assessment. After 14 days of treatment, skin samples from the wound were taken for H&E and Masson staining ( Figure 5 C, D). The results showed that the scar width in the 3D-Exo group was the narrowest, significantly smaller than that in the 2D-Exo and PBS groups. Masson staining revealed that the collagen fibers in the 3D-Exo group were densest and regularly arranged, indicating excellent tissue reconstruction.

[0067] To further explore the molecular mechanism of 3D-Exo in wound healing, immunohistochemical analysis was performed. First, the effect of collagen remodeling was investigated. Compared with the 2D-Exo group and PBS group, the expression of collagen III in the 3D-Exo group was significantly increased ( Figure 5EG), while collagen I expression did not change significantly. In the early stages of wound healing, collagen III is synthesized first, which facilitates cell migration and the initial formation of granulation tissue. In the later stages of healing, collagen I gradually replaces collagen III, which helps to reduce excessive scar formation. The significant increase in collagen III in the 3D-Exo-treated group and the absence of significant changes in collagen I indicate that 3D-Exo may be more inclined to promote the early stages of healing. This tendency may contribute to the rapid establishment of soft granulation tissue, improve the elasticity and cell migration ability of the wound area, and thus accelerate the healing process. Cell proliferation was evaluated by Ki67 immunostaining, and it was found that the proportion of Ki67-positive cells in the 3D-Exo group was significantly higher than that in the 2D-Exo group ( Figure 5 H, I), indicating that 3D-Exo has a stronger ability to promote cell proliferation than 2D-Exo. In terms of promoting angiogenesis, double immunofluorescence staining and quantitative analysis of CD31 and α-smooth muscle actin (αSMA) revealed that the density of new blood vessels in the 3D-Exo group was significantly higher than that in the 2D-Exo group and the PBS group ( Figure 5 J, K), confirming the advantages of 3D-Exo in promoting angiogenesis. It is worth noting that after 3D-Exo treatment, the expression of vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2) in the wound area was significantly upregulated ( Figure 5 Therefore, the activation of the VEGF / Ang2 axis may be one of the key mechanisms by which 3D-Exos promote angiogenesis.

[0068] Example 6 Safety evaluation of 3D-Exo.

[0069] The in vivo safety of exosomes was systematically investigated. After 2D-Exo and 3D-Exo treatment, the blood biochemical indicators, including ALT, AST, BUN and CREA, showed no significant differences compared with the control group ( Figure 6 A), indicating no significant hepatotoxicity or renal toxicity. After treatment, major organs (including heart, liver, lungs, spleen, and kidneys) were collected, and tissue sections were sliced ​​and stained for pathological analysis by HE staining. The cell structure in all tissue samples remained intact, with no obvious signs of cell necrosis or inflammation. The glomeruli and renal tubules in the kidneys were clearly structured, the hepatocytes in the liver were neatly arranged, the alveolar structure in the lung tissue was intact, the lymphoid tissue in the spleen was clearly visible, and the cardiomyocytes in the heart sections were neatly arranged. These results confirm that 3D-Exo obtained by HA-Orn hydrogel culture has high biosafety when applied topically on the wound surface.

[0070] The above is only a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiment. For those skilled in the art, improvements and modifications obtained without departing from the technical concept of the present invention should also be considered as the scope of protection of the present invention.

Claims

1. A method for preparing HA-Orn 3D hydrogel, characterized in that: The following steps are involved: S1. Dissolve sodium hyaluronate in sterile PBS and stir magnetically to dissolve completely. Meanwhile, dissolve L-ornithine methyl ester dihydrochloride in sterile PBS and stir to dissolve. Mix the two ingredients evenly and adjust the pH to 6.

5. S2. DMTMM was added and stirred at room temperature for 3 days to prepare the primary HA-Orn hydrogel product. The primary hydrogel product was placed in a dialysis bag with a relative molecular mass of 14000 D and dialyzed in PBS with stirring to obtain a purified HA-Orn hydrogel; Wherein, the molar ratio of the repeating disaccharide units in the sodium hyaluronate, the DMTMM and the L-ornithine methyl ester dihydrochloride in step S1 is 1:1:2; The HA-Orn 3D hydrogel has a pore size of 50-300 μm and a porosity of 69.37 ± 3.94%.

2. The preparation method according to claim 1, characterized in that The relative molecular mass of the sodium hyaluronate in step S1 is 400,000-800,000.

3. The preparation method according to claim 1, characterized in that The pH of PBS in step S2 is 7.

4.

4. A HA-Orn 3D hydrogel obtained according to the preparation method according to any one of claims 1 to 3.

5. Use of the HA-Orn 3D hydrogel prepared by the preparation method according to any one of claims 1 to 3 in culturing mesenchymal stem cells in vitro.

6. Exosomes obtained by culturing mesenchymal stem cells in vitro using the HA-Orn 3D hydrogel prepared by the preparation method according to any one of claims 1 to 3, characterized in that: The exosomes can be applied externally to promote wound healing.

7. The exosome according to claim 6, characterized in that The method for obtaining exosomes by culturing mesenchymal stem cells in vitro is as follows: constructing a 1 mm thick HA-Orn 3D hydrogel coating on a culture plate, inoculating a mesenchymal stem cell suspension thereon for culturing, and separating the exosomes by ultracentrifugation.

8. The exosome according to claim 7, wherein The ultracentrifugation speed is 10000-20000 g, the time is 90 minutes, and the culture time is 3 days.

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