HA-Orn 3D hydrogel for culturing cells to obtain exosomes and preparation method and application of HA-Orn 3D hydrogel

Abstract

The invention provides HA-Orn 3D hydrogel for culturing cells to obtain exosomes and a preparation method and application of the HA-Orn 3D hydrogel, and belongs to the technical field of cell transplantation, HA and Orn serve as substrates to develop a cross-linking strategy at the normal temperature in an aqueous solvent to form the HA-Orn hydrogel, and the strategy has the advantages of being mild in condition, low in cost and capable of expanding industrialization. The HA-Orn hydrogel has an ideal microstructure, mechanical properties, biosafety and biodegradable characteristics, the low adhesion characteristic of the HA-Orn hydrogel promotes cells to naturally form a 3D sphere structure in the culture process, the yield of the 3D exosome is increased, 3D Exo is further used for wound healing, and the 3D exosome has a good application prospect. The HA-Orn-based 3D hydrogel can play a role through collagen remodeling, cell proliferation promotion, angiogenesis promotion and other mechanisms, and the HA-Orn-based 3D hydrogel system has unique advantages in reduction of 3D cell culture cost, increase of exosome yield and enhancement of exosome biological activity.

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] At present, the development of regenerative medicine is changing the way of treating tissue damage and organ failure. This field combines technologies such as stem cell therapy, tissue engineering, gene editing and exosomes to achieve effective repair or replacement of damaged cells, tissues and organs. Among them, stem cell therapy, with the help of stem cells' own renewal and multidirectional differentiation capabilities, has shown excellent pro-regeneration effects in multiple application scenarios. However, the application of stem cell therapy is still limited by factors such as complex operation, high cost, potential cell heterogeneity and risk of tumor formation. In contrast, cell-free therapy, especially stem cell-derived exosome therapy, shows higher safety and stability. These exosomes carry a variety of active molecules, including growth factors, cytokines, mRNA and miRNA, which can act together on damaged tissues to promote their repair and regeneration. Therefore, exosomes retain the biomedical activity of stem cells, but avoid the potential risks of direct cell transplantation.

[0003] Given the advantages of exosomes in cell-free therapy, developing a high-yield production method that can maintain the original biological properties of exosomes to ensure the effectiveness and economic feasibility of exosome therapy is of great significance for 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 the field of cell-free therapy, especially in wound treatment. They have shown unique regulatory effects in multiple stages of wound repair, such as promoting angiogenesis, accelerating skin cell migration and proliferation, regulating extracellular matrix remodeling, and inhibiting cell apoptosis. However, the traditional two-dimensional culture method has a low yield, and the morphology and behavior of cells in the 2D culture environment are constrained, resulting in the loss of some activities of exosomes. In the study of improving the yield and function of exosomes, three-dimensional (3D) cell culture technology has shown certain potential. By simulating the natural three-dimensional growth environment of cells in the body, it can more realistically reproduce the physiological state of cells. This method can promote the natural display of cell behavior, optimize cell-to-cell interactions and their communication with the extracellular matrix, and thus improve the quality and biological activity of exosomes. Studies have shown that exosomes cultured in 3D systems have functions that surpass 2D systems in promoting tissue repair and regulating immune responses. However, due to the high operating cost of 3D technology and the fact that it has not yet been standardized, differences in culture conditions may lead to inconsistent exosome quality, limiting its promotion in clinical applications. Therefore, although 3D cell culture technology provides a new way to produce and study 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] In order to solve the above technical problems, the present invention provides a HA-Orn 3D hydrogel for obtaining exosomes by culturing cells, and a preparation method and application thereof, wherein the HA-Orn hydrogel is formed by a cross-linking strategy in a water-based solvent at room temperature, and the 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 properties promote the natural formation of 3D spherical structures by cells during the culture process, and increase the yield of 3D exosomes.

[0006] To achieve the above objectives, the present invention first provides a HA-Orn 3D hydrogel for obtaining exosomes by culturing 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 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, the present invention also provides a method for preparing HA-Orn 3D hydrogel, comprising the following steps:

[0010] S1. Dissolve sodium hyaluronate in sterile PBS and completely dissolve it by magnetic stirring. Meanwhile, 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 product of HA-Orn hydrogel. Put the primary product of hydrogel into a dialysis bag with a relative molecular mass of 14000D and stir and dialyze in PBS to obtain 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, the present solution also provides an application of HA-Orn 3D hydrogel in culturing mesenchymal stem cells.

[0015] Based on a general inventive concept, the present solution 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 and obtaining exosomes by ultracentrifugation.

[0017] Preferably, the speed of the ultracentrifugation is 10000-20000 g, the time is 90 minutes, and the culture time is 3 days.

[0018] The mechanism of this scheme's 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 cell environment, promotes interaction between cells and supports the three-dimensional growth of cells; ornithine, as an important cell nutrient, helps maintain cell growth and physiological function. DMTMM acts as a coupling agent to activate the carboxyl group of HA and promotes its reaction with the amine group on L-ornithine methyl ester dihydrochloride (L-Orn) to form an amide bond to form a hydrogel. 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 growing by adhering to the surface of the culture plate.

[0020] HA-Orn hydrogel has a loose and highly interconnected porous network structure, and the pore size is mainly in the range of 50-300μm. This structure is conducive to the effective diffusion of culture medium and cell metabolites. HA / L-Orn hydrogel will form a 3D spherical structure after co-culture with human umbilical cord mesenchymal stem cells (hUC-MSCs). 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, this biological characteristic 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 cell-cell adhesion molecules. The rich 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.

[0021] 3D mesenchymal stem cells are cultured with HA-Orn as a hydrogel matrix. The phenotype and function of the cells largely depend on the complex interactions with neighboring cells, proteins and extracellular matrix (ECM). The 3D environment formed by HA-Orn promotes the secretion of exosomes, and 3D-Exo has advantages in promoting skin cell proliferation, has a stronger ability to promote collagen deposition, and tends to promote the early healing stage. This tendency may help to quickly establish soft granulation tissue, improve the elasticity and cell migration ability of the wound area, and thus accelerate the healing process; the activation of the VEGF / Ang2 axis may be one of the key mechanisms by which 3D-Exos promote angiogenesis.

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

[0023] (1) The newly developed HA-Orn hydrogel 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 collagen remodeling, cell proliferation promotion, angiogenesis promotion and other mechanisms. In addition, 3D-Exo obtained by HA-Orn hydrogel culture has high biosafety when applied topically on the wound.

[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 drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 creative work.

[0028] Figure 1 The 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 spheres, scale bar = 100 μm;

[0029] Figure 2 Characterization of the performance of HA-Orn hydrogel in Example 2, (A) Scanning electron microscope (SEM) image of HA-Orn hydrogel; (B) Infrared spectra of HA and HA-Orn hydrogel; (C) Storage modulus (G'), loss modulus (G") and tanδ value of HA-Orn hydrogel under frequency scanning; (D) Change of complex viscosity of HA-Orn hydrogel with frequency; (E) Swelling behavior of HA-Orn hydrogel in PBS; (F) Degradation behavior of HA-Orn hydrogel in PBS buffer with and without hyaluronidase; (G) Microstructure of 3D mesenchymal stem cells cultured in HA-Orn hydrogel, and live / dead cell staining;

[0030] Figure 3The extraction and characterization of 3D spheroid stem cell exosomes based on HA-Orn hydrogel in Example 3, (A) The extraction process of 3D spheroid stem cell exosomes based on HA-Orn hydrogel, (B) Comparison of 2D cell culture and 3D spheroid stem cell culture, (C) WB characterization 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 to determine the exosome protein concentration and compare the exosome yield;

[0031] Figure 4 To characterize the cell biological properties of 3D-Exo in Example 4, (AD) MTT was used to determine the effects of 2D-Exo and 3D-Exo on the viability of HaCaT and HSF cells at different incubation times and different incubation concentrations, (E) Plate clones of HaCaT and HSF cells treated with 2D-Exo and 3D-Exo, (FI) Quantitative results of crystal violet staining of HaCaT and HSF cell clone formation, (JK) Cell scratch assay results of HaCaT and HSF after treatment with 2D-Exo and 3D-Exo, scale bar = 100 μm, (LM) Quantitative analysis of HaCaT and HSF cell scratch assay, (N) Angiogenesis of HUVEC after treatment with 2D-Exo and 3D-Exo, scale bar = 100 μm, (OP) Quantitative comparison of tube formation length after treatment with 2D-Exo and 3D-Exo. (Q) HYP expression level after 2D-Exo and 3D-Exo treatment;

[0032] Figure 5 For the in vivo wound healing activity study of 3D-Exo in Example 5, (A) The healing process of the full-thickness skin wound model on the back of mice under different treatment groups (PBS, 2D-Exo, 3D-Exo), the wound images from day 0 to day 14 were observed, (B) the changes in wound area at different time points, (C) microscopic images of wound tissues after H&E and Masson staining on day 14 in different treatment groups, and (D) comparison of wound scar width, (E) double immunofluorescence staining images of collagen I and III in skin sections of different treatment groups, (F) quantitative percentage of collagen deposition, and (G) quantitative analysis results after immunofluorescence staining of collagen I and III, (H) Ki67 immunofluorescence showed cell proliferation, (I) quantitative analysis of cell proliferation rate, (JK) double immunofluorescence staining of CD31 and α-SMA was used to mark new blood vessels, (L) quantitative analysis of the number of blood vessels marked by CD31 and α-SMA, (M) VEGF immunofluorescence labeling showed the VEGF expression level 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 is 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 more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0036] The following examples are used 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 amine 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 amount of DMTMM was changed to evaluate the effect of crosslinker concentration on gelation. HA / DMTMM / L-Orn with a molar ratio of 1:1:1 was prepared first. The specific steps are as follows:

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

[0042] S2, adding 0.0037g DMTMM, stirring and reacting at room temperature for 3 days, thus preparing HA-Orn hydrogel. The hydrogel was placed in a dialysis bag with a relative molecular mass of 14000D, and dialyzed with stirring in PBS (pH 7.4) 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, and further addition of DMTMM (1:4:1) will cause the hydrogel texture to be too stiff ( Figure 1 B). Based on the principle of minimizing the use of crosslinkers, the ratio of HA / DMTMM was fixed at 1:1, and the effect of the amount of L-Orn on gelation was explored. When the amount of L-Orn input was insufficient (1:1:0.5), the reaction was incomplete and no hydrogel could be formed. Based on the above results, HA / DMTMM / L-Orn can form hydrogels with ideal morphology at molar ratios of 1:1:1, 1:1:2 and 1:2:1.

[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 the cell viability was evaluated by MTT assay. Among the three formulations with good gel forming properties, HA / DMTMM / L-Orn (1:1:2) had the best biocompatibility, with a cell survival rate of more than 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 the culture plate and the hUC-MSCs suspension was inoculated 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 1D). After culturing for 3 days, hUC-MSCs 3D spheroids with uniform shapes began to form, while other groups showed irregular elliptical structures. After 7 days of culture, the differences between the groups further expanded. The HA / DMTMM / L-Orn (1:1:2) group continued to form uniform and complete spheroid structures, while other groups showed significant cell fragments at the periphery, indicating an increase in cell outer layer mortality. Therefore, the hydrogels in the HA / DMTMM / L-Orn (1:1:2) group 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. In comparison, the hydrogels in other groups performed poorly and had obvious cytotoxicity. Considering the gel morphology, biocompatibility and support function of 3D cell culture, HA / DMTMM / L-Orn has the best performance at a molar ratio of 1:1:2, which is 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, and 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 the 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 3397.43cm -1 The stretching vibrations of -OH and -NH groups are shown at 1610 cm -1 The 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 2.82-4.04ppm, while HA-Orn polymer showed new resonance peaks at 4.52ppm (α-H), 1.92ppm (β-H, 2H), 2.95ppm (δ-H, -CH2-) and 1.70ppm (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 decrease trend with the increase of 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 is reached within 24 hours, and the equilibrium swelling ratio is 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, showing controllable enzymatic degradation characteristics.

[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 supporting the culture of 3D cell spheroids was evaluated. After 3 days of culture of hUC-MSCs, the formation of 3D spheroids was observed ( Figure 2 G), and the spheres increase in size as the culture time increases. The formation of this 3D cell spheroid may be driven mainly by two factors: on the one hand, the self-assembly ability of stem cells, which 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 through cell-to-cell adhesion molecules. The rich 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), the phenotype and function of cells largely depend on complex interactions with neighboring cells, proteins and extracellular matrix (ECM). Compared with traditional 2D culture systems, 3D cell culture can better simulate the in vivo microenvironment and provide richer cell-cell and cell-ECM interactions. Based on this, the 3D spheroid stem cells cultured under HA-Orn hydrogel conditions were studied, taking the traditional 2D cultured hUC-MSCs as a control, and compared the differences in 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, the cells formed spheres of uniform size. Next, the exosomes were separated by ultracentrifugation and characterized. 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 production of exosomes 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 43 Characterization of the cell biological properties of D-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, and 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 can significantly promote the proliferation of HaCaT and HSF ( Figure 4 AD), and has time and concentration dependence. It is worth noting that 3D-Exo has a better proliferation-promoting effect than 2D-Exo, and the difference is more obvious with the increase of concentration and the extension of time.

[0060] To further verify the long-term effect of exosomes on cell proliferation, crystal violet staining was used to evaluate their cloning 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 experiment 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). In particular, in HSF cells, the 3D-Exo treatment group achieved complete closure of the scratch within 48 hours, while only a few cells in the 2D-Exo treatment group migrated at the same time point. Quantitative characterization of scratch closure showed that the migration-promoting effect of 3D-Exo was significantly stronger than that of 2D-Exo ( Figure 4 L, M).

[0062] In order to evaluate the potential of 3D exosomes in tissue repair, their effects on angiogenesis and collagen synthesis were investigated. Using the human umbilical vein endothelial cell (HUVEC) in vitro tube formation model, all exosome-treated groups showed a tube formation trend after 4 hours of culture on Matrigel matrix ( Figure 4 N); After 8 hours of continuous culture, the 3D-Exo treatment group showed a significant advantage, and the induced cell tubular connections were significantly longer than those of the 2D-Exo treatment group and the control group. Quantitative analysis of the length of the lumen formation further confirmed that the promotion 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 evaluated by measuring the hydroxyproline (HYP) content. HYP, as a characteristic amino acid of collagen, is a key indicator for measuring the level of collagen 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. It is worth noting that the 3D-Exo-treated group showed a slightly higher level of HYP, 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 53 Study on the in vivo wound healing activity of D-Exo

[0066] After clarifying 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 speed 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 faster than 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 evaluation. 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 group and the PBS group. Masson staining revealed that the collagen fiber density in the 3D-Exo group was the highest and arranged regularly, indicating an excellent tissue reconstruction effect.

[0067] To further explore the molecular mechanism of 3D-Exo in wound healing, immunohistochemical analysis was performed. First, the effect of collagen remodeling was explored. Compared with the 2D-Exo group and the PBS group, the expression of collagen III in the 3D-Exo group was significantly increased ( Figure 5 EG), 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 lack of significant changes in collagen I indicate that 3D-Exo may be more inclined to promote the early healing stage. This tendency may help to quickly establish 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 6 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 5J, 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 LO). Therefore, the activation of VEGF / Ang2 axis may be one of the key mechanisms by which 3D-Exos promote angiogenesis.

[0068] Example 63 Safety Assessment of D-Exo

[0069] The in vivo safety of exosomes was systematically investigated. After 2D-Exo and 3D-Exo treatment, the blood biochemical indexes, including ALT, AST, BUN and CREA, were not significantly different from those in the control group ( Figure 6 A), indicating no obvious hepatotoxicity. After treatment, major organs (including heart, liver, lungs, spleen and kidneys) were collected, and pathological analysis was performed by HE staining after tissue sectioning. The cell structure in all tissue samples remained intact, and no obvious signs of cell necrosis or inflammation were observed. The glomeruli and tubules in the kidneys were clearly structured, the hepatocytes in the liver were arranged regularly, 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. The above results confirm that the 3D-Exo obtained by HA-Orn hydrogel culture has high biosafety when applied locally on the wound surface.

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

Claims

1. A HA-Orn 3D hydrogel for obtaining exosomes by culturing cells, characterized in that: The HA-Orn 3D hydrogel includes hyaluronic acid, L-ornithine methyl ester dihydrochloride and DMTMM.

2. The HA-Orn 3D hydrogel according to claim 1, characterized in that The molar ratio of the hyaluronic acid, DMTMM and L-ornithine methyl ester dihydrochloride is 1:1:

2.

3. The HA-Orn 3D hydrogel according to claim 1, characterized in that The HA-Orn 3D hydrogel has a pore size of 50-300 μm and a porosity of 69.37±3.94%.

4. A method for preparing the HA-Orn 3D hydrogel according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Dissolve sodium hyaluronate in sterile PBS and stir to dissolve it completely by magnetic stirring. Meanwhile, dissolve L-ornithine methyl ester dihydrochloride in sterile PBS and stir to dissolve. Mix the two evenly and adjust the pH to 6.

5. S2. Add DMTMM and stir the reaction at room temperature for 3 days to prepare the primary product of HA-Orn hydrogel. Put the primary product of hydrogel into a dialysis bag with a relative molecular mass of 14000D and stir and dialyze in PBS to obtain purified HA-Orn hydrogel.

5. The preparation method according to claim 4, characterized in that: The relative molecular mass of the sodium hyaluronate in step S1 is 400,000-800,000.

6. The preparation method according to claim 4, characterized in that: The pH of PBS in step S2 is 7.

4.

7. Use of the HA-Orn 3D hydrogel prepared by the preparation method according to any one of claims 1 to 3 or claims 4 to 6 in culturing mesenchymal stem cells.

8. Exosomes obtained by culturing mesenchymal stem cells using HA-Orn 3D hydrogel prepared by the preparation method according to any one of claims 1 to 3 or claims 4 to 6, characterized in that: The exosomes can be applied externally to promote wound healing.

9. The exosome according to claim 8, characterized in that The method for culturing mesenchymal stem cells to obtain exosomes 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 and obtaining exosomes by ultracentrifugation.

10. The exosome according to claim 9, characterized in that The speed of the ultracentrifugation is 10000-20000 g, the time is 90 minutes, and the culture time is 3 days.

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