Composite hydrogel system with functions of promoting angiogenesis and / or repairing skin defects as well as preparation method and application of composite hydrogel system

Abstract

The invention provides a composite hydrogel system with a function of promoting angiogenesis and / or skin defect repair as well as a preparation method and application of the composite hydrogel system. The composite hydrogel system comprises methacrylated gelatin, hypoxia-induced human umbilical vein endothelial cell exosome and a photoinitiator. Compared with a conventionally cultured human umbilical vein endothelial cell exosome, the hypoxia-induced human umbilical vein endothelial cell exosome provided by the invention shows more obvious capabilities of promoting angiogenesis and accelerating skin injury repair.

Description

Technical Field

[0001] The present invention relates to the field of biomaterials, and particularly to a composite hydrogel system having the functions of promoting angiogenesis and / or skin defect repair, and a preparation method and application thereof. Background Art

[0002] The treatment of diabetic skin defects remains a major challenge in the field of regenerative medicine. Currently, commonly used clinical drug treatments and traditional dressings have many limitations, including limited improvement of the local microenvironment, insufficient ability to continuously release bioactive factors, and inability to effectively promote tissue reconstruction. Exosomes have become a research hotspot in the field of tissue regeneration due to their ability to carry a variety of bioactive molecules and their potential to regulate cell behavior and improve the tissue microenvironment. However, problems such as poor in vivo stability and uncontrollable release of exosomes limit their practical application in promoting the repair of diabetic skin defects.

[0003] As a biomaterial with good biocompatibility and adjustable mechanical properties, hydrogel has become an important direction in the research of tissue repair materials because it can simulate the extracellular matrix microenvironment and provide a scaffold for cell adhesion, migration, and growth, offering new possibilities for solving the repair of diabetic skin defects. However, traditional hydrogels face multiple challenges in promoting the repair of chronic wounds, including the lack of targeted repair factor release function, limited antioxidant and anti-inflammatory capabilities, and easy degradation in complex pathological environments. Therefore, developing a hydrogel material with excellent biological activity and good stability, which can significantly improve the repair efficiency of diabetic skin defects, has important scientific value and clinical significance. Summary of the Invention

[0004] Aiming at the defects of the prior art, the purpose of the present invention is to provide a composite hydrogel system having the functions of promoting angiogenesis and / or skin defect repair, and a preparation method and application thereof.

[0005] The technical solution of the present invention is as follows: In the first aspect of the present invention, a composite hydrogel system having the functions of promoting angiogenesis and / or skin defect repair is provided. The composite hydrogel system includes methacrylated gelatin (GelMa), hypoxia-induced human umbilical vein endothelial cell exosomes, and a photoinitiator.

[0006] Further, in the composite hydrogel system, the concentration of hypoxia-induced human umbilical vein endothelial cell exosomes is 50 - 400 ug / ml.

[0007] Further, in the composite hydrogel system, the concentration of methacrylated gelatin is 0.10 - 0.15 g / ml.

[0008] The second aspect of the present invention provides a method for preparing a composite hydrogel system having the function of promoting angiogenesis and / or repairing skin defects, comprising the following steps: S1. Preparation of hypoxia-induced human umbilical vein endothelial cell exosome solution: hypoxia-induced human umbilical vein endothelial cells, ultracentrifugation to extract hypoxia-induced human umbilical vein endothelial cell exosomes, resuspending and diluting the exosomes to obtain hypoxia-induced human umbilical vein endothelial cell exosome solution; wherein the hypoxia induction conditions are: 2 Concentration 1%, CO 2 The concentration was 5%, the culture temperature was 37°C, and the hypoxia induction time was 48 hours; S2, preparing a methacrylic gelatin solution, and adding a photoinitiator to the prepared methacrylic gelatin solution; S3, mixing the hypoxia-induced human umbilical vein endothelial cell exosome solution in step S1 and the methacrylated gelatin solution with a photoinitiator added in step S2 to obtain a mixed solution, and curing the mixed solution into a composite hydrogel by ultraviolet light curing; Furthermore, in step S1, the ultracentrifugation method is to centrifuge at 300×g for 10 minutes and take the supernatant; 3 × g centrifuge for 10 minutes, take the supernatant; 1×10 4 × g centrifuge for 30 minutes, take the supernatant; 1×10 5 × g, centrifuged at 4 °C for 90 min, the supernatant was removed, the remaining pellet was resuspended in PBS, and then resuspended in 1 × 10 5 × g for 90 minutes, the supernatant was removed, and the remaining precipitate was resuspended with PBS to obtain the exosome solution, wherein the concentration of the exosome solution was 2000-5000 ug / ml.

[0009] Furthermore, in step S2, the concentration of the methacrylated gelatin solution is 0.10-0.15 g / ml.

[0010] Furthermore, in step S2, the concentration of the photoinitiator is 2.50-5.00 mg / ml.

[0011] The third aspect of the present invention provides a composite hydrogel having the function of promoting angiogenesis and / or repairing skin defects, wherein the composite hydrogel comprises the above-mentioned composite hydrogel system having the function of promoting angiogenesis and / or repairing skin defects, and the composite hydrogel is prepared according to the above-mentioned method for preparing the composite hydrogel system having the function of promoting angiogenesis and / or repairing skin defects.

[0012] A fourth aspect of the present invention provides a composite hydrogel having the function of promoting angiogenesis and / or repairing skin defects, and its use in the preparation of angiogenesis-promoting products and / or skin damage treatment products.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 1. Compared with conventionally cultured human umbilical vein endothelial cell (HUVEC) exosomes, the hypoxia-induced HUVEC exosomes provided by the present invention show more excellent efficacy in promoting angiogenesis and repairing skin damage.

[0014] 2. The composite hydrogel with the function of promoting angiogenesis and / or repairing skin defects provided by the present invention has good biocompatibility, and its internal porous grid structure can achieve the slow release of loaded exosomes, significantly enhance the action time of exosomes, and improve the biological efficacy of exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 A schematic diagram of transmission electron microscopy results of two types of exosomes prepared in the third embodiment provided in the experimental examples of the present invention (Hp-Exo: hypoxia-induced human umbilical vein endothelial cell exosomes; N-Exo: conventional culture-induced human umbilical vein endothelial cell exosomes); Figure 2 Schematic diagram of the particle size distribution test results of two exosomes prepared in the third embodiment provided in the experimental example of the present invention (Hp-Exo: hypoxia-induced human umbilical vein endothelial cell exosomes; N-Exo: conventional culture-induced human umbilical vein endothelial cell exosomes); Figure 3 A schematic diagram of the detection results of the protein imprint (Western Blot) of two exosomes prepared in the third embodiment provided in the experimental example of the present invention (Hp-Exo: hypoxia-induced human umbilical vein endothelial cell exosomes; N-Exo: conventional culture-induced human umbilical vein endothelial cell exosomes); Figure 4 Schematic diagram of the scanning electron microscopy results of two composite hydrogels prepared in the third embodiment provided in the experimental examples of the present invention (GelMa@Hp-Exo: composite hydrogel obtained by using GelMa@Hp-Exo as the preparation system; GelMa@N-Exo: composite hydrogel obtained by using GelMa@N-Exo as the preparation system); Figure 5Fourier transform infrared spectra (FTIR) of two composite hydrogels prepared in the third embodiment provided in the experimental examples of the present invention (GelMa@Hp-Exo: composite hydrogel prepared with GelMa@Hp-Exo as the preparation system; GelMa@N-Exo: composite hydrogel prepared with GelMa@N-Exo as the preparation system; GelMa: pure GelMa hydrogel without exosomes); Figure 6 Schematic diagram of mechanical test results of two composite hydrogels prepared in the third embodiment provided in the experimental examples of the present invention (GelMa@Hp-Exo: composite hydrogel obtained by using GelMa@Hp-Exo as the preparation system; GelMa@N-Exo: composite hydrogel obtained by using GelMa@N-Exo as the preparation system; GelMa: pure GelMa hydrogel without exosomes); Figure 7 Schematic diagram of the exosome release results of two composite hydrogels prepared in the third embodiment provided in the experimental examples of the present invention (GelMa@Hp-Exo: composite hydrogel obtained by using GelMa@Hp-Exo as the preparation system; GelMa@N-Exo: composite hydrogel obtained by using GelMa@N-Exo as the preparation system); Figure 8 Schematic diagram of the results of detecting cell viability using the CCK8 method for the composite hydrogels prepared in the second to fifth embodiments provided in the experimental examples of the present invention (H: composite hydrogel prepared using GelMa@Hp-Exo as the preparation system; N: composite hydrogel prepared using GelMa@N-Exo as the preparation system); Fig. 9 Schematic diagram of the skin repair results of the two composite hydrogels prepared in the third embodiment provided in the experimental example of the present invention as wound dressings implanted into a mouse skin defect model for repair after 2, 4, 7, 10, and 14 days (GelMa@Hp-Exo: composite hydrogel prepared with GelMa@Hp-Exo as the preparation system; GelMa@N-Exo: composite hydrogel prepared with GelMa@N-Exo as the preparation system; GelMa: GelMa hydrogel without exosomes; Control: no treatment of the wound); Fig.10The two composite hydrogels prepared in the third embodiment provided in the experimental examples of the present invention were used as wound dressings and implanted into a mouse skin defect model for repair. The skin defect samples were repaired 7 and 14 days later by immunofluorescence staining of angiogenic markers CD31 and α-SMA (GelMa@Hp-Exo: composite hydrogel prepared with GelMa@Hp-Exo as the preparation system; GelMa@N-Exo: composite hydrogel prepared with GelMa@N-Exo as the preparation system; GelMa: GelMa hydrogel without exosomes; Control: no treatment was performed on the wound). DETAILED DESCRIPTION

[0016] The present invention is described in detail below with reference to specific embodiments.

[0017] The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

[0018] First embodiment This embodiment provides a composite hydrogel system having the function of promoting angiogenesis and / or repairing skin defects and a preparation method thereof.

[0019] The composite hydrogel system with the function of promoting angiogenesis and / or repairing skin defects provided in this embodiment includes methacrylated gelatin, hypoxia-induced human umbilical vein endothelial cell exosomes and a photoinitiator.

[0020] In the composite hydrogel system, the concentration of hypoxia-induced human umbilical vein endothelial cell exosomes was 50-400 ug / ml.

[0021] In the composite hydrogel system, the concentration of methacryloyl gelatin is 0.10-0.15 g / ml.

[0022] The method for preparing the composite hydrogel system having the function of promoting angiogenesis and / or repairing skin defects provided in this embodiment comprises the following steps: S1. Preparation of hypoxia-induced human umbilical vein endothelial cell exosome solution: hypoxia-induced human umbilical vein endothelial cells, ultracentrifugation to extract hypoxia-induced human umbilical vein endothelial cell exosomes; resuspend and dilute to obtain hypoxia-induced human umbilical vein endothelial cell exosome solution; hypoxia induction conditions are: O 2 Concentration 1%, CO 2 The concentration was 5%, the culture temperature was 37 °C, and the hypoxia induction time was 48 hours; S2, preparing a methacrylic gelatin solution, and adding a photoinitiator to the prepared methacrylic gelatin solution; S3. Evenly mix the hypoxia-induced human umbilical vein endothelial cell exosome solution and the methacrylylated gelatin solution to which a photoinitiator is added to obtain a mixed solution, and solidify the mixed solution into a composite hydrogel by ultraviolet light curing.

[0023] In step S1, the ultracentrifugation method is to centrifuge at 300×g for 10 minutes and take the supernatant; 3 × g centrifuge for 10 minutes, take the supernatant; 1×10 4 × g centrifuge for 30 minutes, take the supernatant; 1×10 5 × g, centrifuged continuously at 4°C for 90 min, the supernatant was removed, and the remaining precipitate was resuspended in PBS; 5 ×g centrifugation for 90 minutes, the supernatant was removed, the remaining precipitate was resuspended with 200 ul PBS, and then the protein concentration was determined by BCA method, and the protein concentration was measured to be 2000-5000 ug / ml; PBS was diluted to obtain hypoxia-induced human umbilical vein endothelial cell exosome solution.

[0024] In step S2, the concentration of the methacrylated gelatin solution is 0.10-0.15 g / ml.

[0025] In step S2, the concentration of the photoinitiator is 2.50-5.00 mg / ml.

[0026] Second embodiment This embodiment provides a composite hydrogel having the function of promoting angiogenesis and / or repairing skin defects. The composite hydrogel is prepared by the preparation method provided in the first embodiment, comprising the following steps: 1) Human umbilical vein endothelial cells (HUVEC) were cultured under hypoxia-induced conditions and conventional induction conditions, and Hp-Exo (hypoxia-induced HUVEC exosomes) and N-Exo (conventional culture-induced HUVEC exosomes) were obtained by ultracentrifugation; Hp-Exo and N-Exo were resuspended in 200 ul PBS, and then the protein concentrations measured by the BCA method were both 4000 ug / ml, and then diluted with PBS to 400 ug / ml HUVEC exosome solution cultured under hypoxia-induced conditions and 400 ug / ml HUVEC exosome solution induced by conventional culture. Hypoxia induction conditions: O 2 Concentration 1%, CO 2 The concentration was 5%, the culture was carried out at 37°C, and the induction time was 48 hours.

[0027] Conventional induction conditions: O 2 Concentration 21%, CO 2 The concentration was 5%, the culture was carried out at 37°C, and the induction time was 48 hours.

[0028] 2) Disperse 0.1 g of methacrylated gelatin (GelMa) and 2.5 mg of photoinitiator (LAP) in 0.9 ml of PBS, dissolve at 60 °C for 40 min to obtain a GelMa solution; 3) 0.1 ml of the exosome solution of human umbilical vein endothelial cells cultured under hypoxia-induced conditions in step 1) was mixed evenly with the GelMa solution in step 2), and allowed to stand until there was no foam, to obtain a hypoxic exosome GelMa composite solution (GelMa@Hp-Exo) containing the exosome solution of human umbilical vein endothelial cells cultured under hypoxia-induced conditions, a GelMa concentration of 10%, and a LAP concentration of 0.25%; 0.1 ml of the human umbilical vein endothelial cell exosome solution cultured under conventional induction conditions in step 1) was mixed evenly with the GelMa solution in step 2), and allowed to stand until there was no foam, to obtain a conventional exosome GelMa composite solution (GelMa@N-Exo) containing conventionally induced human umbilical vein endothelial cell exosome solution, a GelMa concentration of 10%, and a LAP concentration of 0.25%; 4) Extrude or apply GelMa@Hp-Exo and GelMa@N-Exo solutions to the desired parts, or directly form them in a mold, and use 405 nm blue light to cure them into gels for 60-90 s; 5) Characterize and compare the performance of the two composite hydrogels obtained in step 4).

[0029] Third embodiment This embodiment provides a composite hydrogel having the function of promoting angiogenesis and / or repairing skin defects. The composite hydrogel is prepared by the preparation method provided in the first embodiment, comprising the following steps: 1) Human umbilical vein endothelial cells (HUVEC) were cultured under hypoxia-induced conditions and conventional induction conditions, and Hp-Exo (hypoxia-induced HUVEC exosomes) and N-Exo (conventional culture-induced HUVEC exosomes) were obtained by ultracentrifugation; Hp-Exo and N-Exo were resuspended in 200 ul PBS, and then the protein concentrations measured by the BCA method were both 4000 ug / ml, and then diluted with PBS to 200 ug / ml HUVEC exosome solution cultured under hypoxia-induced conditions and 200 ug / ml HUVEC exosome solution induced by conventional culture. Hypoxia induction conditions: O 2Concentration 1%, CO 2 The concentration was 5%, the culture was carried out at 37°C, and the induction time was 48 hours.

[0030] Conventional induction conditions: O 2 Concentration 21%, CO 2 The concentration was 5%, the culture was carried out at 37°C, and the induction time was 48 hours.

[0031] 2) Disperse 0.1 g of methacryloyl gelatin (GelMa) and 2.5 mg of photoinitiator (LAP) in 0.9 ml of PBS and dissolve at 60 °C for 40 min to obtain a GelMa solution; 3) 0.1 ml of the exosome solution of human umbilical vein endothelial cells cultured under hypoxia-induced conditions in step 1) was mixed evenly with the GelMa solution in step 2), and allowed to stand until there was no foam, to obtain a hypoxic exosome GelMa composite solution (GelMa@Hp-Exo) containing the exosome solution of human umbilical vein endothelial cells cultured under hypoxia-induced conditions, a GelMa concentration of 10%, and a LAP concentration of 0.25%; 0.1 ml of the human umbilical vein endothelial cell exosome solution cultured under conventional induction conditions in step 1) was mixed evenly with the GelMa solution in step 2), and allowed to stand until there was no foam, to obtain a conventional exosome GelMa composite solution (GelMa@N-Exo) containing conventionally induced human umbilical vein endothelial cell exosome solution, a GelMa concentration of 10%, and a LAP concentration of 0.25%; 4) Extrude or apply GelMa@Hp-Exo and GelMa@N-Exo solutions to the desired parts, or directly form them in a mold, and use 405 nm blue light to cure them into gels for 60-90 s; 5) Characterize and compare the performance of the two composite hydrogels obtained in step 4).

[0032] Fourth embodiment This embodiment provides a composite hydrogel having the function of promoting angiogenesis and / or repairing skin defects. The composite hydrogel is prepared by the preparation method provided in the first embodiment, comprising the following steps: 1) Human umbilical vein endothelial cells (HUVECs) were cultured under hypoxic induction conditions and conventional induction conditions respectively. Hp-Exo (hypoxia-induced human umbilical vein endothelial cell exosomes) and N-Exo (human umbilical vein endothelial cell exosomes induced by conventional culture) were obtained by ultracentrifugation. Hp-Exo and N-Exo were resuspended with 200 μl of PBS respectively, and then the protein concentrations measured by the BCA method were both 4000 μg / ml. They were further diluted with PBS into a solution of human umbilical vein endothelial cell exosomes cultured under hypoxic induction conditions at 100 μg / ml and a solution of human umbilical vein endothelial cell exosomes induced by conventional culture at 100 μg / ml respectively. Hypoxic induction conditions: O 2 Concentration 1%, CO 2 Concentration 5%, cultured at 37 °C for 48 hours.

[0033] Conventional induction conditions: O 2 Concentration 21%, CO 2 Concentration 5%, cultured at 37 °C for 48 hours.

[0034] 2) 0.1 g of methacrylated gelatin (GelMa) and 2.5 mg of photoinitiator (LAP) were dispersed in 0.9 ml of PBS and dissolved at 60 °C for 40 min to obtain a GelMa solution. 3) 0.1 ml of the solution of human umbilical vein endothelial cell exosomes cultured under hypoxic induction conditions in step 1) was mixed evenly with the GelMa solution in step 2), and left standing until there were no bubbles, to obtain a hypoxic exosome GelMa composite solution (GelMa@Hp-Exo) containing the solution of human umbilical vein endothelial cell exosomes cultured under hypoxic induction conditions, with a GelMa concentration of 10% and an LAP concentration of 0.25%; 0.1 ml of the solution of human umbilical vein endothelial cell exosomes induced by conventional culture in step 1) was mixed evenly with the GelMa solution in step 2), and left standing until there were no bubbles, to obtain a conventional exosome GelMa composite solution (GelMa@N-Exo) containing the solution of human umbilical vein endothelial cell exosomes induced by conventional culture, with a GelMa concentration of 10% and an LAP concentration of 0.25%. 4) The GelMa@Hp-Exo and GelMa@N-Exo solutions were extruded or smeared onto the desired site respectively, or directly formed in a mold, and cured into a gel using 405 nm blue light, with a curing time of 60 - 90 s. 5) The two composite hydrogels obtained in step 4) were characterized and evaluated for performance comparison.

[0035] The fifth embodiment This embodiment provides a composite hydrogel having the function of promoting angiogenesis and / or repairing skin defects. The composite hydrogel is prepared by the preparation method provided in the first embodiment, comprising the following steps: 1) Human umbilical vein endothelial cells (HUVEC) were cultured under hypoxia-induced conditions and conventional induction conditions, and Hp-Exo (hypoxia-induced HUVEC exosomes) and N-Exo (conventional culture-induced HUVEC exosomes) were obtained by ultracentrifugation. Hp-Exo and N-Exo were resuspended in 200 ul PBS, respectively, and then the protein concentrations measured by the BCA method were both 4000 ug / ml, and then diluted with PBS to 50 ug / ml HUVEC exosome solution cultured under hypoxia-induced conditions and 50 ug / ml HUVEC exosome solution induced by conventional culture.

[0036] Hypoxia induction conditions: O 2 Concentration 1%, CO 2 The concentration was 5%, the culture was carried out at 37°C, and the induction time was 48 hours.

[0037] Conventional induction conditions: O 2 Concentration 21%, CO 2 The concentration was 5%, the culture was carried out at 37°C, and the induction time was 48 hours.

[0038] 2) Disperse 0.1 g of methacryloyl gelatin (GelMa) and 2.5 mg of photoinitiator (LAP) in 0.9 ml of PBS and dissolve at 60 °C for 40 min to obtain a GelMa solution; 3) 0.1 ml of the exosome solution of human umbilical vein endothelial cells cultured under hypoxia-induced conditions in step 1) was mixed evenly with the GelMa solution in step 2), and allowed to stand until there was no foam, to obtain a hypoxic exosome GelMa composite solution (GelMa@Hp-Exo) containing the exosome solution of human umbilical vein endothelial cells cultured under hypoxia-induced conditions, a GelMa concentration of 10%, and a LAP concentration of 0.25%; 0.1 ml of the human umbilical vein endothelial cell exosome solution cultured under conventional induction conditions in step 1) was mixed evenly with the GelMa solution in step 2), and allowed to stand until there was no foam, to obtain a conventional exosome GelMa composite solution (GelMa@N-Exo) containing conventionally induced human umbilical vein endothelial cell exosome solution, a GelMa concentration of 10%, and a LAP concentration of 0.25%; 4) Extrude or apply GelMa@Hp-Exo and GelMa@N-Exo solutions to the desired parts, or directly form them in a mold, and use 405 nm blue light to cure them into gels for 60-90 s; 5) Characterize and compare the performance of the two composite hydrogels obtained in step 4).

[0039] Application Examples This application example provides an application of a composite hydrogel having the function of promoting angiogenesis and / or repairing skin defects in the preparation of angiogenesis-promoting products and skin damage treatment products. The composite hydrogel described in this application example is provided by the third embodiment.

[0040] Experimental example The following experiments were conducted to characterize and evaluate the performance of the human umbilical vein endothelial cell exosomes prepared under two culture conditions in the third embodiment and the composite hydrogel having the function of promoting angiogenesis and / or repairing skin defects.

[0041] 1.1 Characterization of exosomes from human umbilical vein endothelial cells under two culture conditions Transmission electron microscopy images of two types of human umbilical vein endothelial cell exosomes prepared in the third embodiment, as shown in Figure 1 As shown in the figure, although the culture conditions are different, the exosomes of human umbilical vein endothelial cells extracted in the third embodiment are all in the shape of round cakes, which is consistent with the morphological characteristics of exosomes, indicating that the third embodiment successfully extracts exosomes and hypoxic treatment does not change the morphology of exosomes.

[0042] The particle size distribution diagram of the two human umbilical vein endothelial cell exosomes prepared in the third embodiment is as follows: Figure 2 As shown in the figure, the particle sizes of the two types of human umbilical vein endothelial cell exosomes extracted in this example are both within the normal range (70-150nm), which is consistent with the particle size characteristics of exosomes, and the exosomes are successfully extracted.

[0043] Western Blot of two human umbilical vein endothelial cell exosomes prepared in the third example, Figure 3 As shown in the figure, the exosome marker proteins lysosomal associated membrane protein 3 (LAMP3, CD63), tumor susceptibility gene 101 (TSG101) and calnexin were successfully expressed, proving that the exosome extraction was successful.

[0044] 1.2 Characterization of composite hydrogels with the function of promoting angiogenesis and / or repairing skin defects GelMa@Hp-Exo: composite hydrogel prepared with GelMa@Hp-Exo as the system; GelMa@N-Exo: composite hydrogel prepared with GelMa@N-Exo as the system; GelMa: Exosome-free hydrogel prepared with GelMa as the preparation system. Preparation method: Disperse 0.1 g of methacrylated gelatin (GelMa) and 2.5 mg of photoinitiator (LAP) in 0.9 ml of PBS, dissolve at 60 °C for 40 min to obtain a GelMa solution; Extrude or apply the GelMa solution to the desired site, or directly mold it in a mold, and cure it with 405 nm blue light for 60 - 90 s to form a gel.

[0045] Scanning electron micrographs of the two composite hydrogels prepared in the third embodiment, as Figure 4 shown. This figure shows that the two composite hydrogels prepared in this embodiment have a porous structure. The internal porous network structure can achieve slow release of loaded exosomes, significantly enhance the action time of exosomes, and improve the biological efficacy of exosomes.

[0046] Fourier transform infrared spectroscopy (FTIR) of the two composite hydrogels prepared in the third embodiment, as Figure 5 shown. This figure shows that the same characteristic peaks as those of the GelMa hydrogel were observed in the two composite hydrogels prepared in this embodiment, indicating that although the culture conditions changed, they did not interfere with the normal gelation process of the composite hydrogels.

[0047] Mechanical test results of the two composite hydrogels prepared in the third embodiment, as Figure 6 shown. This figure shows that the performance of the three hydrogels on the stress-strain curve is quite close, indicating that although their culture conditions are different, these conditions do not have a significant impact on the mechanical properties of the composite hydrogels.

[0048] Exosome release results of the two composite hydrogels prepared in the third embodiment, as Figure 7 shown. This figure shows that the porous structure inside the two composite hydrogels realizes slow release of loaded exosomes, and the exosome release rate gradually increases with the extension of time.

[0049] 1.3 In vitro bioactivity test of composite hydrogels with functions of promoting angiogenesis and / or skin defect repair Regulate the biological properties of the composite hydrogel by changing the exosome content in the composite hydrogel, and use the CCK8 method to detect the promoting effect of the composite hydrogel on cell viability.

[0050] Cell viability diagrams of the composite hydrogels prepared in the second to fifth embodiments, as Figure 8As shown in the figure, the two composite hydrogels with exosomes of 400 μg / ml, 200 μg / ml, 100 μg / ml, and 50 μg / ml have the ability to promote cell viability, and the composite hydrogel prepared with GelMa@Hp-Exo has a stronger biological effect than the composite hydrogel prepared with GelMa@N-Exo. In addition, when the exosome content is 200μg / ml, the difference in biological properties of the two composite hydrogels is most significant, and the introduction of Hp-Exo can enhance cell viability.

[0051] 1.4 Testing of the repair ability of two composite hydrogels in diabetic skin defects in vivo A critical skin defect model was constructed on the back of diabetic C57BL / 6 mice, and the two composite hydrogels were applied to the defect as wound dressings. The mice were photographed 2, 4, 7, 10, and 14 days after surgery, and the images were analyzed using ImageJ to observe the repair of the skin defect. Fig. 9 As shown in the figure, compared with the Control group and the GelMa group, the composite hydrogel with exosomes added as wound dressing can promote wound healing; compared with GelMa@Hp-Exo, the GelMa@N-Exo group (composite hydrogel obtained by GelMa@N-Exo as the preparation system) as wound dressing, the skin defect area of ​​mice healed slowly, while the GelMa@Hp-Exo group (composite hydrogel obtained by GelMa@Hp-Exo as the preparation system) as wound dressing, the skin defect area of ​​mice was significantly reduced. This shows that the composite hydrogel obtained by GelMa@Hp-Exo as the preparation system has a more significant effect on promoting the regeneration of diabetic skin defects.

[0052] GelMa: A hydrogel without exosomes and prepared with GelMa as the preparation system. The preparation method is as follows: 0.1 g of methacryloyl gelatin (GelMa) and 2.5 mg of photoinitiator (LAP) are dispersed in 0.9 ml of PBS, dissolved at 60 °C for 40 min to obtain a GelMa solution; the GelMa solution is extruded or applied to the desired area, or directly formed in a mold, and cured into a gel using 405 nm blue light for 60-90 s.

[0053] Control: No treatment is performed on the wound.

[0054] 1.5 Testing of the ability of two composite hydrogels to promote angiogenesis in diabetic skin defects in vitro A skin critical defect model was constructed on the back of diabetic C57BL / 6 mice, and two composite hydrogels were applied as wound dressings to the defects respectively. At 7 and 14 days after surgery, skin defect tissues of the mice were sampled and detected by immunofluorescence staining for the expression of angiogenesis markers CD31 and ɑ-SMA. ImageJ was used to analyze the pictures to observe the repair of skin defects. The results are as Fig.10 shown. Compared with the Control group and the GelMa group, the composite hydrogels supplemented with exosomes as wound dressings could promote wound angiogenesis. Compared with GelMa@Hp-Exo, as a wound dressing, the GelMa@N-Exo group (the composite hydrogel obtained from the preparation system) had a lower degree of angiogenesis in the defect site of the mice, while with the GelMa@Hp-Exo group (the composite hydrogel obtained from the GelMa@Hp-Exo preparation system) as the wound dressing, the angiogenesis in the skin defect of the mice was significantly enhanced. It shows that the composite hydrogel obtained from the GelMa@Hp-Exo preparation system has a more significant effect on promoting the regeneration of diabetic skin defect sites.

[0055] GelMa: The hydrogel obtained from the GelMa preparation system without exosomes. The preparation method is as follows: 0.1 g of methacrylated gelatin (GelMa) and 2.5 mg of photoinitiator (LAP) were dispersed in 0.9 ml of PBS, dissolved at 60 °C for 40 min to obtain a GelMa solution; the GelMa solution was extruded or applied to the required site, or directly formed in a mold, and cured with 405 nm blue light to form a gel, and the curing time was 60 - 90 s.

[0056] Control: No treatment was given to the wound.

[0057] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. Without conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. A composite hydrogel system having the function of promoting angiogenesis and / or repairing skin defects, characterized in that: The composite hydrogel system comprises methacrylated gelatin, hypoxia-induced human umbilical vein endothelial cell exosomes and a photoinitiator.

2. The composite hydrogel system having the function of promoting angiogenesis and / or repairing skin defects according to claim 1, characterized in that: In the composite hydrogel system, the concentration of hypoxia-induced human umbilical vein endothelial cell exosomes is 50-400 ug / ml.

3. The composite hydrogel system having the function of promoting angiogenesis and / or repairing skin defects according to claim 2, characterized in that: In the composite hydrogel system, the concentration of methacryloyl gelatin is 0.10-0.15 g / ml.

4. A method for preparing a composite hydrogel system having the function of promoting angiogenesis and / or repairing skin defects according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Preparing a hypoxia-induced human umbilical vein endothelial cell exosome solution: inducing human umbilical vein endothelial cells with hypoxia, extracting the hypoxia-induced human umbilical vein endothelial cell exosomes by ultracentrifugation, resuspending and diluting the exosomes to obtain a hypoxia-induced human umbilical vein endothelial cell exosome solution; wherein the hypoxia induction conditions are: O2 concentration 1%, CO2 concentration 5%, culture temperature 37°C, and hypoxia induction time 48 hours; S2, preparing a methacrylic gelatin solution, and adding a photoinitiator to the prepared methacrylic gelatin solution; S3, mixing the hypoxia-induced human umbilical vein endothelial cell exosome solution in step S1 and the methacrylylated gelatin solution to which a photoinitiator is added in step S2 to obtain a mixed solution, and curing the mixed solution into a composite hydrogel by ultraviolet light curing.

5. The method for preparing the composite hydrogel system having the function of promoting angiogenesis and / or repairing skin defects according to claim 4, characterized in that: In step S1, the ultracentrifugation method is to centrifuge at 300×g for 10 minutes and take the supernatant; 3 × g centrifuge for 10 minutes, take the supernatant; 1×10 4 × g centrifuge for 30 minutes, take the supernatant; 1×10 5 × g, centrifuged at 4 °C for 90 min, the supernatant was removed, the remaining pellet was resuspended in PBS, and then resuspended in 1 × 10 5 × g for 90 minutes, the supernatant was removed, and the remaining precipitate was resuspended with PBS to obtain the exosome solution, wherein the concentration of the exosome solution was 2000-5000 ug / ml.

6. The method for preparing the composite hydrogel system having the function of promoting angiogenesis and / or repairing skin defects according to claim 4, characterized in that: In step S2, the concentration of the methacrylated gelatin solution is 0.10-0.15 g / ml.

7. The method for preparing the composite hydrogel system having the function of promoting angiogenesis and / or repairing skin defects according to claim 4, characterized in that: In step S2, the concentration of the photoinitiator is 2.50-5.00 mg / ml.

8. A composite hydrogel having the function of promoting angiogenesis and / or repairing skin defects, characterized in that: The composite hydrogel includes the composite hydrogel system with the function of promoting angiogenesis and / or repairing skin defects as described in any one of claims 1 to 3 or is prepared by the preparation method of the composite hydrogel system with the function of promoting angiogenesis and / or repairing skin defects as described in any one of claims 4 to 7.

9. Use of the composite hydrogel having the function of promoting angiogenesis and / or repairing skin defects according to claim 8 in the preparation of angiogenesis-promoting products.

10. Use of the composite hydrogel having the function of promoting angiogenesis and / or repairing skin defects according to claim 8 in the preparation of a product for treating skin damage.

Images