A decellularized matrix composite hydrogel dressing and methods of making and using same

Abstract

This invention belongs to the field of biomedical materials and tissue engineering technology, and provides a decellularized matrix composite hydrogel dressing, its preparation method, and its application. The decellularized matrix composite hydrogel dressing provided by this invention includes a hydrogel matrix and ginger exosome-like nanoparticles (GELNs) dispersed in the hydrogel matrix; the hydrogel matrix is ​​prepared from raw materials including decellularized dermal matrix and decellularized aortic adventitia matrix. The composite hydrogel dressing of this invention has a three-dimensional porous structure, enabling sustained release of GELNs. It also significantly accelerates the healing process of full-thickness skin defects by downregulating the TLR4 / MyD88 / NF-κB signaling pathway, synergistically regulating macrophage polarization towards the M2 type, and simultaneously promoting angiogenesis and collagen deposition. The dressing has widely available raw materials, good biocompatibility, and broad application prospects in the field of skin wound repair.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and tissue engineering technology, and in particular to a decellularized matrix composite hydrogel dressing, its preparation method, and its application. Background Technology

[0002] Skin injury repair is a significant clinical challenge. Ideal wound dressings should possess good biocompatibility and biodegradability, and be able to actively regulate the healing microenvironment, particularly by modulating macrophage polarization from the pro-inflammatory M1 type to the anti-inflammatory and pro-repair M2 type, and promoting angiogenesis.

[0003] Ginger-Derived Exosome-Like Nanoparticles (GELNs) have been shown to have excellent anti-inflammatory activity, but they are easily and rapidly eliminated in vivo and have a short half-life, which limits their application. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a decellularized matrix composite hydrogel dressing, its preparation method, and its application. The decellularized matrix composite hydrogel dressing provided by this invention can effectively release GELNs, regulate macrophage M2 polarization, and promote angiogenesis.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a decellularized matrix composite hydrogel dressing, comprising a hydrogel matrix and GELNs dispersed in the hydrogel matrix; The hydrogel matrix is ​​made from raw materials including decellularized dermal matrix and decellularized aortic adventitia matrix.

[0006] Preferably, the mass ratio of the decellularized dermal matrix to the decellularized aortic adventitia matrix is ​​1:3 to 3:1.

[0007] Preferably, the concentration of GELNs in the decellularized matrix composite hydrogel dressing is 0.5~1.5 mg / mL.

[0008] This invention also provides a method for preparing the decellularized matrix composite hydrogel dressing described in the above technical solution, comprising the following steps: Acellular dermal matrix, acellular aortic adventitia matrix, and pepsin hydrochloric acid solution were mixed, and the resulting mixture was digested and dissolved to obtain a digested and dissolved system. The digestion and dissolution system was mixed with ginger exosome-like nanoparticles and incubated to obtain a digestion and dissolution system containing ginger exosome-like nanoparticles. The digestion and dissolution system containing ginger exosome-like nanoparticles was adjusted to neutral, and a buffer solution was added to the resulting neutral system to form a gel, thereby obtaining the decellularized matrix composite hydrogel dressing.

[0009] Preferably, the concentration of pepsin in the pepsin hydrochloric acid solution is 2.5~5 mg / mL, and the concentration of hydrochloric acid is 0.01~0.05 mol / L; The total concentration of decellularized dermal matrix and decellularized aortic adventitia matrix in the mixed system is 15-18 mg / mL.

[0010] Preferably, the digestion and dissolution temperature is 4~8℃ and the time is 24~72h.

[0011] Preferably, the incubation temperature is 37°C and the incubation time is 10~30 min.

[0012] Preferably, the buffer solution is 20×PBS; the volume of the buffer solution is 1 / 19 of the volume of the neutral system.

[0013] Preferably, the gelation temperature is 35~40℃.

[0014] The present invention also provides the application of the decellularized matrix composite hydrogel dressing described in the above technical solution or the decellularized matrix composite hydrogel dressing prepared by the preparation method described in the above technical solution in the preparation of skin damage repair materials.

[0015] This invention provides a decellularized matrix composite hydrogel dressing.

[0016] The beneficial effects of this invention are as follows: 1. This invention is the first to combine active nanoparticles (GELNs) derived from ginger with decellularized matrix (dECM), a natural scaffold material derived from animals. The hydrogel matrix (ADMv) made from raw materials including decellularized dermal matrix and decellularized aortic adventitia matrix not only serves as a sustained-release carrier for GELNs, but also promotes angiogenesis itself. It has a synergistic effect with the anti-inflammatory and immunomodulatory effects of GELNs, and together accelerates wound healing.

[0017] 2. Sustained-release properties: The three-dimensional porous structure of the hydrogel matrix can effectively load and sustain the release of GELNs, maintaining an effective concentration at the wound site for up to 14 days, overcoming the short half-life of GELNs.

[0018] 3. Safe and effective: The raw materials used (pigskin, pig aorta, ginger) are widely available and inexpensive. The prepared composite hydrogel has good biocompatibility, no cytotoxicity, and does not damage vital organs in rats.

[0019] 4. Clear Mechanism: The mechanisms promoting wound healing include: regulating the M2 polarization of macrophages in the wound area, reducing the expression of pro-inflammatory factors (such as TNF-α, IL-1β, iNOS), and increasing the expression of anti-inflammatory factors (such as IL-10, Arg-1, VEGF). It also promotes the proliferation, migration, and tubule formation of human umbilical vein endothelial cells (HUVECs), accelerating angiogenesis. Furthermore, it increases the deposition of collagen fibers (especially type III collagen) at the wound site, promoting tissue repair and regeneration. In vitro and in vivo experiments show that the composite hydrogel dressing of this invention can significantly accelerate the healing rate and improve the healing quality of a rat full-thickness skin defect model. This invention, through in vitro and in vivo experiments, clarifies that this composite hydrogel dressing regulates macrophage polarization by downregulating the TLR4 / MyD88 / NF-κB signaling pathway, thereby reducing inflammation, promoting angiogenesis, and promoting tissue repair. Attached Figure Description

[0020] Figure 1 The changes and statistical results of wound size in each group at 0, 3, 7, 10 and 14 days postoperatively; Figure 2 Representative HE staining images of rat wounds treated with PBS (Control), GELNs, ADMv, and ADMv-GELNs on days 7 and 14; Figure 3 Representative Masson staining images of rat wounds treated with PBS (Control), GELNs, ADMv, and ADMv-GELNs on days 7 and 14; Figure 4 Images of Sirius red staining in each group under a polarized light microscope on day 14; Figure 5 Immunohistochemical staining of CD31 and α-SMA was used to assess angiogenesis in the wound area of ​​rats 7 and 14 days post-surgery. Figure 6 To detect macrophage polarization in the wound area using immunohistochemistry and immunofluorescence staining; Figure 7 The expression levels of Nos2, Cd86, Mrc1, and Vegfa genes in the skin of SD rats were detected by qRT-PCR. Figure 8 HE staining images of vital organs from experimental SD rats; Figure 9 Immunofluorescence staining and statistical graphs of iNOS and CD206; Figure 10 Statistical graph of gene expression of Nos2, IL-1β, Arg-1 and Vegfa in macrophages for qRT-PCR assay; Figure 11 Statistical graph showing the expression of TNF-α, IL-1β and IL-10 in macrophage culture supernatant as detected by ELISA; Figure 12 The effects of macrophage supernatants treated with different groups on HUVECs proliferation, migration and angiogenesis; Figure 13 A statistical graph showing the expression levels of key proteins in the TLR4 / MyD88 / NF-κB signaling pathway as detected by Western blotting. Detailed Implementation

[0021] This invention provides a decellularized matrix composite hydrogel dressing, comprising a hydrogel matrix and GELNs dispersed in the hydrogel matrix; The hydrogel matrix is ​​made from raw materials including decellularized dermal matrix and decellularized aortic adventitia matrix.

[0022] The decellularized matrix composite hydrogel dressing provided by this invention comprises a hydrogel matrix, which is prepared from raw materials including decellularized dermal matrix and decellularized aortic adventitia matrix. In this invention, the decellularized dermal matrix is ​​preferably porcine decellularized dermal matrix. In this invention, the decellularized aortic adventitia matrix is ​​preferably porcine decellularized aortic adventitia matrix. In this invention, the mass ratio of the decellularized dermal matrix to the decellularized aortic adventitia matrix is ​​preferably 1:3 to 3:1, specifically preferably 1:1. In this invention, the preparation method of the hydrogel matrix is ​​preferably described in the preparation method section and will not be repeated here.

[0023] The decellularized matrix composite hydrogel dressing provided by the present invention comprises GELNs dispersed in the hydrogel matrix. In the present invention, the concentration of GELNs in the decellularized matrix composite hydrogel dressing is preferably 0.5~1.5 mg / mL, more preferably 1 mg / mL.

[0024] Before introducing the preparation method of the decellularized matrix composite hydrogel dressing in this invention, the preparation methods of decellularized dermal matrix, decellularized aortic adventitia matrix and GELNs will be described first.

[0025] In this invention, the method for preparing the decellularized dermal matrix preferably includes the following steps: Fresh pigskin is pretreated to obtain pretreated pigskin; The pigskin was soaked in a trypsin solution for a first decellularization treatment to obtain the first decellularized pigskin. The first decellularized pigskin was soaked in Triton X-100 solution for a second decellularization treatment to obtain the second decellularized pigskin. The second decellularized pigskin was subjected to freeze-drying, low-temperature pulverization, and irradiation sterilization in sequence to obtain the decellularized dermal matrix.

[0026] This invention pre-treats fresh pigskin to obtain pre-treated pigskin. The pre-treatment preferably includes: first washing the fresh pigskin, removing and cutting it, followed by a second washing to obtain the pre-treated pigskin. In this invention, the reagent used for the first washing preferably includes tap water; the first washing method is preferably rinsing; this invention does not specifically limit the number of times the first washing is performed or the amount of tap water used, as long as it is clean. In this invention, the equipment used to remove the pigskin is preferably a skinning knife; the cut size is preferably 4cm × 6cm. In this invention, the reagent used for the second washing is preferably deionized water; this invention does not specifically limit the number of times the second washing is performed or the amount of deionized water used, until no blood remains.

[0027] After obtaining the pretreated pigskin, the present invention soaks the pretreated pigskin in a trypsin solution for a first decellularization treatment to obtain the first decellularized pigskin. In the present invention, the mass concentration of the trypsin solution is preferably 0.25~0.5%; the temperature of the first decellularization treatment is preferably 35~40℃, more preferably 37℃, and the time is preferably 4~8h; the first decellularization treatment is preferably carried out under shaking conditions, and the shaking is preferably provided by a constant temperature shaker; after the first decellularization treatment, the present invention preferably further includes sequential washing with tap water and washing with distilled water; the number of times the tap water washing and distilled water washing are independently preferably 5 times, and the time of each washing is independently preferably 10min.

[0028] After obtaining the first decellularized pigskin, the present invention soaks the first decellularized pigskin in Triton X-100 solution for a second decellularization treatment to obtain the second decellularized pigskin. In the present invention, the mass concentration of the Triton X-100 solution is preferably 0.5-1.5%, more preferably 1%; the temperature of the second decellularization treatment is preferably 35-40℃, and the time is preferably 12-36 hours; the second decellularization treatment is preferably carried out under shaking conditions, and the shaking is preferably provided by a constant temperature shaker. In the present invention, the second decellularization treatment preferably includes the following steps: soaking the first decellularized pigskin in Triton X-100, changing the solution every 4 hours, and after the liquid becomes clear, changing the solution every 8 hours, for a total of 24 hours. After the second decellularization treatment, the present invention preferably further includes: sequentially washing with tap water and washing with distilled water, wherein the number of times the tap water washing and distilled water washing are independently preferably 5 times, and the time for each washing is independently preferably 10 minutes.

[0029] After obtaining the second decellularized pigskin, the present invention sequentially performs freeze-drying, low-temperature pulverization, and irradiation sterilization on the second decellularized pigskin to obtain the decellularized dermal matrix. In the present invention, the freeze-drying temperature is preferably -40°C, the freeze-drying time is preferably 24 hours, and the freeze-drying is preferably carried out in a freeze dryer. In the present invention, the low-temperature pulverization temperature is preferably below 15°C, and the low-temperature pulverization is preferably carried out in a cell wall blender. In the present invention, the irradiation sterilization intensity is preferably 5 K Gly. In the present invention, the decellularized dermal matrix is ​​preferably stored in a sealed environment at -20°C for later use.

[0030] In this invention, the method for preparing the decellularized aortic adventitia matrix preferably includes the following steps: Fresh pig aortas were pretreated to obtain the aortic adventitia; After the aortic adventitia was broken up, it was subjected to freeze-thaw treatment and degreasing treatment in sequence to obtain a degreased aortic adventitia; The defatted aortic adventitia was immersed in CHAPS-NaCl-EDTA solution and incubated to obtain the incubated defatted aortic adventitia; The incubated defatted aortic adventitia was immersed in trypsin-EDTA solution for a first decellularization treatment to obtain a first decellularized aortic adventitia. The first decellularized aortic adventitia was immersed in Triton X-100 solution for a second decellularization treatment to obtain the second decellularized aortic adventitia. The second decellularized aortic adventitia was sequentially freeze-dried, cryogenically pulverized, and irradiated to obtain the decellularized aortic adventitia matrix.

[0031] This invention pre-treats fresh porcine aorta to obtain the aortic adventitia. In this invention, the pre-treatment preferably includes the following steps: thoroughly washing the fresh porcine aorta, carefully peeling off the adventitia with tweezers, removing the adhered media and adipose tissue, to obtain the aortic adventitia.

[0032] After obtaining the aortic adventitia, the present invention breaks down the aortic adventitia and then performs freeze-thaw treatment and degreasing treatment sequentially to obtain a defatted aortic adventitia. In the present invention, the breaking method is preferably shearing. In the present invention, the parameters of the freeze-thaw treatment include: a freezing temperature preferably of -40°C and a thawing temperature preferably of 37°C. In the present invention, the freeze-thaw treatment is preferably performed 5 times. In the present invention, the reagent used for the degreasing treatment is preferably isopropanol. In the present invention, the degreasing treatment preferably includes the following steps: immersing the material obtained from the freeze-thaw treatment in isopropanol, replacing the isopropanol according to the turbidity of the solution until the isopropanol becomes clear to obtain the defatted aortic adventitia.

[0033] After obtaining the defatted aortic adventitia, the present invention immerses the defatted aortic adventitia in a CHAPS-NaCl-EDTA solution for incubation (referred to as the first incubation) to obtain an incubated defatted aortic adventitia. In the present invention, the concentration of CHAPS in the CHAPS-NaCl-EDTA solution is preferably 8 mM, the concentration of NaCl is preferably 1 M, and the concentration of EDTA is preferably 25 mM. In the present invention, the temperature of the first incubation is preferably 37°C, and the time is preferably 24 h. After the first incubation, the present invention preferably further includes thorough washing with PBS.

[0034] After obtaining the de-fatted aortic adventitia, the present invention immerses the de-fatted aortic adventitia in a trypsin-EDTA solution for a first decellularization treatment to obtain a first decellularized aortic adventitia. In the present invention, the mass concentration of trypsin in the trypsin-EDTA solution is preferably 0.05-0.15%, more preferably 0.1%; the mass concentration of EDTA is preferably 0.01-0.07%, specifically preferably 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, or 0.07%. In the present invention, the temperature of the first decellularization treatment is preferably 35-40°C, the time is preferably 0.5-1.5 h, and the first decellularization treatment is preferably performed under shaking conditions, preferably provided by a constant temperature shaker. After the first decellularization treatment, the present invention preferably further includes: sequential washing with PBS and washing with distilled water.

[0035] After obtaining the first decellularized aortic adventitia, the present invention immerses the first decellularized aortic adventitia in Triton X-100 solution for a second decellularization treatment to obtain the second decellularized aortic adventitia. In the present invention, the mass concentration of the Triton X-100 solution is preferably 0.5-1.5%, more preferably 1%. In the present invention, the temperature of the second decellularization treatment is preferably 35-40°C, the time is preferably 12-36 hours, and the second decellularization treatment is preferably carried out under shaking conditions, preferably provided by a constant temperature shaker. In the present invention, the second decellularization treatment preferably includes the following steps: immersing the tissue in Triton X-100 solution and shaking at 37°C for 24 hours, changing the solution every 12 hours. After the second decellularization treatment, the present invention preferably further includes washing with PBS.

[0036] After obtaining the second decellularized aortic adventitia, the present invention sequentially performs freeze-drying, cryogenic pulverization, and irradiation sterilization on the second decellularized aortic adventitia to obtain the decellularized aortic adventitia matrix. In the present invention, the freeze-drying temperature is preferably -40°C, the time is preferably 24 hours, and the freeze-drying is preferably carried out in a freeze dryer. In the present invention, the cryogenic pulverization temperature is preferably below 15°C, and the cryogenic pulverization is preferably carried out in a cell wall blender. In the present invention, the irradiation sterilization intensity is preferably 5 K Gly. In the present invention, the decellularized aortic adventitia matrix is ​​preferably stored in a sealed condition at -20°C for later use.

[0037] In this invention, the GELNs are preferably dispersed in a PBS solution. In this invention, the preparation method of the GELNs preferably includes the following steps: Fresh ginger is pre-treated to obtain pre-treated ginger; The pretreated ginger was successively ground and separated into solid and liquid components to obtain ginger juice; The ginger juice was centrifuged for the first time to obtain the first supernatant; The first supernatant was centrifuged a second time to obtain the second supernatant; The second supernatant was centrifuged a third time, and the precipitate was collected. The precipitate was resuspended for the first time to obtain a crude nanoparticle suspension; The crude extracted nanoparticle suspension was subjected to sucrose density gradient centrifugation to collect the target bands; The target band was diluted and centrifuged for the fourth time, the supernatant was discarded, and the resulting precipitate was resuspended for the second time to obtain the GELNs.

[0038] This invention pre-processes fresh ginger to obtain pre-treated ginger. In this invention, the ginger is preferably from Weifang, Shandong Province. The pre-processing preferably includes: peeling, washing, and slicing in sequence.

[0039] After obtaining pretreated ginger, the present invention sequentially grinds and separates the solid and liquid components to obtain ginger juice. In this invention, the grinding time is preferably 10 minutes; the grinding is preferably carried out in a juicer, and the grinding preferably includes the following steps: grinding in the forward direction for 1 minute, then grinding in the reverse direction for 1 minute, and repeating this cycle. In this invention, the solid-liquid separation method is preferably gauze filtration.

[0040] After obtaining ginger juice, the present invention performs a first centrifugation on the ginger juice to obtain a first supernatant. In the present invention, the preferred speed of the first centrifugation is 3,000 g, the preferred temperature is 4°C, and the preferred time is 20 min.

[0041] After obtaining the first supernatant, the present invention performs a second centrifugation on the first supernatant to obtain a second supernatant. In the present invention, the rotation speed of the second centrifugation is preferably 10,000 g, the temperature is preferably 4°C, and the time is preferably 2 h.

[0042] After obtaining the second supernatant, the present invention performs a third centrifugation on the second supernatant to collect the precipitate. In the present invention, the preferred speed of the third centrifugation is 120,000 g, the preferred temperature is 4°C, and the preferred time is 2 hours.

[0043] After collecting the precipitate, the precipitate is resuspended for the first time to obtain a suspension of crudely extracted nanoparticles. In this invention, the reagent used for the first resuspension is preferably PBS solution.

[0044] After obtaining the crude extracted nanoparticle suspension, the present invention performs sucrose density gradient centrifugation on the crude extracted nanoparticle suspension to collect the target band. In the present invention, the parameters of the sucrose density gradient centrifugation include: the mass concentration of the sucrose solution used from the bottom to the top is 45%, 30%, 15%, and 8% respectively; the temperature is preferably 4°C, the centrifugation speed is preferably 120,000g, and the time is preferably 90min. In the present invention, the sucrose density gradient centrifugation preferably includes the following steps: using a long needle, 45%, 30%, 15%, and 8% sucrose solutions are added sequentially to the bottom of the ultracentrifuge tube, and finally the crude extracted nanoparticle suspension is gently added; centrifuged at 4°C, 120,000g for 90min; the ultracentrifuge tube is carefully removed, at which point clearly layered yellow bands can be seen in the ultracentrifuge tube, and the band between the 15% and 30% layers is harvested.

[0045] After collecting the target band, the present invention sequentially dilutes and centrifuges the target band a fourth time, discards the supernatant, and resuspends the resulting precipitate a second time to obtain the GELNs. In the present invention, the reagent used for dilution is preferably PBS solution. In the present invention, the temperature of the fourth centrifugation is preferably 4°C, the rotation speed is preferably 100,000g, and the time is preferably 1 hour. In the present invention, the reagent used for the second resuspension is preferably PBS solution.

[0046] In this invention, the GELNs are preferably stored at -80°C. In this invention, the GELNs are preferably filtered before use, and the filter membrane used for filtration preferably has a pore size of 0.45 μm, and the filter membrane is preferably a sterile microporous filter membrane.

[0047] This invention also provides a method for preparing the decellularized matrix composite hydrogel dressing described in the above technical solution, comprising the following steps: Acellular dermal matrix, acellular aortic adventitia matrix, and pepsin hydrochloric acid solution were mixed, and the resulting mixture was digested and dissolved to obtain a digested and dissolved system. The digestion and dissolution system was mixed with ginger exosome-like nanoparticles and incubated to obtain a digestion and dissolution system containing ginger exosome-like nanoparticles. The digestion and dissolution system containing ginger exosome-like nanoparticles was adjusted to neutral, and a buffer solution was added to the resulting neutral system to form a gel, thereby obtaining the decellularized matrix composite hydrogel dressing.

[0048] This invention involves mixing decellularized dermal matrix, decellularized aortic adventitia matrix, and a pepsin hydrochloric acid solution, and then digesting and dissolving the resulting mixture to obtain a digested and dissolved system. In this invention, the concentration of pepsin in the pepsin hydrochloric acid solution is preferably 2.5~5 mg / mL, specifically preferably 5 mg / mL, and the concentration of hydrochloric acid is preferably 0.01~0.05 mol / L, specifically preferably 0.01 mol / L. In this invention, the total concentration of decellularized dermal matrix and decellularized aortic adventitia matrix in the mixture is preferably 15~18 mg / mL, specifically preferably 15 mg / mL, 16 mg / mL, 17 mg / mL, or 18 mg / mL; the mass ratio of the decellularized dermal matrix to the decellularized aortic adventitia matrix is ​​preferably 1:3~3:1, specifically preferably 1:1.

[0049] In this invention, the digestion and dissolution temperature is preferably 4~8℃, specifically 4℃, and the time is preferably 24~72h, specifically 48h. In this invention, the digestion and dissolution are preferably carried out under stirring conditions, and the stirring is preferably provided by a magnetic stirrer.

[0050] After obtaining the digestion and dissolution system, the present invention mixes the digestion and dissolution system with GELNs and incubates it (referred to as the second incubation) to obtain a digestion and dissolution system containing GELNs. In the present invention, the mixing of the digestion and dissolution system and GELNs is preferably carried out under stirring conditions, the temperature is preferably 4°C, and the time is preferably 20 min. In the present invention, the temperature of the second incubation is preferably 37°C, and the time is preferably 10~30 min.

[0051] After obtaining the digestion and dissolution system containing ginger exosome-like nanoparticles, this invention adjusts the digestion and dissolution system to neutral. A buffer solution is then added to the neutral system to form a gel, resulting in the decellularized matrix composite hydrogel dressing. In this invention, the preferred pH value of the neutral solution is 7.2-7.4. The reagent used to adjust the digestion and dissolution system to neutral is preferably sodium hydroxide solution, with a preferred concentration of 0.1 mol / L. The preferred buffer solution is 20×PBS, and its volume is preferably 1 / 19 of the neutral system volume. The preferred gelation temperature is 35℃-40℃, specifically 37℃. The gelation is preferably performed in a constant temperature incubator. This invention does not specify a particular gelation time, as long as the hydrogel can solidify.

[0052] The present invention also provides the application of the decellularized matrix composite hydrogel dressing described in the above technical solution or the decellularized matrix composite hydrogel dressing prepared by the preparation method described in the above technical solution in the preparation of skin damage repair materials.

[0053] The present invention does not impose specific limitations on the application method of the decellularized matrix composite hydrogel dressing; any operation known to those skilled in the art can be used.

[0054] The following detailed description, in conjunction with embodiments, illustrates the decellularized matrix composite hydrogel dressing, its preparation method, and its application, but these should not be construed as limiting the scope of protection of this invention.

[0055] Example 1 The preparation of acellular dermal matrix (ADM) powder is as follows: Fresh pigskin is repeatedly rinsed with tap water, and a 0.6 mm thick piece of pigskin is removed with a skinning knife and cut into 4 cm × 6 cm pieces. The cut pig skin was thoroughly washed with deionized water until no blood was left, and then decellularized. The specific steps are as follows: (1) Soak the pig skin in a 0.25% trypsin solution and shake it in a shaker at 37°C for 6 hours; (2) Wash it with tap water and distilled water 5 times each for 10 minutes each time; (3) Soak the pig skin washed in step (2) in a 1% Triton X-100 solution and shake it at 37°C. Change the solution every 4 hours. After the solution becomes clear, change the solution every 8 hours for a total of 24 hours. At this time, the epidermis can be seen to have peeled off; (4) Wash it with tap water and distilled water 5 times each for 10 minutes each time to obtain decellularized pig skin; Dry the decellularized pig skin in a freeze dryer for 24 hours; (5) Put the freeze-dried decellularized pig skin into a wall-breaking machine to pulverize it; (6) Seal the pulverized tissue powder and sterilize it by 5K Gly irradiation. Store it at -20°C for later use.

[0056] The preparation of decellularized aortic adventitia matrix (Adv) powder is as follows: (1) The fresh porcine aorta is thoroughly washed, the adventitia is carefully peeled off with tweezers, and excess adipose tissue is removed; (2) The peeled aortic adventitia is cut into small pieces as much as possible, and the freeze-thaw cycle is performed between -40℃ and 37℃ 5 times; (3) The cut tissue is immersed in isopropanol for defatting. The solution is changed when the isopropanol becomes turbid until the liquid is clear; (4) The tissue is immersed in a solution containing 8 mM CHAPS (zwitterionic non-denaturing detergent), 1M NaCl, and 25 mM EDTA, and incubated at 37℃ for 24h; (5) After being thoroughly washed with PBS, the tissue is placed in a solution containing 0.1% trypsin and 0.04% EDTA and shaken on a shaker at 37℃ for 1h; (6) The tissue is thoroughly washed with distilled water and then immersed in 1% Triton solution. In X-100 solution, shake at 37°C for 24 hours, and change the solution every 12 hours; (7) Wash thoroughly with PBS, dry in a freeze dryer for 24 hours, and then grind into powder using a wall-breaking machine; (8) Seal the pulverized tissue and sterilize by 5K Gly irradiation, and store at -20°C for later use.

[0057] The specific process for preparing GELNs is as follows: (1) Fresh ginger (origin: Weifang, Shandong) is peeled, washed, and sliced; (2) The processed ginger is put into a juicer and ground for 10 min, stirring in the opposite direction for 1 min after each 1 min of stirring; (3) The ginger juice is filtered through gauze and then subjected to gradient centrifugation at 4℃, centrifuged at 3,000g for 20 min, the supernatant is collected, centrifuged at 10,000g for 2 h, the supernatant is collected, centrifuged at 120,000g for 2 h, and the precipitate is collected; (4) The precipitate is resuspended in PBS to obtain a crude nanoparticle suspension; (5) Sucrose density gradient centrifugation: 45%, 30%, and 15% sucrose are added sequentially to the bottom of the ultracentrifuge tube using a long needle. Add 8% sucrose solution and finally gently add the crude ginger-derived nanoparticle suspension; (6) Centrifuge at 120,000g for 90 min at 4℃; (7) Carefully remove the centrifuge tube. At this time, you can see the yellow band with obvious layering in the centrifuge tube. Harvest the band between the 15% and 30% layers; (8) Add an appropriate amount of PBS to dilute, centrifuge at 100,000g for 1 h at 4℃, discard the supernatant, and resuspend the precipitate in PBS to obtain the purified GELNs suspension; (9) Use the BCA protein detection kit to determine the protein concentration in the GELNs sample, aliquot and store in a -80℃ refrigerator for later use. Filter through a 0.45μm sterile microporous membrane before use.

[0058] Preparation of ADM, Adv, and ADMv hydrogels: ADM, Adv, and ADMv (ADM and Adv were mixed at a mass ratio of 1:1) were dissolved in 5 mg / mL pepsin solution (prepared with 0.01 M hydrochloric acid) at a concentration of 15 mg / mL, and stirred at 4 °C on a magnetic stirrer for 48 h. The pH of the hydrogels was adjusted to 7.2–7.4 using 0.1 M NaOH solution to obtain a neutral system; then, 1 / 19 volume of 20×PBS was added to the neutral system, and the system was placed in a 37 °C incubator until the hydrogels solidified.

[0059] Preparation and characterization of ADMv-GELNs hydrogels Prepare ADMv hydrogel for later use. GELNs were fluorescently labeled with exosome red fluorescent dye (PKH26) using the following steps: (1) Take an appropriate amount of GELNs suspension and mix it with an equal amount of Diluent C reagent, and incubate at 37°C for 30 min; (2) Stop staining with 2 mL of 1% BSA for 1 min; (3) Add PBS to the mixture, centrifuge at 4°C and 100,000 g for 2 h to remove free dye; (4) Resuspend the precipitate at the bottom of the tube with PBS to obtain GELNs labeled with PKH26; (5) Incorporate the PKH26-labeled GELNs into ADMv hydrogel (the preparation method of ADMv hydrogel is as follows: ADMv (ADM and Adv are mixed at a mass ratio of 1:1) is dissolved in 5 mg / mL pepsin solution (prepared with 0.01 M hydrochloric acid) at a concentration of 15 mg / mL, and stirred at 4°C with a magnetic stirrer). (6) After stirring for 48 hours, the mixture was stirred at 4°C for 20 minutes to ensure that the GELNs were in full contact with the hydrogel, and then added to a confocal dish; (7) The mixture was placed in a 37°C incubator for 10 minutes to obtain a digestion and dissolution system containing ginger exosome-like nanoparticles; (8) The digestion and dissolution system containing ginger exosome-like nanoparticles was adjusted to neutral, and a buffer solution (20×PBS) of 1 / 19 of the volume of the neutral system was added to the obtained neutral system to form a gel. After solidification, the three-dimensional distribution of GELNs in ADMv was observed by confocal microscopy.

[0060] Performance testing: (1) The ADMv-GELNs composite hydrogel dressing was tested in a rat full-thickness skin wound repair experiment.

[0061] The specific process is as follows: This experiment uses male SD rats weighing between 180 and 200 g to construct a full-thickness skin defect model on their backs.

[0062] Experimental groups: Control group: 100 μL PBS; GELNs group: 100 μL PBS suspension containing 100 μg GELNs; ADMv group: 100 μL ADMv hydrogel with a concentration of 15 mg / mL; ADMv-GELNs group: 100 μL ADMv hydrogel with a concentration of 15 mg / mL (containing 100 μg GELNs).

[0063] Animal surgical procedure: Twelve SD rats were randomly selected. After anesthesia with isoflurane (40 mg / kg), the hair on their backs was shaved with electric clippers, and the exposed skin was disinfected three times with povidone-iodine. Four circular full-thickness skin defects with a diameter of 8 mm were drilled in the disinfected area on the back of the rats using a disposable sterile skin punch. The spacing between each pair of holes was 1.5 cm. The materials of each group were applied to the skin defects in sequence according to different groups. The wounds were photographed at 0, 3, 7, 10, and 14 days, and the wound area was measured using ImageJ image analysis software. The wound contraction rate (Formula 1) and wound healing rate (Formula 2) were calculated at 0, 3, 7, 10, and 14 days.

[0064] Formula 1.

[0065] Formula 2.

[0066] PBS, GELNs, ADMv, and ADMv-GELNs were applied to dorsal wounds in rats, with PBS serving as a control, to investigate the role of each material in the skin wound healing process. Figure 1 The changes in wound size and statistical results for each group at postoperative days 0, 3, 7, 10, and 14 are shown. A: Representative images of wound healing in rats treated with PBS (control), GELNs, ADMv, and ADMv-GELNs; B: Statistical graphs of wound residual rate at days 0, 3, 7, 10, and 14; C: Statistical graphs of wound healing rate at days 0, 3, 7, 10, and 14 (Note: P <0.05, P <0.01, ns indicates no significant difference, # P <0.05, ## P <0.01, # indicates the difference between each group and the control group. Representing the differences between groups, n=6). From Figure 1 It can be seen that the wound size of rats in all groups decreased significantly over time after surgery. Among them, the wound healing speed was fastest in the ADMv-GELNs group, followed by the GELNs group and the ADMv group, while the wound healing speed was slowest in the control group.

[0067] (2) Histological morphology (HE staining, Masson staining and Sirius red staining) tests were performed on the ADMv-GELNs composite hydrogel dressing.

[0068] The specific procedure was as follows: On days 7 and 14 of the experiment, rats were euthanized, and skin tissue was excised around the wound. The excised skin tissue was cut into two equal halves. One half was frozen at -80°C for later use, while the other half was fixed in 10% paraformaldehyde. The fixed specimens were embedded in paraffin and cut into 3μm thick sections. Subsequently, HE staining, Masson staining, and Sirius red staining were performed to compare the tissue repair and collagen fiber deposition in the wound area after treatment with different materials in experimental groups 1 and 2.

[0069] Tissue paraffin embedding and slide preparation: (1) Fixation with paraformaldehyde for 48 h; (2) Dehydration: 2 h each of 75% ethanol, 80% ethanol, 85% ethanol and 90% ethanol, and 1.5 h each of anhydrous ethanol I and anhydrous ethanol II; (3) Clearing: 10 min each of xylene I and xylene II; (4) Paraffin infiltration: 90 min each of paraffin I and paraffin II at 65℃; (5) Embedding; (6) Slicing: Slice the embedded paraffin block using a tissue microtome, and adjust the slice thickness parameter to 3 μm; (7) (8) Slicing: Spread the sliced ​​tissue in 42°C warm water and gently lift it with a clean glass slide to avoid wrinkling; (9) Baking: Place the slices in a 70°C oven and bake for about 30 minutes, then store at room temperature for later use; (10) Dewaxing: Immerse the slices in xylene I and xylene II for 10 minutes each, anhydrous ethanol I and anhydrous ethanol II for 10 minutes each, 95% ethanol and 90% ethanol for 5 minutes each, 80% ethanol and 70% ethanol for 2 minutes each, and wash with tap water for 2 minutes.

[0070] HE staining: (1) Stain the nucleus with hematoxylin solution for 5-10 min, and wash with water for 3-5 min; (2) Differentiate with hydrochloric acid ethanol for 3 s, and rinse with running water for 2 min; (3) Blue back for 2 min, and rinse with running water for 2 min; (4) Stain with eosin solution for 3-5 min, and rinse with running water for 2 min; (5) Dehydration: Place the section in 70% and 80% ethanol for 10 s each, and in anhydrous ethanol I and anhydrous ethanol II for 5 min each; (6) Clearing: Place the section in xylene I and xylene II for 5 min each; (7) Mount with neutral resin, observe under a microscope, and take pictures.

[0071] Sirius red staining: (1) Stain the sections in Sirius red staining solution for 10-30 min; (2) Dehydrate with graded alcohol; (3) Clear with xylene; (4) Mount with neutral resin, observe under a microscope, and take pictures.

[0072] Masson staining: (1) Stain the nucleus with Weigert iron hematoxylin for 5 min; (2) Wash thoroughly with water and examine under a microscope after blue staining. If overstained, use 0.5% hydrochloric acid ethanol solution for color separation; (3) Wash with distilled water for 1-2 min; (4) Stain with Ponceau S acid fuchsin solution for 5-10 min; (5) Immerse in 0.2% glacial acetic acid solution for a while; (6) Treat with 1% phosphomolybdic acid solution for color separation for 3 min; (7) Immerse in 0.2% glacial acetic acid solution for a while; (8) Stain with 2% aniline blue solution for 5 min; (9) Immerse in 0.2% glacial acetic acid solution for a while; (10) Dehydrate and clear, and mount with neutral resin.

[0073] Figure 2 Representative HE staining images of rat wounds treated with PBS (Control), GELNs, ADMv, and ADMv-GELNs on days 7 and 14, scale bar = 2 mm. Figure 2 As shown, there were significant differences in wound area among the groups on days 7 and 14. The ADMv-GELNs group had the smallest wound area, followed by the GELNs group, the ADMv group, and the control group. On day 14, compared with other groups, the wound in the ADMv-GELNs group was significantly smaller, and the ECM remodeling was more significant.

[0074] Masson staining results are as follows: Figure 3 As shown, Figure 3 In the image, A: Representative images of rat skin sections stained with Masson staining at 7 and 14 days post-surgery, with a magnified view below. Scale bar = 100 μm; B: Statistical chart of collagen fiber deposition ratio in each group on day 14. Note: P <0.05, P <0.01, ns indicates no significant difference, # P <0.05, ## P <0.01, # indicates the difference between each group and the control group. This represents the differences between groups, n=6. For example... Figure 3 As shown, during the healing process, the control group had a lower level of collagen fiber deposition, while the collagen fibers in the GELNs group and the ADMv-GELNs group were larger and denser, with the ADMv-GELNs group showing the best effect. Type I and Type III collagen fibers are two major components in the skin ECM, and the deposition and remodeling of collagen fibers are beneficial to tissue repair and regeneration.

[0075] Figure 4The images show Sirius red staining of each group under a polarized light microscope on day 14. A: Representative image of Sirius red staining in the rat wound area under a polarized light microscope on day 14 post-surgery; the bottom image is a magnified view. Type I collagen: yellow or red; Type III collagen: green; scale bar = 50 μm. B: Statistical chart of the proportion of Type I / III collagen fibers in each group on day 14. Note: P <0.05, P <0.01, ns indicates no significant difference, # P <0.05, ## P <0.01, # indicates the difference between each group and the control group. This represents the differences between groups, n=6. For example... Figure 4 As shown, the number of type I and type III collagen fibers was statistically analyzed ( Figure 4 Experiment B) found that, compared with the control group, the proportion of type I / III collagen fibers in the three treatment groups was significantly reduced, which is beneficial to scarless wound repair.

[0076] (3) Test the angiogenesis effect of ADMv-GELNs composite hydrogel dressing.

[0077] The specific process is as follows: the expression of CD31, α-SMA and iNOS in rat skin tissue is detected by immunohistochemical staining. The specific steps of immunohistochemical staining are as follows: (1) use sodium citrate antigen retrieval solution to repair the slides under high temperature and high pressure for 2 min; (2) after the slides are cooled to room temperature, rinse with running water for 1 min, carefully absorb the water stains on the slides with absorbent paper, and do not touch the tissue. Use a histological pen to circle the tissue to be tested on the slides, and then rinse with PBS solution for 3 min × 3 times; (3) remove the PBS solution, add endogenous peroxidase inhibitor (reagent 1) to the area circled by the histological pen, and incubate at room temperature for 10 min; (4) wash with PBS solution for 3 min × 3 times; (5) remove the PBS solution, add non-specific staining inhibitor (reagent 2), and incubate at room temperature for 10 min; (6) remove the non-specific staining inhibitor, do not wash, and apply primary antibodies (CD31, α-SMA and iNOS, anti-) respectively. (7) The dilution ratio was 1:200. The slides were placed in a humidified box and incubated overnight at 4°C. The next day, the slides were taken out and washed with PBS solution for 3 min × 3 times. (8) The PBS solution was removed and biotin-labeled goat anti-mouse / rabbit IgG polymer (reagent 3) was added and incubated at room temperature for 10 min. (9) The slides were washed with PBS solution for 3 min × 3 times. (10) The PBS solution was removed and streptomycin-peroxidase (reagent 4) was added and incubated at room temperature for 10 min. (11) The slides were washed with PBS solution for 3 min × 3 times. (12) The PBS solution was removed and DAB chromogenic solution was added and incubated at room temperature for 5 min. (13) After rinsing with running water, the slides were counterstained in hematoxylin staining solution for 8 min. (14) The slides were dehydrated with gradient ethanol, cleared with xylene, and mounted with neutral resin.

[0078] The results are as follows Figure 5 As shown, A: Representative image of CD31 immunohistochemical staining, scale bar = 50μm; B: Representative image of α-SMA immunohistochemical staining, scale bar = 50μm; C: Statistical chart of the percentage of CD31-positive areas; D: Statistical chart of the percentage of α-SMA-positive areas. Note: P <0.05, P <0.01, ns indicates no significant difference, # P <0.05, ## P <0.01, # indicates the difference between each group and the control group. This represents the differences between groups, n=6. For example... Figure 5As shown, compared to other groups, the ADMv-GELNs group showed the highest CD31 expression in the vascular region on days 7 and 14, followed by the GELNs group and the ADMv group. Mature blood vessels possess complete vascular structure and function, and nutrients and oxygen are mainly transported through mature blood vessels; therefore, the formation of mature blood vessels plays a crucial role in tissue repair. This experiment used α-SMA immunohistochemical staining to assess the number of mature blood vessels formed in the wound of each treatment group. The results showed that α-SMA expression in the ADMv-GELNs group was higher than that in other groups, followed by the GELNs group (…). Figure 5 (B and D in the text). This indicates that the ADMv-GELNs group has more mature angiogenesis, which is more conducive to the wound healing process.

[0079] (4) Macrophage polarization test of ADMv-GELNs composite hydrogel dressing.

[0080] The specific procedure was as follows: Immunofluorescence (to detect the expression of F4 / 80 and CD206) and immunohistochemical staining (to detect the expression of iNOS) were used to observe the polarization effect of ADMv-GELNs on macrophages during wound healing.

[0081] Immunofluorescence staining: (1) Citrate antigen retrieval; (2) Rinse with running water for 1 min, then circle the tissue area to be tested on the slide with a histochemical pen, and rinse with PBS solution for 3 min × 3 times; (3) Add 10% goat serum to block for 30 min, remove excess liquid, and do not wash; (4) Add primary antibody (F4 / 80 and CD206 are prepared together, and the antibody dilution ratio is 1:200), and place in a humidified chamber at 4°C overnight; (5) Wash with PBS for 5 min × 3 times, add fluorescent secondary antibody (antibody dilution ratio is 1:250), and incubate at room temperature in the dark for 1 h; (6) Wash with PBS for 5 min × 3 times, add mounting medium containing DAPI to prevent quenching, and observe under an immunofluorescence microscope as soon as possible.

[0082] The immunohistochemical staining procedure is the same as above.

[0083] The results are as follows Figure 6 As shown, A: Representative images of iNOS immunohistochemical staining of M1 macrophages in the wound area on days 7 and 14. The scale bar of the top row is 50 μm, and the bottom row is a magnified view of the corresponding area within the black box in the top row; B: Representative images of immunofluorescence staining of F4 / 80 (red) and CD206 (green) macrophages in the wound area on days 7 and 14. The scale bar of the top row is 20 μm, and the bottom row is a magnified view of the corresponding area within the white box in the top row; C: Percentage chart of iNOS positive areas; D: Percentage chart of CD206 positive areas. Note: P <0.05, P <0.01, ns indicates no significant difference, # P <0.05, ## P <0.01, # indicates the difference between each group and the control group. This represents the differences between groups, n=6. For example... Figure 6 As shown, on days 7 and 14, compared with the control group, the expression of iNOS in both the ADMv and ADMv-GELNs groups increased, while the expression in the GELNs group did not increase significantly. Figure 6 Figures B and D show that CD206 expression was highest in the ADMv-GELNs group, followed by the GELNs group and the ADMv group. Overall, the experimental results indicate that ADMv can simultaneously induce both M1 and M2 macrophages and can synergistically regulate M2 macrophage polarization with GELNs.

[0084] (5) The polarization of macrophages in the skin wound area of ​​SD rats was detected by qRT-PCR experiment.

[0085] The relative mRNA expression of M1 macrophage markers iNos (Nos2) and Cd86, and M2 macrophage markers Cd206 (Mrc1) and Vegf (Vegfa) was detected.

[0086] Specific steps of qRT-PCR experiment: (1) Take out the skin tissue from the -80℃ refrigerator, cut off the excess normal skin around it, retain the tissue at the wound site and cut it into pieces as much as possible. (1) Weigh 50 mg of skin tissue for each group and add 1 mL of RNAiso Plus to completely cover the skin tissue; (2) After standing at room temperature for 10 min, add 200 μL of chloroform to the above lysis buffer, tighten the centrifuge tube cap, shake vigorously for 30 s, and mix until the solution emulsifies and turns milky white; (3) After standing at room temperature for 15 min, centrifuge at 12,000 g and 4 °C for 15 min; (4) Carefully remove the centrifuge tube from the centrifuge and use a 200 μL pipette to transfer the supernatant to another new centrifuge tube; (5) Add isopropanol at a ratio of supernatant:isopropanol = 1:1, invert the centrifuge tube to mix thoroughly, and then stand at room temperature for 10 min; (6) Centrifuge at 12,000 g and 4 °C for 10 min, with the centrifuge tubes placed in the same position; (7) Carefully discard the supernatant, being careful not to touch the bottom precipitate, and add RNAiso Plus. Plus an equal amount of 75% ethanol to disperse the precipitate, gently invert the centrifuge tube to wash the wall; (8) Centrifuge at 7,500g, 4℃ for 5min and carefully discard the supernatant, being careful not to touch the precipitate; (9) Repeat step (8); (10) Open the centrifuge tube cap and dry the precipitate at room temperature for 3min; (11) After the precipitate is dried, add 20μL of RNase-free water to dissolve the precipitate; (12) Heat in a 60℃ metal bath for 10min to make the RNA dissolve more fully; (13) After measuring the sample concentration using a micro-ultraviolet-visible spectrophotometer, perform the reverse transcription reaction. The reverse transcription reaction system is shown in the following table (Table 1 and Table 2), and then Real-time PCR is performed (Table 3). The primers for the target gene used in the reaction were all synthesized by Sangon Biotech (primer sequences are shown in Table 4), with GAPDH as the internal reference gene, according to 2 -ΔΔCt The calculation results are obtained by [method name].

[0087] Table 1. Removal of Genomic DNA

[0088] Table 2 Reverse transcription reaction

[0089] Table 3 Real-time PCR reactions

[0090] Table 4. Target gene name and primer sequence

[0091] See results Figure 7 , Figure 7In the table, A and B: qPCR analysis of M1 macrophage-related genes Nos2 and Cd86; C and D: qPCR analysis of M2 macrophage-related genes Mrc1 and Vegfa. Note: P <0.05, P <0.01, ns indicates no significant difference, # P <0.05, ## P <0.01, # indicates the difference between each group and the control group. This represents the differences between groups, n=6. For example... Figure 7 As shown in A, compared to the control group, the expression of Nos2 was increased in both the ADMv and ADMv-GELNs treatment groups, while the expression was slightly decreased in the GELNs group. P >0.05, the difference is not statistically significant. Figure 7 As shown in B, compared with the control group, Cd86 expression was increased in the ADMv group and the ADMv-GELNs group, and decreased in the GELNs group. P >0.05, the difference is not statistically significant. Figure 7 As shown in C and D, compared with the control group, the expression of Mrc1 and Vegfa was significantly increased in the ADMv-GELNs group, followed by the GELNs group and the ADMv group.

[0092] (6) Visceral toxicity test of ADMv-GELNs composite hydrogel dressing.

[0093] The specific process is as follows: Fourteen days after the experiment, the rats were euthanized, and their hearts, livers, spleens, lungs, and kidneys were completely removed. After paraffin embedding, the organs were stained with hematoxylin and eosin (HE) to observe the toxic effects of GELNs and ADMv on the rat viscera. The HE staining procedure was the same as above. The results are shown in [Figure 1]. Figure 8 , Figure 8 In the text, the scale bar is 50 μm. For example... Figure 8 As shown, compared with the control group, the cells in the organs of each experimental group were intact, without obvious deformation, and the arrangement was normal. No obvious inflammatory cells or necrotic cells and tissues were observed.

[0094] (7) The effects of ADMv-GELNs on macrophage M2 polarization in vitro were examined by flow cytometry, immunofluorescence staining and qRT-PCR.

[0095] The specific process is as follows: The experimental methods and grouping (see Table 5 for details) are as follows: (1) RAW264.7 cells were seeded in cell culture dishes and cultured overnight; (2) LPS and IFN-γ were added to the culture dishes the next day and incubated for 24 h; (3) After 24 h, the Control group was replaced with normal culture medium, and the other groups were treated as follows: the GELNs group was replaced with complete culture medium containing GELNs (GELNs concentration was 50 μg / mL); the ADMv group was replaced with complete culture medium containing ADMv (ADMv extract concentration was 10 mg / mL); the ADMv-GELNs group was replaced with complete culture medium containing ADMv-GELNs (ADMv extract concentration was 10 mg / mL, GELNs concentration was 50 μg / mL); (4) After 24 h, the cells were collected, washed once with Cell Staining Buffer, centrifuged at 350 g, 4 ℃ for 5 min, and the supernatant was discarded; (5) Blocking Fc receptors and reducing non-specific fluorescent staining: 100 μL Resuspend cells in PBS, add 1 μL TruStain fcX™ (anti-mouse CD16 / 32), mix thoroughly by pipetting, and incubate on ice in the dark for 10 min; (6) Centrifuge at 350g, 4℃ for 5 min, and discard the supernatant. Add FITC-CD11b antibody, stain on ice in the dark for 30 min, and do not add antibody to the negative control; (7) Wash cells twice with Cell Staining Buffer, centrifuge at 350g, 4℃ for 5 min, and discard the supernatant; (8) Add cell fixation solution, fix cells at room temperature in the dark for 20 min, centrifuge at 350g, 4℃ for 5 min, and discard the supernatant; (9) Add Cyto-Fast™ Perm Wash Solution to resuspend the fixed cells, centrifuge at 350g, 4℃ for 5 min, and discard the supernatant. Repeat 3 times, without centrifugation on the last time. After resuspending the cells, incubate at 4℃ in the dark overnight; (10) The next day, centrifuge directly at 350g, 4℃ for 5 min, and discard the supernatant. Wash cells twice with Cell Staining Buffer and discard the supernatant; (11) Add APC-CD206 antibody and incubate at room temperature in the dark for 40 min. No antibody is added for the negative control; (12) Wash cells twice with Cell Staining Buffer, centrifuge at 350g and 4℃ for 5 min and discard the supernatant; (13) Resuspend cells in Cell Staining Buffer, filter through a 300-mesh filter, and perform flow cytometry analysis.

[0096] Table 5 Experimental Grouping and Treatment Methods

[0097] The immunofluorescence staining experiment grouping and experimental methods are the same as above; the specific methods for qRT-PCR are the same as above, and the experimental primer sequences are shown in Table 6.

[0098] Table 6. Target gene name and primer sequence

[0099] See results Figure 9 , Figure 9 In the image, A: Representative image of iNOS and CD206 immunofluorescence staining, iNOS: red, CD206: green, Merge represents the fusion of iNOS and CD206, DAPI staining of cell nuclei, scale bar = 50μm; B: Statistical graph of relative fluorescence intensity of iNOS; C: Statistical graph of relative fluorescence intensity of CD206; D: Statistical graph of M2 / M1 ratio. Note: P <0.01, ns indicates no significant difference. P <0.01, # indicates the difference between each group and the control group. This represents the differences between groups, n=6. For example... Figure 9 As shown, compared with the GELNs and ADMv-GELNs groups, the iNOS fluorescence of the ADMv group was the strongest, indicating that the ADMv group induced higher iNOS expression. Figure 9 (A and B in the text). Compared to other groups, the ADMv-GELNs group showed the strongest CD206 staining fluorescence, followed by the GELNs and ADMv groups. P <0.01, indicating a statistically significant difference. Figure 9 (A and C in the text). Figure 9 D represents the ratio of M2 to M1 macrophages. Compared with the control group, the M2 / M1 ratio increased in the experimental group, with the highest ratio in the ADMv-GELNs group, followed by the GELNs group.

[0100] Figure 10 This is a statistical graph showing the gene expression of Nos2, IL-1β, Arg-1, and Vegfa in macrophages using qRT-PCR. In the graph, A and B represent qPCR analysis of Nos2 and IL-1β, genes associated with macrophage M1 type; C and D represent qPCR analysis of Arg-1 and Vegfa, genes associated with macrophage M2 type. Note: P <0.05, P <0.01, ns indicates no significant difference, # P <0.05, ## P <0.01, # indicates the difference between each group and the control group. This represents the difference between the groups, n=6. Figure 10Figures A and B show that, compared with the control group, the expression of Nos2 and IL-1β (markers of M1 macrophages) was downregulated in the GELNs group and the ADMv-GELNs group, with the effect of GELNs being the most significant. P <0.01). Conversely, Nos2 expression was upregulated in the ADMv group ( P <0.01), IL-1β expression was slightly upregulated, but the difference was not statistically significant. P >0.05). Figure 10 Figures C and D show that, compared with the control group, the expression of Arg-1 and Vegfa (a marker of M2 macrophages) was upregulated in the other three groups, with the most significant upregulation in the ADMv-GELNs group. P <0.01).

[0101] (8) The effect of GELNs on the expression of pro-inflammatory and anti-inflammatory factors in macrophages was detected by ELISA.

[0102] The specific process is as follows: The cell processing method and experimental grouping are the same as above. The ELISA experimental steps are as follows: (1) Collect the cell culture supernatant and centrifuge at 4,000 rpm for 20 min to take the supernatant; (2) Set up standard wells, zero value wells, blank wells and sample wells. Add 50 μL of different concentrations of standard to each standard well, add 50 μL of sample diluent to the zero value well, do not add to the blank well, and add 50 μL of the sample to be tested to the sample well; (3) Add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to the standard wells and sample wells, and incubate at 37°C in the dark for 60 min; (4) Discard the liquid and wash 5 times with washing buffer; (5) Mix substrate A and substrate B thoroughly at a ratio of 1:1, add 100 μL to each well, and incubate at 37°C in the dark for 15 min; (6) Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm.

[0103] Figure 11 Statistical graph showing the expression of TNF-α, IL-1β, and IL-10 in macrophage culture supernatant as detected by ELISA; Note: P <0.05, P <0.01, ns indicates no significant difference, # P <0.05, ## P <0.01, # indicates the difference between each group and the control group. This represents the difference between the groups, n=3. For example... Figure 11As shown, compared with the control group, the levels of pro-inflammatory cytokines TNF-α and IL-1β were decreased in both the GELNs group and the ADMv-GELNs group, while the level of anti-inflammatory cytokines IL-10 was increased. Compared with the control group, the expression levels of TNF-α, IL-1β, and IL-10 were increased in the ADMv group.

[0104] (9) The effects of macrophage supernatant treated with GELNs on HUVECs proliferation, migration and angiogenesis were detected by cell proliferation assay, Transwell cell migration assay and tubule formation assay.

[0105] The specific process is as follows: 1) CCK-8 Cell Proliferation Assay: Cell culture supernatant was collected and centrifuged at 4,000 rpm for 20 min. The supernatant from each group was then applied to HUVECs, and cell proliferation was detected using the CCK-8 assay. The specific method is as follows: 2 × 10⁶ cells per well. 3 HUVECs were seeded into 96-well plates at a density of 100 cells and cultured overnight. The next day, the culture medium was aspirated and 100 μL of the supernatant from each group was added. After incubation for 1, 3, and 5 days (with medium changed every other day), 10 μL of CCK-8 solution was added, and the plates were incubated at 37°C for 3 hours. The OD value was then measured at a wavelength of 450 nm.

[0106] 2) Transwell cell migration assay: (1) HUVECs in good growth condition were starved for 24 h in low serum medium containing 2% FBS; (2) HUVECs were removed, cells were digested with trypsin, and cells were resuspended in FBS-free medium; (3) 200 μL of HUVECs suspension (containing 3×10⁻⁶ FBS) was added to the upper chamber of the Transwell (8 μm pore size). 4 (1) Add 500 μL of supernatant from different treatment groups to the lower chamber; (4) Incubate at 37℃ for 24 h; (5) Remove the cells, move the upper chamber to other wells, and observe under a microscope whether any cells fall off from the original wells. If cells fall off, staining can be performed. (6) Remove the cells, aspirate the culture medium from the upper and lower chambers, and wash once with PBS; (7) Fix the cells with 4% paraformaldehyde for 20 min; (8) Wash twice with PBS, 2 min each time; (9) Stain with crystal violet at room temperature for 20 min; (10) Wash twice with distilled water, and wipe the inside of the upper chamber clean with a clean cotton swab; (11) Place the upper chamber of the Transwell into a clean well plate, randomly select 3-5 fields of view under a microscope, and count the number of cells that migrate to the lower membrane side.

[0107] 3) Tube formation experiment: (1) Pre-cool the pipette tips, centrifuge tubes and well plates required for the experiment, and transfer the matrix gel from -80℃ to 4℃ to thaw slowly; (2) Starve the HUVECs in good growth condition with low serum medium containing 2% FBS for 24h; (3) Place the matrix gel completely thawed at 4℃ on ice, dilute the matrix gel to 10mg / mL with medium and add it vertically to the 96-well plate (avoiding the generation of air bubbles) so that the matrix gel covers the bottom of the well; (4) Transfer the 96-well plate to the cell culture incubator and incubate at 37℃ for 1h to allow the basement membrane to form a hydrogel; (5) Digest the starved HUVECs, centrifuge and count them; (6) Resuspend the cells with complete medium to prepare a single-cell suspension (3~5×10⁻⁶ cells / well). 5 (7) Take out the 96-well plate and add 100 μL of cell suspension to each well (resuspend using the cell culture supernatant of each treatment group); (8) Place the 96-well plate in a cell culture incubator and continue to culture for 5 h, and take pictures under a microscope for observation.

[0108] Figure 12 The effects of different treatments of macrophage supernatant on HUVEC proliferation, migration, and angiogenesis are shown in the following figures: A: Statistical graph of HUVEC proliferation results after 1, 2, and 3 days of growth; B: Representative graph of HUVEC migration after 24 hours of co-culture of macrophage supernatant and HUVECs using Transwell assay (scale bar = 50 μm); C: Statistical graph of HUVEC migration number; D: Representative graph of tube formation after 4 hours of co-culture of macrophage supernatant and HUVECs (scale bar = 50 μm); E: Statistical graph of tube formation number. Note: P <0.01, ns indicates no significant difference. P <0.01, # indicates the difference between each group and the control group. This represents the differences between groups, n=6. For example... Figure 12 As shown, compared with the control group, GELNs, ADMv, and ADMv-GELNs all promoted the proliferation, migration, and angiogenesis of HUVECs. The ADMv-GELNs group had the strongest effect, followed by the GELNs group.

[0109] (10) The effect of GELNs on the TLR4 / MyD88 / NF-κB signaling pathway was detected by Western Blot experiment.

[0110] The specific process is as follows: The expression of MyD88, p-TLR4, p-P65, P65 and TRAF6 proteins in macrophages after treatment with GELNs was detected by Western blotting. The experimental steps were as follows: (1) RAW264.7 cells were seeded in cell culture dishes and cultured overnight; (2) LPS and IFN-γ were added to the culture dishes the next day, but not to the M0 group (Control), and the cells were placed in a cell culture incubator for 24 h; (3) After 24 h, the M1 group was replaced with normal culture medium, and the GELNs group was replaced with complete culture medium containing GELNs (GELNs concentration was 50 μg / mL); (4) The M1 group was replaced with complete culture medium containing GELNs ( (5) Take out the cells, wash them 3 times with PBS, and after absorbing the PBS, add an appropriate amount of RIPA lysis buffer to each group of cell samples, sonicate on ice for 10 times (sonicate for 3 seconds, stop for 1 second), and sonicate on ice for 2 hours; (6) Use a cell scraper to collect the cells into a centrifuge tube, centrifuge at 12,000 rpm and 4°C for 20 minutes, take the supernatant, and detect the protein concentration in the supernatant by BCA method; (7) Add loading buffer to the sample supernatant according to the ratio of sample:loading buffer = 4:1, incubate at 105°C for 5 minutes to denature the protein, and store the sample in a -80°C refrigerator after it has returned to room temperature; (8) Electrophoresis: Prepare 5% stacking gel and 10% separating gel, add marker and sample in sequence, and the sample loading amount is 20 μg / well. Set the voltage to 80V and electrophoresis at room temperature for 30 minutes. After the protein reaches the junction of the stacking gel and the separating gel, adjust the voltage to 120V and continue electrophoresis for 40 minutes; (9) Transfer: Transfer at 4℃ and 100V for 90 minutes; (10) Cut the membrane: Carefully remove the transfer membrane. At this time, the protein in the pre-cast gel has been transferred to the membrane. Cut the desired band according to the molecular weight of the protein; (11) Block: Immerse the band in 5% skim milk powder and block at room temperature for 2 hours; (12) Wash 3 times with TBST (Tris-Borate-Sodium Tween-20 buffer), 5 ml each time. min; (13) Add primary antibody β-actin (1:3000), MyD88 (1:2000), p-TLR4 (1:1000), P65 (1:1000), p-P65 (1:1000) and TRAF6 (1:2000), and incubate overnight at 4℃; (14) Wash 3 times with TBST, 5 min each time; (15) Add secondary antibody (dilution ratio 1:10,000), and incubate at room temperature for 2 h; (16) Prepare ECL luminescent solution according to the ratio of solution A: solution B = 1:1, place the strip on the luminescent plate, add appropriate luminescent solution and luminescently photograph on the device.

[0111] Figure 13This is a statistical plot of expression levels of key proteins in the TLR4 / MyD88 / NF-κB signaling pathway detected by Western blotting. A: Western blot bands of β-actin, MyD88, p-TLR4, p-P65, P65, and TRAF6; B, D, and E: relative density plots of TRAF6, p-TLR4, and MyD88 to β-actin, respectively; C: relative density plot of p-P65 to P65. Note: P <0.01, ns indicates no significant difference, n=3. For example... Figure 13 As shown in the BE diagram, compared to the control group (M0), LPS+IFN-γ induction significantly induced the overexpression of TRAF6, p-TLR4, and MyD88, as well as the hyperphosphorylation of NF-κB p65. P <0.05). Compared with the M1 group, GELNs significantly downregulated the expression of TRAF6, p-TLR4, and MyD88, and inhibited the superphosphorylation of NF-κB p65. P <0.05).

[0112] In summary, the decellularized matrix hydrogel matrix (ADMv) prepared in this invention, with a concentration of 15 mg / mL, exhibits good biocompatibility and degradability, and can promote the proliferation, migration, and secretion of FGF and VEGF by HUVECs. This invention utilizes the ADMv hydrogel matrix to sustain the release of GELNs for up to 14 days, effectively increasing the residence time and efficacy of GELNs on wounds. In the decellularized matrix composite hydrogel dressing (ADMv-GELNs) of this invention, GELNs at concentrations of 0.5–1.5 mg / mL significantly promote skin wound healing in rats, increase collagen fiber deposition and angiogenesis, and regulate M2 polarization of macrophages in the skin wound area. GELNs at 50 μg / mL significantly alleviate LPS+IFN-γ-induced macrophage inflammation, reduce the expression levels of pro-inflammatory factors, and increase the expression levels of anti-inflammatory factors. This effect may be related to the downregulation of key protein expression in the TLR4 / MyD88 / NF-κB signaling pathway by GELNs. The hydrogel matrix (ADMv) can induce higher expression of inflammatory factors and regulate the polarization of macrophage M2 type, and can synergistically regulate macrophage polarization with GELNs.

[0113] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A decellularized matrix composite hydrogel dressing, characterized in that, It includes a hydrogel matrix and ginger exosome-like nanoparticles dispersed in the hydrogel matrix; The hydrogel matrix is ​​made from raw materials including decellularized dermal matrix and decellularized aortic adventitia matrix.

2. The decellularized matrix composite hydrogel dressing according to claim 1, characterized in that, The mass ratio of the decellularized dermal matrix to the decellularized aortic adventitia matrix is ​​1:3 to 3:

1.

3. The decellularized matrix composite hydrogel dressing according to claim 1, characterized in that, The concentration of ginger exosome-like nanoparticles in the decellularized matrix composite hydrogel dressing is 0.5~1.5 mg / mL.

4. The method for preparing the decellularized matrix composite hydrogel dressing according to any one of claims 1 to 3, characterized in that, Includes the following steps: Acellular dermal matrix, acellular aortic adventitia matrix, and pepsin hydrochloric acid solution were mixed, and the resulting mixture was digested and dissolved to obtain a digested and dissolved system. The digestion and dissolution system was mixed with ginger exosome-like nanoparticles and incubated to obtain a digestion and dissolution system containing ginger exosome-like nanoparticles. The digestion and dissolution system containing ginger exosome-like nanoparticles was adjusted to neutral, and a buffer solution was added to the resulting neutral system to form a gel, thereby obtaining the decellularized matrix composite hydrogel dressing.

5. The preparation method according to claim 4, characterized in that, The concentration of pepsin in the pepsin hydrochloric acid solution is 2.5~5 mg / mL, and the concentration of hydrochloric acid is 0.01~0.05 mol / L; The total concentration of decellularized dermal matrix and decellularized aortic adventitia matrix in the mixed system is 15-18 mg / mL.

6. The preparation method according to claim 4 or 5, characterized in that, The digestion and dissolution process takes place at a temperature of 4-8°C for 24-72 hours.

7. The preparation method according to claim 4, characterized in that, The incubation temperature is 37°C, and the time is 10~30 minutes.

8. The preparation method according to claim 4, characterized in that, The buffer solution is 20×PBS; the volume of the buffer solution is 1 / 19 of the volume of the neutral system.

9. The preparation method according to claim 4 or 8, characterized in that, The gelation temperature is 35~40℃.

10. The use of the decellularized matrix composite hydrogel dressing according to any one of claims 1 to 3 or the decellularized matrix composite hydrogel dressing prepared by the preparation method according to any one of claims 4 to 9 in the preparation of skin damage repair materials.

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