A kind of acellular dermal hydrogel and its preparation method and application

Abstract

The invention relates to the field of biotechnology, and provides a decellularized dermal hydrogel and a preparation method and application thereof; the decellularized dermal hydrogel of the invention exerts multiple effects on tissue regeneration by regulating cell proliferation, migration and immune response, thereby promoting full-thickness repair of wounds, and plays an important role in the development of tissue processes and regenerative medicine; the decellularized dermal hydrogel of the invention regulates the immune microenvironment in vivo by simulating the time-sequential release of cytokines, promotes the growth and proliferation of vascular endothelial cells and fibroblasts, promotes the regeneration of skin appendages, and significantly promotes wound healing. Compared with commercial dressings, the decellularized dermal hydrogel of the invention greatly shortens the wound healing time and accelerates the reconstruction of skin structure and function.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a decellularized dermal hydrogel and a preparation method and application thereof. Background Art

[0002] Skin wound healing is the process by which the skin repairs itself after being damaged by various external factors. Therefore, promoting the healing of these accidental and intentional injuries, minimizing the aesthetic impact on patients, and maximizing the restoration of tissue function remain central issues in clinical care.

[0003] In clinical practice, skin defects are mainly treated with flap surgery, but flap reconstruction and replantation often require a long period of fixation and recovery, and are not suitable for large-scale skin defects. Traditional bioengineering solutions focus on growth factors, cells and scaffolds separately, lack overall consideration of wound healing, and have limited results. Existing bioengineering, from traditional seed cells to simulated extracellular matrix (ECM), provides strong interactions between natural cells and different tissue structures, providing a better solution for wound healing.

[0004] Decellularized bioscaffold (dECM) is a natural biomaterial that removes cellular components from tissues and organs, retains the unique structure of the extracellular matrix, and contains active ingredients such as peptides, which provide physical and chemical support for cell survival and activity, and effectively promote tissue and organ remodeling and regeneration. dECM also has good biological activity, biocompatibility, and non-immunogenicity. These advantages make dECM one of the most promising ECM-mimicking tissue regeneration scaffolds in the field of translational medicine. However, the geometric and mechanical properties of dECM greatly limit its clinical application.

[0005] At present, materials that promote wound healing mainly include: hydrogels, bandages, sponges and drug powders, among which hydrogel materials are widely used in wound healing because they help control bleeding and keep wounds moist.

[0006] Hydrogel is a three-dimensional network structure material composed of polymers and water molecules, including natural hydrogels and synthetic hydrogels. The former is widely used in the biomedicine field due to its good biocompatibility and the hemostatic and tissue regeneration properties of some hydrogels themselves.

[0007] Wound healing is divided into four stages: hemostasis, inflammation, proliferation, and remodeling. Each stage is not completely independent, but proceeds simultaneously. In the early stage of wound healing, activated skin tissue macrophages and recruited bone marrow-derived monocytes are polarized into proinflammatory (M1) macrophages, which produce proinflammatory chemokines, cytokines (such as tumor necrosis factor (TNF), interleukin-1β (IL-1β), IL-6, IL-33, interferon (IFN)-γ), growth factors, antimicrobial peptides, and reactive oxygen species, and phagocytose cell debris and necrotic cells to prevent pathogen infection. As wound healing enters the proliferation stage, M1 macrophages transform into an anti-inflammatory (M2) phenotype, which secretes a variety of angiogenic growth factors, cytokines, and chemokines (such as metalloproteinases, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), IL-8, transforming growth factor-β (TGF-β), IL-10), promoting wound blood flow reconstruction, matrix production, and re-epithelialization. However, due to the poor local conditions of the wound, the loss of macrophage function leads to ineffective macrophage polarization, especially the impaired or premature phenotype switch from macrophage M1 to macrophage M2, which hinders the formation and transformation of the pro-inflammatory and pro-healing cytokine environment, resulting in impaired wound healing process. Therefore, the mixture of pro-inflammatory and pro-healing cytokines in skin wounds to simulate the macrophage-mediated immune microenvironment may have a positive regulatory effect on wound healing.

[0008] However, flap reconstruction and replantation often require a long period of fixation and recovery, and are not suitable for large-scale skin defects. Traditional dry wound dressings (bandages, sponges, and drug powders, etc.) cannot provide a moist wound environment and have problems such as low cost-effectiveness, low healing rate, and high wound infection rate. Ordinary natural hydrogels and synthetic hydrogels often target the wound healing rate of a single period in wound healing, have poor mechanical properties, and are sensitive to water.

[0009] Therefore, there is an urgent need for a time-dependent extracellular matrix-free hydrogel with adjustable physical and mechanical properties, low cost, wide source, non-immunogenicity, biodegradability, photosensitivity, customizable to any wound shape, and the ability to regulate the in vivo immune microenvironment. Summary of the invention

[0010] The purpose of the present invention is to provide a decellularized dermal hydrogel and a preparation method and application thereof in view of the deficiencies in the prior art.

[0011] To achieve the above object, the technical solution adopted by the present invention is:

[0012] The first aspect of the present invention is to provide a method for preparing acellular dermal hydrogel, comprising the steps of:

[0013] S1. preparing conditioned medium of macrophages polarized with M1 phenotype and conditioned medium of macrophages polarized with M2 phenotype;

[0014] S2, taking a dermis layer from an animal, and sequentially performing a decellularization process, a first freeze-drying process, and a pulverization process on the dermis layer to prepare a dermis decellularized scaffold;

[0015] S3, dissolving the decellularized dermis scaffold in a PBS solution containing hydrochloric acid and pepsin to obtain a decellularized dermis scaffold solution, and sequentially performing digestion treatment, acylation treatment, dialysis treatment and a second freeze-drying treatment to obtain a photosensitive decellularized dermis freeze-dried powder;

[0016] S4, mixing the M1 phenotype polarized macrophage conditioned medium, the photosensitive acellular dermis freeze-dried powder and a photoinitiator to prepare a cell-free pre-gel of M1 immune factor;

[0017] S5, mixing the M2 phenotype polarized macrophage conditioned medium, the photosensitive acellular dermis freeze-dried powder and a photoinitiator to prepare a cell-free pre-gel of M2 immune factors;

[0018] S6. Covering the decellularized pre-gel of the M1 immune factor on the outside of the decellularized pre-gel of the M2 immune factor, and curing it under ultraviolet irradiation to obtain the decellularized dermal hydrogel.

[0019] Preferably, step S1 comprises:

[0020] S11, separating the tibia and femur of the mouse, and after disinfection, flushing the bone marrow in the tibia and femur into a centrifuge tube using DMEM complete medium;

[0021] S12, adding red blood cell lysis solution to the centrifuge tube, repeatedly blowing and then letting it stand, centrifuging and removing the supernatant after standing;

[0022] S13, adding DMEM cell culture medium to the centrifuge tube to suspend the cells, filtering and centrifuging, removing the supernatant and repeating this step once;

[0023] S14, adding stimulation medium into the centrifuge tube to induce the bone marrow cells to differentiate into the macrophages; adjusting the cell density to 1×10 6 / mL and placed at 37°C, 5% CO 2 Cultivate in an incubator for subsequent processing; the stimulation medium is the DMEM complete medium containing 10 ng / mL macrophage colony stimulating factor;

[0024] S15, replacing the stimulation medium and continuing the culture for 4 days, and then replacing the stimulation medium again; adding 100 ng / mL lipopolysaccharide or 100 ng / mL lipopolysaccharide containing 50 ng / mL IFNγ to the stimulation medium to perform the M1 polarization treatment; or

[0025] adding 10 ng / mL IL-4 and / or 10 ng / mL IL-13 to the stimulation medium to perform the M2 polarization treatment;

[0026] S16, collecting the culture medium after the M1 polarization treatment and the M2 polarization treatment respectively, centrifuging and taking the supernatant to obtain the M1 phenotype polarized macrophage conditioned medium and the M2 phenotype polarized macrophage conditioned medium.

[0027] Preferably, in step S2, the decellularization treatment comprises: subjecting the dermis layer to agitation treatment for several times in sequence;

[0028] The stirring process includes:

[0029]

[0030] Preferably, in step S3, the digestion treatment comprises: stirring the dermal decellularized scaffold solution at 35°C-40°C for 48h-96h, filtering to remove large particles and adding 10×PBS solution to terminate the digestion; the volume ratio of the 10×PBS solution to the dermal decellularized scaffold solution is 1:(8-10);

[0031] The acylation treatment comprises: adding an alkaline solution to the dermal decellularized scaffold solution after the digestion treatment to adjust the pH to 8-9, adding methacrylic acid, stirring continuously at 35° C.-40° C. for 24 h-72 h, and then adding 1×PBS solution to terminate the acylation reaction; the volume ratio of the 1×PBS solution to the dermal decellularized scaffold solution is (4-6):1;

[0032] The dialysis treatment comprises: adding an alkaline solution to the dermal decellularized scaffold solution after the acylation treatment to adjust the pH to 7.35-7.45, and dialyzing for 48h-96h;

[0033] The freeze-drying treatment includes: stirring the dermal decellularized scaffold solution after the dialysis treatment at a temperature of 35° C.-40° C. for 48 h-96 h, and freezing it at -85° C. to -75° C. for 0.5 h-1.5 h.

[0034] More preferably, in the PBS solution, the concentration of the dermal decellularized scaffold is 8 mg / mL-12 mg / mL, the concentration of the hydrochloric acid is 0.005 mol / L-0.015 mol / L, and the concentration of the pepsin is 0.5 mg / mL-2 mg / mL; the concentration of the alkaline solution is 8 mol / L-12 mol / L; the ratio of the methacrylic acid to the dermal decellularized scaffold is 0.5 mL / g-1.5 mL / g; and the dripping acceleration of the methacrylic acid is 0.3 mL / min-0.8 mL / min.

[0035] Preferably, the photoinitiator is photoinitiator I2959, and the concentration of the photoinitiator is 0.5 mg / mL-1.5 mg / mL; in the decellularized pregel of the M1 immune factor and in the decellularized pregel of the M2 immune factor, the concentration of the photosensitive decellularized dermis freeze-dried powder is 40 mg / mL-60 mg / mL.

[0036] Preferably, in step S6, the volume ratio of the decellularized pre-gel of the M1 immune factor to the decellularized pre-gel of the M2 immune factor is 1:(1-3); in the curing treatment, the wavelength of the ultraviolet light is 365nm-395nm.

[0037] The second aspect of the present invention is to provide a decellularized dermal hydrogel prepared by the above preparation method.

[0038] The third aspect of the present invention is to provide a dressing for promoting wound healing, comprising: the above-mentioned decellularized dermal hydrogel.

[0039] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0040] The acellular dermal hydrogel of the present invention has a good gelation time and is suitable for dynamic wounds; it has good shape adaptability, making it suitable for wounds with irregular shapes; the acellular dermal hydrogel of the present invention exerts multiple effects on tissue regeneration by regulating cell proliferation, migration and immune response, thereby promoting full-thickness repair of wounds, and plays an important role in the development of tissue processes and regenerative medicine; the acellular dermal hydrogel of the present invention regulates the immune microenvironment in the body by simulating the time-sequential release of cytokines, promotes the growth and proliferation of vascular endothelial cells and fibroblasts, promotes the regeneration of skin appendages, and significantly promotes wound healing. Compared with commercial dressings, the acellular dermal hydrogel of the present invention greatly shortens the wound healing time and accelerates the reconstruction of skin structure and function. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is a diagram showing the results of macrophage polarization; Figure 1A This is the fluorescence staining result of M1 phenotype polarized macrophages; Figure 1B This is the fluorescence staining result of macrophages polarized to M2 phenotype; Figure 1C The results were verified by flow cytometry analysis of macrophages polarized to the M1 phenotype; Figure 1D The results were verified by flow cytometry analysis of macrophages polarized to the M2 phenotype; Figure 1E RT-PCR validation results for macrophages polarized to the M1 phenotype; Figure 1F RT-PCR validation results for macrophages polarized to the M2 phenotype;

[0042] Figure 2 Design pattern diagram for decellularized dermal hydrogel;

[0043] Figure 3 is a hydrogel performance test diagram; wherein, Figure 3A is the NMR spectrum of dECM@1:3MC hydrogel; Figure 3B This is a graph showing the test results of the hydrogel storage modulus; Figure 3C This is the result diagram of the photocuring time of dECM@1:3MC hydrogel; Figure 3D The microstructure of the hydrogel. Figure 3E This is the pore size result diagram of the hydrogel; Figure 3F The degradation test results of the hydrogel;

[0044] Figure 4 is a graph showing the test results of acellular dermal hydrogel releasing factors; Figure 4A is the expression heat map of macrophage-related cytokines; Figure 4B is the expression of TNF-α; Figure 4C is the expression of IL-6; Figure 4D is the expression of IL-12; Figure 4E is the expression of CCL5; Figure 4F is the expression of TGF-β; Figure 4G is the expression of 1L-1Ra; Figure 4H is the expression of IL-10; Fig. 4I is the expression of CCL1; Figure 4J is the expression of CCL17; Figure 4K is the expression of CCL22; Figure 4L is the expression of VEGFA; Figure 4M is the expression of PDGF;

[0045] FIG5 is a diagram showing the results of culturing L929 cells and HUVEC cells on the hydrogel surface; wherein, Figure 5A Fluorescence images of L929 cells cultured on the hydrogel surface; Figure 5B This is the live-dead staining result of L929 cells;

[0046] Figure 5C Fluorescence images of HUVEC cells cultured on the hydrogel surface; Figure 5D This is the live-dead staining result of HUVEC cells;

[0047] Figure 6 shows the hydrogel wound treatment results; Fig. 6A This is a diagram of the wound treatment pattern; Figure 6B This is the general appearance of the hydrogel-repaired rat skin wound; Figure 6C This is the analysis result diagram of wound healing; Fig.6D This is a comparison chart of wound healing time. DETAILED DESCRIPTION

[0048] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0050] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0051] Example

[0052] This embodiment provides a method for preparing acellular dermal hydrogel, the steps comprising:

[0053] S1. Take 4-8 week old C57BL / 6 mice and kill them by cervical dislocation, and disinfect them thoroughly with 75% alcohol; separate and remove the tibia and femur of the mice under sterile conditions, and put them into a cell culture dish containing 75% alcohol; move the separated tibia and femur into a biosafety cabinet, further separate and remove the remaining tissues around the tibia and femur, and then put the tibia and femur into a cell culture dish containing PBS and wash them 2-3 times, and finally transfer them to a cell culture dish containing DMEM complete medium (1% penicillin-streptomycin double antibody + 10% fetal bovine serum + DMEM basal medium), cut the two ends of the tibia and femur with ophthalmic scissors, and then use a 1mL syringe to absorb the DMEM complete medium, and the medium The bone marrow cells were washed from one end of the bone into a 50 mL sterile centrifuge tube, and the process was repeated several times until the bone turned white; 5 times the volume of red blood cell lysis solution was added to the 50 mL centrifuge tube, and the tube was repeatedly blown with a Pasteur pipette, and the tube was allowed to stand for 15 minutes, and then centrifuged at 1000 rpm / min for 10 minutes, and the supernatant was discarded; an appropriate amount of DMEM cell culture medium was added to resuspend the cells, and then the cells were filtered with a 200-mesh filter, and the tube was centrifuged at 1000 rpm / min for 10 minutes, and the supernatant was discarded. After repeating the centrifugation twice, a stimulation medium (DMEM complete medium containing 10 ng / mL macrophage colony stimulating factor) was added to the centrifuge tube to induce the bone marrow cells to differentiate into the macrophages; the cell density was adjusted to 1×10 6 / mL and placed at 37°C, 5% CO 2 After culturing in the incubator for 3 days, the stimulation medium was replaced and the culture was continued for 4 days, and then the stimulation medium was replaced again; 100 ng / mL lipopolysaccharide or 100 ng / mL lipopolysaccharide containing 50 ng / mL IFNγ was added to the stimulation medium to perform the M1 polarization treatment; 10 ng / mL IL-4 and / or 10 ng / mL IL-13 were added to the stimulation medium to perform the M2 polarization treatment; within 24 h to 48 h after successful polarization, the medium after the M1 polarization treatment and the medium after the M2 polarization treatment were collected, centrifuged at 1000 rpm for 5 min, and the supernatant was taken to obtain the M1 phenotype polarized macrophage conditioned medium and the M2 phenotype polarized macrophage conditioned medium; the fluorescence staining results after macrophage polarization are as shown Figure 1A As shown, the polarized M1 and M2 macrophages were subjected to flow cytometry analysis and RT-PCR to verify the polarization effect. The results are shown in Figure 1B-Figure 1C As shown, macrophages were successfully polarized to M1 and M2 phenotypes respectively.

[0054] S2, mechanically stratify the full-thickness skin of the pig, remove the subcutaneous fat, connective tissue and epidermis of the full-thickness skin of the pig, obtain the dermis, and sequentially place the dermis in a trypsin solution and stir continuously for 5h-7h; place it in deionized water and stir continuously for 10min-20min, repeat 1-3 times; place it in an ethanol solution and stir continuously for 8h-12h; place it in a hydrogen peroxide solution and stir continuously for 10min-20min; place it in deionized water and stir continuously for 10min-20min, repeat 1-3 times; place it in a Triton X-100 / EDTA / Tris solution and stir continuously for 5h-7h; place it in a replaced Triton X-100 / EDTA / Tris solution, stirring continuously for 15h-20h; placed in deionized water, stirring continuously for 10min-20min, repeated 1-3 times; placed in peracetic acid / ethanol solution, stirring continuously for 1h-3h; placed in PBS solution, stirring continuously for 10min-20min, repeated 1-3 times; placed in deionized water, stirring continuously for 10min-20min, repeated 1-3 times; finally, the first freeze-drying treatment was performed to obtain the dermal decellularized scaffold (dECM);

[0055] S3, cutting the decellularized dermis scaffold into very small pieces or grinding it into powder, dissolving it in a PBS solution containing hydrochloric acid and pepsin to obtain a decellularized dermis scaffold solution, and sequentially performing digestion treatment, acylation treatment, dialysis treatment and a second freeze-drying treatment to obtain a photosensitive decellularized dermis freeze-dried powder (dECMMA);

[0056] The digestion treatment comprises: stirring the dermal decellularized scaffold solution at 35° C.-40° C. for 48 h-96 h, filtering to remove large particles and adding 10×PBS solution to terminate the digestion; the volume ratio of the 10×PBS solution to the dermal decellularized scaffold solution is 1:(8-10);

[0057] The acylation treatment comprises: adding an alkaline solution to the dermal decellularized scaffold solution after the digestion treatment to adjust the pH to 8-9, adding methacrylic acid, stirring continuously at 35° C.-40° C. for 24 h-72 h, and then adding 1×PBS solution to terminate the acylation reaction; the volume ratio of the 1×PBS solution to the dermal decellularized scaffold solution is (4-6):1;

[0058] The dialysis treatment comprises: adding an alkaline solution to the dermal decellularized scaffold solution after the acylation treatment to adjust the pH to 7.35-7.45, and dialyzing for 48h-96h;

[0059] The second freeze-drying treatment comprises: stirring the dermal decellularized scaffold solution after the dialysis treatment at a temperature of 35°C-40°C for 48h-96h, and freezing at -85°C--75°C for 0.5h-1.5h;

[0060] In the dermal decellularized scaffold solution, the concentration of the dermal decellularized scaffold is 8 mg / mL-12 mg / mL, the concentration of the hydrochloric acid is 0.005 mol / L-0.015 mol / L, and the concentration of the pepsin is 0.5 mg / mL-2 mg / mL; the concentration of the alkaline solution is 8 mol / L-12 mol / L; the ratio of the methacrylic acid to the dermal decellularized scaffold is 0.5 mL / g-1.5 mL / g; the dripping speed of the methacrylic acid is 0.3 mL / min-0.8 mL / min;

[0061] S4, mixing the M1 phenotype polarized macrophage conditioned medium, the photosensitive decellularized dermis freeze-dried powder and the photoinitiator I2959 to prepare the M1 immune factor decellularized pre-gel (dECM@M1C pre-gel); the concentration of the photoinitiator I2959 is 0.5 mg / mL-1.5 mg / mL;

[0062] S5, mixing the M2 phenotype polarized macrophage conditioned medium, the photosensitive decellularized dermis freeze-dried powder and the photoinitiator I2959 to prepare the M2 immune factor decellularized pre-gel (dECM@M2C pre-gel); the concentration of the photoinitiator I2959 is 0.5 mg / mL-1.5 mg / mL;

[0063] In the decellularized pregel of the M1 immune factor and the decellularized pregel of the M2 immune factor, the concentration of the photosensitive decellularized dermis freeze-dried powder is 40 mg / mL-60 mg / mL;

[0064] S6, placing the decellularized pregel of the M2 immune factor into Figure 2 In the mold shown, after photocuring with ultraviolet light, the decellularized pre-gel of the M1 type immune factor is coated on the outside of the decellularized pre-gel of the M2 type immune factor and exposed to ultraviolet light to obtain the decellularized dermal hydrogel; the volume ratio of the decellularized pre-gel of the M1 type immune factor to the decellularized pre-gel of the M2 type immune factor is 1:3 (i.e., dECM@1:3MC is obtained); the wavelength of the ultraviolet light is 365nm-395nm.

[0065] The dECM@M1C hydrogel was obtained by curing the dECM@M1C pre-gel under UV light;

[0066] The dECM@M2C hydrogel was obtained by curing the dECM@M2C pre-gel under UV light;

[0067] The dECM@1:1MC hydrogel was obtained by curing dECM@M1C pre-gel and dECM@M2C pre-gel in a volume ratio of 1:1 under UV light;

[0068] The dECM@1:2MC hydrogel was obtained by curing dECM@M1C pre-gel and dECM@M2C pre-gel in a volume ratio of 1:2 under UV light;

[0069] The NMR spectra of dECM@1:3MC hydrogel are shown in Figure 2. Figure 3A As shown, the NMR curve of dECM@1:3MC hydrogel has obvious double peaks at δ between 5.0ppm and 6.0ppm, which is H 2 C=C(CH 3 ) proton peak, indicating that the methacrylic group was successfully grafted onto the gelatin molecular chain; the network structure has an important influence on the storage modulus and loss modulus of the hydrogel. Figure 3B It can be seen that the storage modulus of dECM@1:3MC hydrogel is improved compared with dECMMA hydrogel, dECM@M1C hydrogel or dECM@M2C hydrogel, indicating that dECM@1:3MC hydrogel has stronger ability to resist deformation; Figure 3C It can be seen that under 365nm ultraviolet light irradiation, dECM@1:3MC hydrogel can be completely cured in about 40s; the microstructure comparison of dECM@1:3MC hydrogel with dECMMA hydrogel, dECM@M1C hydrogel and dECM@M2C hydrogel is shown in Figure 2. Figure 3D shown; from Figure 3E It can be seen that the pore sizes of dECMMA hydrogel, dECM@M1C hydrogel, dECM@M2C hydrogel, dECM@1:1MC hydrogel, dECM@1:2MC hydrogel and dECM@1:3MC hydrogel are mainly distributed in 80μm-120μm, 40μm-50μm, 80μm-120μm, 60μm-100μm, 60μm-100μm, 80μm-120μm, respectively; the degradation of hydrogel in collagenase I is shown in Figure 3F As shown in the figure, the degradation rate of dECM@1:3MC hydrogel was significantly slower than that of pure dECMMA hydrogel, and the degradation rate of the hydrogel accelerated with time. Under the action of collagenase I, dECMMA hydrogel was degraded by half on the 3rd day, while dECM@1:3MC hydrogel was completely degraded on the 7th day.

[0070] To estimate the spatiotemporal release of cytokines in dECM@M1C hydrogel, dECM@M2C hydrogel, and dECMA@1:3MC hydrogel, RT-PCR experiments were used to analyze the expression of macrophage-related cytokines during the degradation of the hydrogels. Figure 4B -M, heat map from real-time PCR data (2 -Δct ) conversion generates, such as Figure 4A The expression of macrophage-related cytokines during hydrogel degradation is shown in Figure 4. Figure 4B-Figure 4E The expression of cytokines related to M1 phenotype polarized macrophages. Figure 4F-Figure 4K The expression of cytokines related to M2 phenotype polarized macrophages. Figure 4L-4M Figure 3 is the expression of angiogenesis-related cytokines in M1 phenotype polarized macrophages and M2 phenotype polarized macrophages; in the early stage, the release of growth factors reached a peak on the first day, and the expression levels of TNF-α, IL-6, CCL5 and VEGFA in dECMA@1:3MC hydrogel were higher, which were 2.12 times, 1.83 times and 1.56 times that of dECM@M2C hydrogel, respectively; on the second day, the curve continued to decline slowly but steadily, and the expression levels of TNF-α, IL-6, CCL5 and VEGFA in dECM@1:3MC hydrogel were higher, which were 3.80 times, 1.21 times, 1.70 times and 1.88 times that of dECM@M2C hydrogel, respectively; however, TGF-β expression was not affected. Compared with dECM@M1C hydrogel and dECMA@1:3MC hydrogel, dECM @M2C hydrogel TGF-β expression was significantly increased; on the third day, the high curves of TNF-α, IL-6, IL-10, IL-12 and CCL5 began to decline rapidly, the TGF-β curve flattened, and dECMA@1:3MC hydrogel began to exceed the daily declining dECM@M1C hydrogel and dECM@M2C hydrogel; in comparison, 3 days after release, the TGF-β expression of dECMA@1:3MC hydrogel was 1.94 times higher than that of dECM@M1C hydrogel, and the gene expression was 1.13 times higher than that of dECM@M2C hydrogel, suggesting the existence of a macrophage-related spatiotemporal immune cytokine environment, that is, in the early stage, dECM@1:3MC hydrogel released pro-inflammatory cytokines during the early degradation process, and then released pro-healing cytokines, which highly simulated the immune response required in the wound healing process.

[0071] The results of culturing L929 cells and HUVEC cells on the hydrogel surface are shown in Figure 5. After one day of culture, L929 cells and HUVEC cells proliferated on the surfaces of dECMMA hydrogel, dECM@M1C hydrogel, dECM@M2C hydrogel, dECM@1:1MC hydrogel, dECM@1:2MC hydrogel and dECM@1:3MC hydrogel. The live-dead staining results showed that the above hydrogels all had good biocompatibility.

[0072] The wound healing results of the hydrogel are shown in Figure 6. The wound healing effect of dECM@1:3MC hydrogel is significantly higher than that of Ctrl, dECM@M1C hydrogel and dECM@M2C hydrogel. The analysis results of wound healing further verified that on the 7th and 14th days, the wound healing effect of dECM@1:3MC hydrogel is significantly higher than that of Ctrl, dECM@M1C hydrogel and dECM@M2C hydrogel.

[0073] In summary, the acellular dermal hydrogel of the present invention has a good gelation time and is suitable for dynamic wounds; it has good shape adaptability, making it suitable for wounds with irregular shapes; the acellular dermal hydrogel of the present invention exerts multiple effects on tissue regeneration by regulating cell proliferation, migration and immune response, thereby promoting full-thickness repair of wounds, and plays an important role in the development of tissue processes and regenerative medicine; the acellular dermal hydrogel of the present invention regulates the immune microenvironment in the body by simulating the time-sequential release of cytokines, promotes the growth and proliferation of vascular endothelial cells and fibroblasts, promotes the regeneration of skin appendages, and significantly promotes wound healing. Compared with commercial dressings, the acellular dermal hydrogel of the present invention greatly shortens the wound healing time and accelerates the reconstruction of skin structure and function.

[0074] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing acellular dermal hydrogel, It is characterized in that the steps include: S1. preparing conditioned medium of macrophages polarized with M1 phenotype and conditioned medium of macrophages polarized with M2 phenotype; S2, taking a dermis layer from an animal, and sequentially performing a decellularization process, a first freeze-drying process, and a pulverization process on the dermis layer to prepare a dermis decellularized scaffold; S3, dissolving the decellularized dermis scaffold in a PBS solution containing hydrochloric acid and pepsin to obtain a decellularized dermis scaffold solution, and sequentially performing digestion treatment, acylation treatment, dialysis treatment and a second freeze-drying treatment to obtain a photosensitive decellularized dermis freeze-dried powder; S4, mixing the conditioned medium of macrophages polarized by M1 phenotype, the photosensitive decellularized dermis freeze-dried powder and the photoinitiator to prepare the decellularized pre-gel of M1 immune factor; S5, mixing the conditioned medium of macrophages polarized by M2 phenotype, the photosensitive decellularized dermis freeze-dried powder and the photoinitiator to prepare the decellularized pre-gel of M2 immune factor; S6, placing the decellularized pregel of the M2 immune factor into a mold, and performing a photocuring treatment under ultraviolet light to obtain the decellularized hydrogel of the M2 immune factor, and coating the decellularized pregel of the M1 immune factor on the decellularized hydrogel of the M2 immune factor, and performing a curing treatment under ultraviolet light to obtain the decellularized dermal hydrogel; Step S1 includes: S11, separating the tibia and femur of the mouse, and after disinfection, flushing the bone marrow in the tibia and femur into a centrifuge tube using DMEM complete medium; S12, adding red blood cell lysis solution to the centrifuge tube, repeatedly blowing and then letting it stand, centrifuging and removing the supernatant after standing; S13, adding DMEM cell culture medium to the centrifuge tube to suspend the cells, filtering and centrifuging, removing the supernatant and repeating this step once; S14. Add stimulation medium into the centrifuge tube to induce bone marrow cells to differentiate into macrophages; adjust the cell density to 1×10 6 / mL and placed at 37°C, 5% CO 2 Cultured in an incubator until subsequent processing; the stimulation medium was DMEM complete medium containing 10 ng / mL macrophage colony stimulating factor; S15, replace the stimulation medium and continue culturing for 4 days, then replace the stimulation medium again; add 100 ng / mL lipopolysaccharide containing 50 ng / mL IFNγ to the stimulation medium for M1 polarization treatment; 10 ng / mL IL-4 and / or 10 ng / mL IL-13 were added to the stimulation medium for M2 polarization; S16. Collect the culture medium after M1 polarization treatment and M2 polarization treatment respectively, centrifuge and take the supernatant to obtain M1 phenotype polarized macrophage conditioned medium and M2 phenotype polarized macrophage conditioned medium.

2. The preparation method according to claim 1, It is characterized in that In step S2, the decellularization process includes: successively placing the dermis layer in trypsin solution and continuously stirring for 5 h - 7 h; placing it in deionized water and continuously stirring for 10 min - 20 min, repeating 1 - 3 times; placing it in ethanol solution and continuously stirring for 8 h - 12 h; placing it in hydrogen peroxide solution and continuously stirring for 10 min - 20 min; placing it in deionized water and continuously stirring for 10 min - 20 min, repeating 1 - 3 times; placing it in Triton X - 100 / EDTA / Tris solution and continuously stirring for 5 h - 7 h; placing it in the replaced Triton X - 100 / EDTA / Tris solution and continuously stirring for 15 h - 20 h; placing it in deionized water and continuously stirring for 10 min - 20 min, repeating 1 - 3 times; placing it in peracetic acid / ethanol solution and continuously stirring for 1 h - 3 h; placing it in PBS solution and continuously stirring for 10 min - 20 min, repeating 1 - 3 times; placing it in deionized water and continuously stirring for 10 min - 20 min, repeating 1 - 3 times.

3. The preparation method according to claim 1, characterized in that, in step S3, the digestion process includes: continuously stirring the dermal decellularized scaffold solution at 35°C - 40°C for 48 h - 96 h, then filtering to remove large particles and adding 10×PBS solution to terminate digestion; the volume ratio of 10×PBS solution to the dermal decellularized scaffold solution is 1:(8 - 10); the acylation process includes: adding an alkaline solution to the dermal decellularized scaffold solution after the digestion process to adjust the pH to 8 - 9, and adding methacrylic acid, continuously stirring at 35°C - 40°C for 24 h - 72 h, then adding 1×PBS solution to terminate the acylation reaction; the volume ratio of 1×PBS solution to the dermal decellularized scaffold solution is (4 - 6):1; the dialysis process includes: adding an alkaline solution to the dermal decellularized scaffold solution after the acylation process to adjust the pH to 7.35 - 7.45, and dialysis for 48 h - 96 h; the second freeze - drying process includes: continuously stirring the dermal decellularized scaffold solution after the dialysis process at a temperature of 35°C - 40°C for 48 h - 96 h, and freezing at - 85°C ~ - 75°C for 0.5 h - 1.5 h.

4. The preparation method according to claim 3, characterized in that, in the dermal decellularized scaffold solution, the concentration of the dermal decellularized scaffold is 8 mg / mL - 12 mg / mL, the concentration of hydrochloric acid is 0.005 mol / L - 0.015 mol / L, the concentration of pepsin is 0.5 mg / mL - 2 mg / mL; the concentration of the alkaline solution is 8 mol / L - 12 mol / L; the ratio of methacrylic acid to the dermal decellularized scaffold is 0.5 mL / g - 1.5 mL / g; the dropping rate of methacrylic acid is 0.3 mL / min - 0.8 mL / min.

5. The preparation method according to claim 1, characterized in that, The photoinitiator is photoinitiator I2959, and the concentration of the photoinitiator is 0.5 mg / mL-1.5 mg / mL; in the decellularized pregel of the M1 immune factor and the decellularized pregel of the M2 immune factor, the concentration of the photosensitive decellularized dermis freeze-dried powder is 40 mg / mL-60 mg / mL.

6. The preparation method according to claim 1, It is characterized in that In step S6, the volume ratio of the decellularized pre-gel of the M1 immune factor to the decellularized pre-gel of the M2 immune factor is 1:(1-3); during the curing process, the wavelength of the ultraviolet light is 365nm-395nm.

7. A decellularized dermal hydrogel prepared by the preparation method according to any one of claims 1 to 6.

8. A dressing for promoting wound healing, It is characterized in that include: The decellularized dermal hydrogel according to claim 7.

Images