Preparation method of acellular matrix skin scaffold for 3D printing slow-release mechanical stimulation exosome

Abstract

The invention discloses a preparation method of a 3D printing acellular matrix skin scaffold for slowly releasing a mechanical stimulation exosome. The preparation method comprises the following steps: (1) preparing acellular dermal matrix powder; (2) mixing acellular dermal matrix powder with methacrylated chitosan, the mass of the acellular dermal matrix powder being 10% of that of the methacrylated chitosan, and then mixing the acellular dermal matrix powder with the fibroblast exosome prepared in the claim 1 to obtain bio-ink, the concentration of the exosome being 150 [mu] g / ml; and (3) printing the bio-ink obtained in the step (2) into a skin stent by adopting a 3D bio-printer, and soaking the stent in a 1% tannic acid solution for 15 minutes to finish surface modification, wherein a printing model is a 2cm * 2cm * 2mm rectangular stent. The acellular matrix skin scaffold provided by the invention provides a good slow-release carrier for the fibroblast exosome subjected to mechanical stimulation, and the slow release and long-term play of the exosome can be realized through the scaffold.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological tissue engineering, and specifically designs a method for preparing a 3D-printed acellular matrix skin scaffold that slowly releases mechanically stimulated exosomes. Background Art

[0002] As the largest organ in the outer layer of the human body, the skin constitutes an important barrier for the human body, protecting it from various threats such as physical damage, interstitial fluid loss and infection. When a large area of skin is lost (>30% of the body surface area) due to severe burns, trauma or diabetic ulcers, the wound healing ability is poor in the early stage of wound healing due to local inflammation, ischemia, infection and oxidative stress. Various dressings are often used clinically for long-term segmented treatment, but the treatment cycle is long and the efficacy is limited. Patients with severe skin loss require surgical treatment with skin transplantation. However, skin grafts are currently difficult to obtain, expensive and accompanied by risks such as immune rejection and disease infection. Therefore, how to promote the healing of large-scale skin wounds and develop skin substitutes with good bioactivity and biodegradability that can promote skin healing is a challenge in the field of wound repair.

[0003] Fibroblasts play a crucial role in the repair of skin injuries. Skin injury repair is a complex biological process involving the collaboration of multiple cell types, with fibroblasts primarily responsible for key steps in wound healing, including cell migration, proliferation, matrix remodeling, and scar formation. Furthermore, it is well known that skin stretching can stimulate tissue growth through cell proliferation and deposition of new extracellular matrix, a principle exploited in reconstructive surgery. Previous studies have shown that mechanical stimulation of fibroblasts can encourage them to help repair skin tissue and restore its structure and function. Exosomes are tiny, unilamellar vesicles approximately 30 to 200 nanometers in diameter. Their topology mirrors that of cells and are rich in specific proteins, lipids, nucleic acids, and glycoconjugates. Once released, exosomes possess a variety of functions, including remodeling the extracellular matrix and transmitting signals and molecules to other cells. However, the use of mechanically stimulated fibroblast exosomes to accelerate skin healing has been rarely reported.

[0004] 3D bioprinting technology provides a basis for the preparation of skin scaffolds loaded with mechanically stimulated fibroblast exosomes. However, the key challenge of 3D bioprinting is to develop bio-inks with good printing performance, good biocompatibility and bioactivity. The bio-inks with collagen, polysaccharides and other protein components similar to the dermal matrix obtained through decellularization technology provide a feasible way out for this challenge. The decellularized matrix can provide cells with a cellular microenvironment similar to that of the skin, promote cell proliferation and migration, and is a good endogenous regeneration guide for stem cells. By mixing chitosan, which has antibacterial properties and can be rapidly photocured, in a certain proportion, it can become a bio-ink with excellent biological properties and rapid photocuring ability, and at the same time provide a good sustained-release carrier for fibroblast exosomes after mechanical stimulation. In order to solve the above challenges, the present invention prepares a 3D printed decellularized matrix skin scaffold for sustained-release of mechanically stimulated exosomes.

[0005] SUMMARY OF THE INVENTION

[0006] In order to solve the above technical problems, the purpose of the present invention is to provide a 3D printed acellular matrix skin scaffold that slowly releases mechanically stimulated exosomes. By creatively extracting fibroblast exosomes after mechanical stimulation and obtaining dermal extracellular matrix through an improved decellularization method with high retention of extracellular matrix, the problem of insufficient repair ability of dermal scaffolds in the existing technology is solved. By simulating the extracellular environment and slowly releasing fibroblast exosomes after mechanical stimulation, the wound cell proliferation, migration and angiogenesis are promoted, thereby repairing the wound.

[0007] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0008] A first aspect of the present invention provides a method for producing high-performance fibroblast exosomes after mechanical stimulation, comprising the following steps:

[0009] Primary fibroblasts were obtained from the dermis and expanded. After being passaged to the P3 generation, the fibroblasts were seeded in a cell stretching dish. A cell mechanical loading instrument was used to stretch the fibroblasts to 5% at a frequency of 1 Hz. After stretching for 12 hours, the cells were further cultured for 12 hours and the supernatant was extracted. The supernatant was extracted using an exosome extraction kit to obtain exosomes from fibroblasts after mechanical stimulation.

[0010] In a second aspect, the present invention provides an improved method for decellularizing a highly retained extracellular matrix to obtain a dermal extracellular matrix, the specific steps of which are as follows:

[0011] (1) Using a microneedle with a diameter of 1 μm, the skin tissue is uniformly punched, and the microneedle penetrates the entire layer of the skin; after uniform punching, the skin is washed with PBS, and then a freeze-thaw cycle is repeated 3-5 times;

[0012] (2) After freeze-thaw cycles, the skin was eluted in cell elution solutions A, B, C, and D, respectively. The elution process was carried out at room temperature. During the elution process, the skin tissue was placed in a 50 ml centrifuge tube and placed on a shaker. Cell elution solution A was a mixed solution containing 10 mM / L Tris and 0.5 mM / L EDTA, cell elution solution B was a mixed solution containing 1% Triton X-100, 5 M / L KCI, and 10 mM / L Tris, solution C was a nuclease solution containing 300 UI / ml deoxyribonuclease I and 500 μg / mL ribonuclease A, and solution D was a sterile 1% penicillin-streptomycin PBS solution.

[0013] (3) The decellularized dermis after washing 3-5 times with D solution in step (2) is pre-frozen in a -80 degree Celsius refrigerator and placed in a freeze dryer for freeze drying for 24-48 hours; after being completely dried, it is placed in a pre-cooled grinder for a second full grinding; a 0.5M / L HCL solution is used to fully mix with the freeze-dried powder and placed on a shaker at room temperature for 24 hours, after 24 hours, 10% pepsin is added and the mixture is fully reacted on a shaker for 72 hours; after 72 hours, the pH of the solution is adjusted to 7.0 using a NaOH standard solution; the solution after pH adjustment is placed in a dialysis bag with a MWCO of 1,000-3,000Da to remove impurities in the mixed solution, and finally the dialyzed solution is freeze-dried again to obtain the decellularized dermal matrix powder.

[0014] In a third aspect, the present invention provides a method for 3D printing a scaffold capable of sustained-release exosomes, comprising the following steps:

[0015] The acellular dermal matrix powder was mixed with methacryloyl chitosan at a ratio of 10%, and then mixed with the fibroblast exosomes prepared above to obtain a bio-ink, wherein the exosome concentration was 150 μg / ml;

[0016] The above-mentioned bio-ink was printed into a skin scaffold using a 3D bioprinter. The printed model was a 2cm×2cm×2mm rectangular scaffold. The scaffold was soaked in 1% tannic acid solution for 15 minutes to complete the surface modification.

[0017] The beneficial effects of this application are:

[0018] The acellular matrix skin scaffold provided by the present invention provides a good sustained-release carrier for fibroblast exosomes after mechanical stimulation, solving the problem that exosomes are difficult to exert sustained effects due to flushing due to local inflammation and other reasons. The sustained release and long-term effect of exosomes can be achieved through the scaffold. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1Figure 1 shows a general image of the freeze-dried decellularized skin matrix (Panel A), a statistical diagram of DNA content before and after decellularization (Panel B), H&E staining, Masson staining (Panel C), Shanghai First-Line Synchrotron Radiation Infrared Spectroscopy (Panel D), scanning electron microscopy (Panel E), and immunofluorescence of key proteins such as type I collagen, elastin, and laminin (Panel F). In Panel B, *** indicates P < 0.001;

[0020] Figure 2 Figure 1 shows the mechanical stimulation of fibroblasts (Figure A) and the general image before and after stimulation (Figure B), exosome transmission electron microscopy (Figure C), particle size analysis (Figure D), and surface marker Western blotting (Figure E).

[0021] Figure 3 The decellularized matrix hydrogel preparation process (Figure A), verification of photocrosslinking efficiency after mixing the decellularized matrix hydrogel with methacryloyl chitosan (Figure B), 3D printed scaffolds and related parameters (Figure C), Fourier transform infrared spectra of scaffold components (Figure D), and scanning electron microscopy before and after surface modification after immersion in 1% tannic acid solution for 15 minutes (Figure E).

[0022] Figure 4 Live and dead cell staining of acellular matrix skin scaffold (Figure A), cell proliferation assay (Figure B), corresponding statistical graph (Figure C), and fluorescence and statistical graph of exosomes sustained release from the scaffold (Figure D);

[0023] Figure 5 This is a comparison chart of the ability and effect of cells under exosome intervention under different conditions.

[0024] Figure 6 This is a general diagram and analysis diagram of the acellular matrix skin scaffold used for rat skin wound repair. DETAILED DESCRIPTION

[0025] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further described below in conjunction with specific examples, but the following examples are only preferred embodiments of the present invention, not all. Based on the examples in the embodiments, other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0026] Example 1:

[0027] The preparation method of the acellular matrix skin scaffold for obtaining and identifying exosomes and 3D printing sustained-release mechanically stimulated exosomes described in this embodiment includes:

[0028] (1) Preparation of acellular dermal matrix:

[0029] 1. Dermal Tissue Acquisition

[0030] Obtain allogeneic or xenogeneic dermal tissue (the samples in this example are from adult male rats) and wash it 3-5 times with sterile PBS containing 1% penicillin-streptomycin in a sterile operating cabinet to remove hair and subcutaneous fat. The cleaned dermal tissue sample is stored at -80°C for later use.

[0031] 2. Decellularization of dermal tissue

[0032] Using a 1-micron diameter microneedle, the skin tissue was uniformly perforated, penetrating the entire skin layer. After uniform perforation, the tissue was rinsed with PBS, wrapped in sterile tin foil, and subjected to five freeze-thaw cycles. A single freeze-thaw cycle involved a 4-minute immersion in liquid nitrogen and thawing in a 37°C water bath.

[0033] After freeze-thaw treatment, samples were immersed in a mixture of solution A containing 10 mM / L Tris and 0.5 mM / L EDTA, sealed, and stirred continuously on a shaker at 80 rpm / min at room temperature for 24 hours. Solution A was discarded, and the samples were immersed in a mixture of solution B containing 1% Triton X-100, 5 M / L KCl, and 10 mM / L Tris, sealed, and stirred continuously on a shaker at 80 rpm / min at room temperature for 3 days. Solution B was discarded, and the samples were placed in sterile PBS and washed three times for 2 hours each at 4°C on a shaker at 150 rpm / min. The wash solution was discarded, and the samples were placed in sterile saline solution (solution C) containing 300 UI / mL DNase (Sigma-Aldrich) and 500 μg / mL RNase A (Sigma-Aldrich) and incubated at 37°C on a shaker at 100 rpm / min for 24 hours. The sample was removed and rinsed three times with sterile 1% penicillin-streptomycin in PBS (solution D), each for 6 hours. Acellular dermal matrix (ADM) was obtained, pre-frozen in a -80°C refrigerator, and then freeze-dried in a freeze dryer for 24 hours.

[0034] (2) Acquisition of exosomes:

[0035] Fibroblasts were isolated from neonatal rat skin tissue and obtained as primary cells using enzymatic digestion. They were cultured to passage 3 (P3) in a medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Fibroblasts were seeded onto a dedicated cell stretching dish and subjected to cyclic mechanical stretching with a 5% strain amplitude and a 1 Hz frequency for 12 hours using a mechanical cell loading system (Celload-300, Haomiao Technology, China).

[0036] Replace the cell culture medium with medium containing 10% exosome-free serum and continue culturing for 48 hours. Collect the supernatant, filter impurities through a 22-μm filter, and centrifuge at 3000g for 30 minutes to remove cells and cell debris. Transfer the supernatant from the centrifuge tube to four ultrafiltration tubes (UFC910096 [15ml 100KD]) using a Pasteur pipette, 15 ml per tube. Centrifuge at 3000g for 15-20 minutes. Discard the lower layer and fill the upper chamber of each tube with the remaining supernatant. Resuspend the concentrate by pipetting approximately 10 times and centrifuge again at 3000g for 40-50 minutes, minimizing the volume in the upper chamber to less than 500 μl. After ultrafiltration, add the concentrate in the upper chamber into a 15ml centrifuge tube using a 200μl tip. Add a certain volume of extract according to the ratio of the exosome extract. Pipette and draw several times to mix thoroughly. After mixing, place it at 4 degrees for 7 hours or overnight. After overnight, the precipitate is visible to the naked eye. Centrifuge at 1500g for 30 minutes, carefully remove the supernatant and place it in a sterile EP tube, and store it at -80 degrees.

[0037] (3) 3D printed skin scaffolds

[0038] The freeze-dried acellular dermal matrix obtained in step (1) was placed in a cryogenic grinder and fully ground into powder. The powder was completely dissolved in a 0.5 M hydrochloric acid solution and stirred continuously for 24 hours. 10% pepsin was then added for a 72-hour enzymatic hydrolysis reaction. The pH was adjusted to 7.0 using a standard sodium hydroxide solution (type A211, Thermo Fisher Scientific, USA). Impurities were removed using a dialysis bag with a molecular weight cutoff of 1000-3000 Da, and finally, ADM purified powder was obtained by freeze drying.

[0039] ADM powder was thoroughly mixed with CSMA solution at a mass ratio of 10% as bio-ink and the exosomes obtained in step (2) were added to the mixture at a concentration of 150 μg / ml. The photocrosslinking efficiency was verified. A 3D bioprinter (Architect-PX, China Ruinuo Company) with a temperature-controlled photopolymerization nozzle was used to prepare the composite scaffold. The parameters were set as follows: nozzle diameter 0.21 mm, barrel temperature 22°C, printing platform temperature 4°C, air pressure 0.35 MPa, printing speed 9 mm / s, photopolymerization power 15%, and the printed model was a 2 cm × 2 cm × 2 mm rectangular scaffold. An interlaced printing path was used with a printing pitch of 1.2 mm. The scaffold was soaked in 1% tannic acid solution for 15 minutes to complete the surface modification.

[0040] like Figure 3As shown, after acid hydrolysis and enzymatic hydrolysis, acellular matrix powder was successfully obtained (Figure A), which had good photocross-linking properties when added to CSMA solution in proportion (Figure B). Figure C shows the printing path and basic parameters of the scaffold; a Fourier transform infrared spectrometer (iS50, Thermo Fisher Scientific, USA) was used to analyze the chemical composition of the scaffold (Figure C); the freeze-dried scaffold was fractured in liquid nitrogen and then gold-sprayed, and the surface / cross-section microstructure was observed by scanning electron microscopy, indicating that the porosity was reduced after immersion in 1% tannic acid solution for 15 minutes.

[0041] Example 2:

[0042] This example characterizes the acellular dermal matrix prepared in Example 1:

[0043] The non-decellularized dermis and acellular dermis were dehydrated, embedded, and sectioned, and then subjected to DNA content detection, H&E staining, Masson staining, and Shanghai First-Line Station Synchrotron Radiation Infrared Spectroscopy Microscopy.

[0044] The non-decellularized dermis and acellular dermis were embedded in OCT and sectioned. After washing away the OCT, immunofluorescence staining of key proteins such as type I collagen, elastin, and laminin was performed.

[0045] like Figure 1 As shown in the figure, after decellularization, the cellular components in the dermis have been completely removed, the residual DNA content is less than 50ng / mg dry weight, and key proteins such as type I collagen, elastin, laminin, and dermal matrix components such as proteoglycans are fully retained.

[0046] Example 3:

[0047] This example characterizes the exosomes prepared in Example 1:

[0048] Transmission electron microscopy (TEM): Hitachi HT-7700 was used to observe the morphology of exosome vesicles (copper grid negative staining method), such as Figure 2 The exosomes shown are typical spherical in shape.

[0049] Nanoparticle Tracking Analysis (NTA): ZetaView system was used to detect particle size distribution (detection mode: Brownian motion tracking). The peak particle size of NTA was 80-150 nm.

[0050] A. Western blot: Detection of marker proteins TSG101, CD63, CD81 and negative indicator Calnexin (antibody source: Abcam), TSG101 / CD63 / CD81 positive, Calnexin negative.

[0051] Comparative Example 1:

[0052] The difference from Example 1 is that the fibroblasts in the control group in this comparative example were cultured for the same period of time without mechanical stimulation.

[0053] like Figure 5 As shown in the figure, M-Exos are exosomes extracted after mechanical stimulation of fibroblasts, Exos are exosomes extracted after mechanical stimulation of fibroblasts, PBS is a blank control group (equal dose buffer without exosomes), Fibroblasts are fibroblasts, and Vascular Endothelial Cells are vascular endothelial cells.

[0054] Figure 5 A reflects the proliferation capacity of cells in each group after different interventions. M-Exos intervention significantly increased the proliferation capacity of fibroblasts, and the C-Exos intervention group also showed improved cell proliferation capacity compared to the blank control group. M-Exos intervention also significantly improved the proliferation capacity of vascular endothelial cells, while the C-Exos intervention group showed no significant improvement compared to the blank control group.

[0055] Figure 5 B reflects the migration ability of cells in each group after different interventions. The migration ability of fibroblasts in the M-Exos exosome intervention group was significantly enhanced compared with the C-Exos intervention group and the blank control group.

[0056] Figure 5 C-Exos evaluated the wound healing ability of each group of cells after different interventions. M-Exos exosome intervention significantly enhanced the horizontal migration of fibroblasts, and the C-Exos intervention group also showed improved cell proliferation compared to the blank control group. However, a significant enhancement in the horizontal migration of endothelial cells was observed only in the M-Exos intervention group; C-Exos intervention did not improve the horizontal migration of endothelial cells.

[0057] Figure 5 D reflects the tube-forming ability of each group of cells after different interventions. The tube-forming ability of vascular endothelial cells in the M-Exos intervention group was significantly enhanced, as evidenced by the formation of more tubular structures and more complete tubular network connections. In contrast, the C-Exos intervention group showed no significant improvement compared to the blank control group, with no significant difference in tube-forming ability.

[0058] The overall results show that compared with traditional directly extracted exosomes, M-Exos intervention not only further enhances the proliferation and migration ability of fibroblasts, but also promotes the migration of endothelial cells and vascular remodeling in the wound, and has stronger wound repair potential.

[0059] Test Example 1

[0060] In this test example, the performance of the acellular matrix skin scaffold prepared in Example 1 was measured.

[0061] M-Exos were mixed with bioink at a concentration of 150 μg / ml, and exosome-loaded composite scaffolds were fabricated using a 3D bioprinter. The scaffolds were co-cultured with fibroblasts in a Transwell chamber, and the in vitro biocompatibility of the composite scaffolds was assessed on days 3 and 7 using CCK-8 assays, live-dead cell staining, and EdU staining.

[0062] Subsequently, laser confocal microscopy was used to evaluate the sustained exosome release of the composite scaffold. Specifically, PKH26-labeled M-Exos were mixed with bioink according to established methods to create a bifunctional composite scaffold. The scaffold was then placed in complete culture medium containing 10% exosomes. Three-dimensional confocal microscopy was used to image the exosomes within the scaffold on days 0, 7, and 14 to assess exosome release.

[0063] like Figure 4 As shown in Figure 1 , there was no significant difference in cell viability after 1, 3, or 7 days of co-culture (Figures A and C). Cell proliferation experiments showed no change in cell proliferation after 1 or 3 days of co-culture, while 7 days of co-culture promoted fibroblast proliferation (Figures B and C). Confocal microscopy results also showed that exosomes exhibited sustained release from the scaffold, with exosomes still present after 14 days (Figure D). *p<0.05, **p<0.01, ***p<0.001, ns: no significant difference.

[0064] like Figure 6 As shown, CSMA is photosensitive chitosan, TA is tannic acid (scaffold modification material, immersion treatment can reduce the pores of the scaffold and make it tougher, and it also has a certain antibacterial effect), ADM is acellular matrix, and M-Exos are exosomes extracted from fibroblasts after mechanical stimulation.

[0065] The wounds were divided into groups according to different conditions, with the control group being the natural healing group without any wound dressing.

[0066] The healing of skin wounds in rats in each group was observed at different time points, as shown in the figure:

[0067] (A) General observations:

[0068] The healing status of skin wounds in rats in each group was recorded at different time points (0, 6, 12, and 18 days), as shown in the figure:

[0069] Control group: No dressing was used on the wound, and the natural healing speed was slower. The wound area gradually shrank, but the healing process was slower. The wound was still not completely closed on day 18, and the granulation tissue proliferation was less.

[0070] CSMA+TA group: The wound healing speed was faster than that of the control group. The wound shrank significantly on the 6th day, but a large wound surface still existed. The wound shrank further on the 12th and 18th days, but the degree of closure was limited.

[0071] ADM+CSMA+TA group: The wound contracted faster than the previous two groups. On day 6, the wound was smaller than that of the CSMA+TA group. On day 12, the wound area was significantly reduced. On day 18, most of the wound was closed, and the epidermal repair was relatively complete.

[0072] ADM+CSMA+TA+M-Exos group: The wound healing speed was the fastest, the wound area was significantly reduced on the 6th day, the wound was mostly closed on the 12th day, and was almost completely repaired on the 18th day. The healing quality was the best, and skin tissue regeneration was obvious.

[0073] (B) Wound area change trend:

[0074] The schematic diagram of the changes in wound area over time in each group showed that the wound area of the ADM+CSMA+TA+M-Exos group shrank the fastest, and the wound areas on days 6, 12, and 18 were significantly smaller than those of the other groups, indicating that the treatment in this group can more effectively promote wound repair.

[0075] (C) Wound reduction rate analysis:

[0076] The bar graph quantitatively analyzed the wound reduction rate of each group, and the results showed:

[0077] The CSMA+TA group showed a slight improvement compared with the control group (P<0.05).

[0078] The ADM+CSMA+TA group showed a more significant promoting effect (P<0.01).

[0079] The wound reduction rate in the ADM+CSMA+TA+M-Exos group was the highest, significantly higher than that in the other groups (P<0.001).

[0080] From the above analysis, it can be seen that the combination of ADM+CSMA+TA+M-Exos significantly accelerated wound closure and improved healing quality.

Claims

1. A method for preparing high-performance fibroblast exosomes after mechanical stimulation, characterized in that: Here are the steps: Primary fibroblasts were obtained from the dermis and expanded. After being passaged to the third generation, the fibroblasts were seeded in a cell stretching dish. A cell mechanical loading instrument was used to stretch the fibroblasts to 5% at a frequency of 1 Hz. After stretching for 12 hours, the cells were further cultured for 48 hours and the supernatant was extracted. The supernatant was extracted using an exosome extraction kit to obtain exosomes from fibroblasts after mechanical stimulation.

2. A method for preparing a 3D printed acellular matrix skin scaffold for sustained release of mechanically stimulated exosomes, characterized in that: The steps include: (1) preparing acellular dermal matrix powder; (2) mixing acellular dermal matrix powder with methacryloyl chitosan, wherein the mass of the acellular dermal matrix powder is 10% of the mass of the methacryloyl chitosan, and then mixing with the fibroblast exosomes prepared according to claim 1 to obtain a bio-ink, wherein the concentration of the exosomes is 150 μg / ml; (3) The bio-ink obtained in step (2) was printed into a skin scaffold using a 3D bioprinter. The printed model was a 2 cm × 2 cm × 2 mm rectangular scaffold. The scaffold was soaked in 1% tannic acid solution for 15 minutes to complete the surface modification.

3. The preparation method according to claim 2, characterized in that The specific steps of step (1) preparing acellular dermal matrix powder are: (1) Using a microneedle with a diameter of 1 μm, the skin tissue was uniformly perforated, followed by freeze-thaw cycles in liquid nitrogen; (2) After freeze-thaw cycles, the skin was eluted in cell elution solutions A, B, C, and D, respectively. The elution process was carried out at room temperature and placed on a shaker for full contact. Cell elution solution A was a mixed solution containing 10 mM / L Tris and 0.5 mM / EDTA. Cell elution solution B was a mixed solution containing 1% Triton X-100, 5 M / L KCI, and 10 mM / L Tris. Solution C was a nuclease solution containing 300 UI / ml deoxyribonuclease I and 500 μg / mL ribonuclease A. Solution D was a sterile 1% penicillin-streptomycin PBS solution. (3) The acellular dermal matrix is freeze-dried and then ground, subjected to acid hydrolysis, enzymatic hydrolysis, and dialysis, and then freeze-dried again to obtain acellular dermal matrix powder.

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