A cell-compounded sheep acellular dermal biological dressing and preparation method thereof

Abstract

The present invention proposes a cell-compounded sheep acellular dermis biological dressing and a preparation method thereof, specifically relating to the technical field of medical biological dressing materials. The cell-compounded sheep acellular dermis biological dressing of the present invention is prepared from sheepskin through depilation, splitting, disinfection, decellularization, freeze-drying, sterilization and co-culturing with cells. It retains the collagen fiber scaffold of the skin and has no cytotoxicity. It can provide a clinical dermis substitute that can carry cells and promote wound healing.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biological dressings, in particular to a sheep acellular dermis biological dressing capable of compounding cells and a preparation method thereof. Background Art

[0002] Skin and soft tissue defects often occur in trauma, burns, scars or after tumor resection, as well as chronic skin ulcers caused by systemic diseases. During the treatment of severe, large-area deep burns, due to the lack of autologous skin sources, various autologous skin substitutes are often needed to cover the wound. Among the substitutes, allogeneic skin has the best clinical application effect. However, the source of allogeneic skin is becoming increasingly difficult and expensive, and its use is subject to many restrictions.

[0003] At present, acellular dermal matrix (ADM) is commonly used in clinical practice as a dermal substitute. After special treatment, the xenogeneic skin can remove the immunogenic cell components in the tissue, retain the three-dimensional structure of the collagen fibers in the skin tissue, induce the growth of fibroblasts and vascular endothelial cells, and promote tissue regeneration. The current methods for preparing and processing acellular dermis mainly include chemical methods, physical methods and biological methods. Among them, the chemical method usually uses acid-base lysis combined with surfactants to remove cells and reduces immunogenicity through glutaraldehyde cross-linking, but its residues still have cytotoxic effects. In response to the residual cytotoxicity prepared by chemical methods, physical methods are also used to prepare acellular dermal matrix. Physical methods usually use repeated freezing and thawing methods to repeatedly freeze the skin slices. The cells are removed by alternating between -80°C and 37°C, forming ice crystals through the freezing process, which expand and destroy the cells. However, this method has a poor decellularization effect and cell fragments are not easy to completely remove. Biological methods often use dispase and trypsin to digest skin tissue, but the cell digestion is incomplete and more immunogenic substances remain. The acellular dermal matrix prepared by these two methods still has cytotoxicity, or the cells are not removed cleanly. The tissue biocompatibility of the obtained acellular dermal matrix is poor and cells cannot grow. In view of the shortcomings of these methods for preparing acellular dermal matrix, it is urgently needed to improve the preparation method and prepare acellular dermal matrix with low immunogenicity, good tissue compatibility and the ability to support cell growth.

[0004] In view of this, the present invention proposes a cell-compounded sheep acellular dermal biological dressing and a preparation method thereof, which has good compatibility and low immunogenicity. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a cell-compounded sheep acellular dermal biological dressing and a preparation method thereof.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides a method for preparing a cell-compounded sheep acellular dermal biological dressing, comprising the following steps:

[0008] S1. Obtain fresh sheep skin aged 3-6 months, soak the sheepskin surface with soap solution, and remove the wool from the sheepskin surface;

[0009] S2. The sheepskin was stripped of wool and the subcutaneous tissue was removed to obtain a skin graft having a thickness of 0.5-0.8 mm. The skin graft was then rinsed with distilled water and soaked in a new Gill solution for 30 min, repeated at least twice, and then soaked in hydrogen peroxide for 30 min, repeated at least twice to obtain a sterilized skin graft.

[0010] S3. Perform overnight digestion with 0.25% pancreatic enzyme solution at 37°C for 12 hours. The next day, rinse the skin piece with distilled water and soak it in distilled water, replacing the distilled water every 12-24 hours.

[0011] S4. Two days later, the samples were digested again with 0.25% pancreatic enzyme solution overnight and then soaked again in distilled water, changing the mixture every 12-24 hours for about ten days until the sheepskin cells were completely removed.

[0012] S5. Place the decellularized sheepskin in a freeze dryer until it is completely dry. Then, place the freeze-dried sheepskin in a clean bench and irradiate both sides of the freeze-dried sheepskin with ultraviolet light for 3 hours each to obtain a cell-reinforced sheep acellular dermal biological dressing.

[0013] Furthermore, the mass fraction of the new gynecomastide solution is 0.1%.

[0014] Furthermore, the mass fraction of the hydrogen peroxide is 3%.

[0015] Furthermore, the second distilled water is sterile distilled water containing 1% biresistant antibiotics.

[0016] Furthermore, the dual antibiotics are 100 U / mL penicillin and 100 U / mL streptomycin.

[0017] Furthermore, the step of completely removing cells from the sheepskin in step S4 includes: taking sheepskin from different parts for H&E staining to observe whether the cells are completely removed; if not, repeatedly digesting with a 0.25% mass fraction trypsin solution overnight until the cells are completely removed.

[0018] Furthermore, the first distilled water is sterile distilled water.

[0019] A cell-compounded sheep acellular dermis biological dressing is prepared from sheepskin through depilation, splitting, disinfection, decellularization, freeze drying, sterilization and co-culturing with cells.

[0020] Beneficial effects of the present invention:

[0021] The cell-compounded sheep acellular dermis biological dressing proposed in the present invention removes the original immunogenic cellular components while retaining the skin's collagen fiber scaffold. It has no cytotoxicity. After freeze-drying, the gaps in the collagen fiber scaffold of the sheep acellular dermis are enlarged, allowing cells to enter the interior of the collagen fiber scaffold, providing a clinical dermis substitute that can carry cells and promote wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The sheep skin without cells removed according to the H&E staining results of the cell-compoundable sheep acellular dermal biological dressing of the present invention;

[0023] Figure 2 The H&E staining result of the cell-compoundable sheep acellular dermal biological dressing of the present invention is the sheep skin with cells removed;

[0024] Figure 3 This is a schematic diagram of the cell-compounded sheep acellular dermal biological dressing of the present invention after the sheepskin is completely dried;

[0025] Figure 4 This is a diagram showing the cytotoxicity test results of Example 1 of the cell-compoundable sheep acellular dermal biological dressing of the present invention;

[0026] Figure 5 This is a co-cultivation diagram of Example 2 of the cell-compoundable sheep acellular dermal biological dressing of the present invention;

[0027] Figure 6 This is the H&E staining result of the co-culture of Example 2 of the cell-compoundable sheep acellular dermal biological dressing of the present invention;

[0028] Figure 7 This is a schematic diagram of the compounding results of Example 3 of the cell-compoundable sheep acellular dermal biological dressing of the present invention;

[0029] The implementation, functional features and advantages of this application will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0030] In order to more clearly and completely illustrate the technical solution of the present invention, the present invention will be further described below with reference to the accompanying drawings.

[0031] Please refer to Figure 1-Figure 7The present invention provides a method for preparing a cell-compounded sheep acellular dermal biological dressing, comprising the following steps:

[0032] S1. Obtain fresh sheep skin aged 3-6 months, soak the sheepskin surface with soap solution, and remove the wool from the sheepskin surface;

[0033] S2. The sheepskin was stripped of wool and the subcutaneous tissue was removed to obtain a skin graft having a thickness of 0.5-0.8 mm. The skin graft was then rinsed with distilled water and soaked in a new Gill solution for 30 min, repeated at least twice, and then soaked in hydrogen peroxide for 30 min, repeated at least twice to obtain a sterilized skin graft.

[0034] S3. Perform overnight digestion with 0.25% pancreatic enzyme solution at 37°C for 12 hours. The next day, wash the skin piece with distilled water and soak it in distilled water. Replace the distilled water every 12-24 hours.

[0035] S4. Two days later, 0.25% pancreatic enzyme solution was used for overnight digestion, and the samples were soaked again in distilled water, which was replaced every 12-24h for about ten days until the sheepskin cells were completely removed.

[0036] S5. Place the decellularized sheepskin in a freeze dryer until it is completely dry. Then, place the freeze-dried sheepskin in a clean bench and irradiate both sides of the freeze-dried sheepskin with ultraviolet light for 3 hours each to obtain a cell-reinforced sheep acellular dermal biological dressing.

[0037] In this embodiment, the cell-compounded sheep acellular dermal biological dressing is prepared from sheepskin by depilation, splitting, disinfection, decellularization, freeze-drying, sterilization and co-culture with cells. The first distilled water is sterile distilled water, and the second distilled water is sterile distilled water containing 1% biresistant antibiotics. The difference between the first distilled water and the second distilled water is that the second distilled water contains 1% biresistant 100U / mL penicillin and 100U / mL streptomycin. The step of completely removing sheepskin cells in step S4 includes: taking sheepskin from different parts for H&E staining to observe whether the cells are completely removed. If not, repeatedly using 0.25% mass fraction trypsin solution for overnight digestion until the cells are completely removed. See attached. Figure 1 -Attached Figure 2 , attached Figure 1 For sheepskin that has not completely removed cells, Figure 2 To completely remove the cells from the sheepskin, the sheepskin is completely dried in step S5. Figure 3 shown. Example

[0038] Cytotoxicity test of cell-compounded sheep acellular dermal biological dressing;

[0039] After the revived L929 cells were passaged twice, the third generation was used to test the cytotoxicity of the sheepskin extract. The cells were digested with trypsin and then neutralized with serum-containing culture medium. The cells were centrifuged at 1000 rpm for 5 minutes and resuspended in culture medium. The density was adjusted to 10 5 cells / mL, and 100 μL was taken with a density of 1×10 5 cells / mL (10 4 The cell suspension of 100 cells was plated in a 96-well cell culture dish, with a total of 30 wells inoculated (5 groups, 6 parallel experiments in each group). The carbon dioxide content was controlled at 5%, the temperature was 37°C, and the humidity was greater than 90% to ensure that the cells recovered and attached to exponential growth. Each well was checked under a microscope to ensure that the growth of cells in each well was relatively equal to reduce experimental errors. 10 mL of complete culture medium was placed in a 50 mL centrifuge tube, and the cell-compoundable sheep acellular dermal biological dressing in step S5 was punched into several 2 cm and 1.5 cm circular skin pieces (controlled weight of about 0.44 g). Place in culture medium and let it stand at 37°C for 24 hours to obtain the extract; after incubation for 24 hours, aspirate the culture medium and replace it. The experimental group used 100% extract and the control group used complete culture medium. Add 100 μL to each well; control the carbon dioxide content to 5%, the temperature to 37°C, and the humidity to greater than 90%. After incubating the cells for 24 hours, replace the new culture medium and add 20 μL of MTS reagent to each well. Control the carbon dioxide content to 5%, the temperature to 37°C, and the humidity to greater than 90%. Incubate in a cell culture incubator for 2 hours and use a microplate reader to detect the absorbance of each well at 490 nm as shown in the reference. Figure 4 As shown, the calculation formula for calculating the survival rate of cells in the extract is:

[0040] Survival rate (%) = 100 × OD of experimental group 490 / control group OD 490 ,

[0041] from Figure 4 It can be seen that 100% extract has no significant effect on cell proliferation, the cell survival rate is 95.2%, and the effect on cell growth is relatively small. Example

[0042] The cell-composite sheep acellular dermal biological dressing was co-cultured with cells to obtain a cell-composite sheep acellular dermal biological dressing; the freeze-dried and decellularized sheepskin in step S5 was cut into circular skin pieces with a diameter of about 1.5 cm, placed in PBS (phosphate buffered saline), then taken out and placed in a 12-well cell culture dish, the cells were digested with trypsin, neutralized with serum-containing culture medium, and then centrifuged at 1000 rpm for 5 minutes, the cells were resuspended in culture medium, and the density was adjusted to 10 6 cells / mL, 500 μL of cell resuspension was added to the culture dish with the skin slice and incubated for 24 h. The carbon dioxide content in the culture dish was controlled to be 5%, the temperature to be 37°C, and the humidity to be greater than 90%. The results were as follows: Figure 5 As shown, after 72 hours of culture, H&E staining was performed and the results were as follows Figure 6 shown. Example

[0043] The sheep decellularized dermal biological dressing after cell composite was observed by scanning electron microscopy. The cell-compounded sheep decellularized dermal biological dressing and the cell-compounded sheep decellularized dermal biological dressing obtained in Example 2 were fixed with a 2.5% glutaraldehyde solution, and the temperature was controlled at 4°C for 24 hours. Subsequently, a gradient alcohol solution was prepared, and the specimens were immersed in alcohol solutions with volume fractions of 30%, 50%, 75%, 90%, 100%, and 100% in sequence for dehydration, with each dehydration time being 20 minutes. A medical vacuum freeze dryer was used for freeze drying for about 12 hours. The cell-compounded sheep decellularized dermal biological dressing and the cell-compounded sheep decellularized dermal biological dressing were cut into 5 mm × 5 mm squares, adhered to the sample stage with conductive glue, placed in a vacuum coating machine for gold spraying, and placed in a scanning electron microscope for observation and photography. The results are shown in FIG. Figure 7 , where A is a sheep acellular dermis biological dressing that can be compounded with cells, B is a schematic diagram of a sheep acellular dermis biological dressing compounded with cells for 18 hours, and C is a schematic diagram of a sheep acellular dermis biological dressing compounded with cells for 7 days.

[0044] As can be seen from Examples 1-3, the cell-compounded sheep acellular dermal biological dressing provided by the present invention is non-cytotoxic, and at the same time, the gaps between the collagen fiber scaffolds are increased, allowing cells to enter the interior of the scaffold, which can provide a clinical dermal substitute that can carry cells and promote wound healing.

[0045] Of course, the present invention may have many other implementations. Based on this implementation, other implementations obtained by ordinary technicians in this field without any creative work are all within the scope of protection of the present invention.

Claims

1. A method for preparing a cell-compounded sheep acellular dermal biological dressing, characterized in that: The following steps are involved: S1. Obtain fresh sheep skin aged 3-6 months, soak the sheepskin surface with soap solution, and remove the wool from the sheepskin surface; S2. Remove the subcutaneous tissue from the sheepskin after removing the wool to obtain a skin graft with a thickness of 0.5-0.8 mm. Then rinse the skin graft with sterile distilled water, soak it in 0.1% syngeramine solution for 30 minutes, repeat at least twice, and then soak it in 3% hydrogen peroxide for 30 minutes, repeat at least twice, to obtain a sterilized skin graft. S3. Digest the skin overnight with a 0.25% pancreatic enzyme solution at 37°C for 12 hours. The next day, wash the skin piece with sterile distilled water containing 1% amphotericin. Then, soak the skin piece in sterile distilled water containing 1% amphotericin, changing the solution every 12-24 hours for two days. The amphotericin solution consists of 100 U / mL penicillin and 100 U / mL streptomycin. S4. Digest the sheepskin again with 0.25% trypsin solution overnight at 37°C for 12 hours. Then, continue soaking in sterile distilled water containing 1% biresistant strain, changing the solution every 12-24 hours for ten days. Verify that the sheepskin cells are completely removed by H&E staining. If not, repeat the overnight digestion with 0.25% trypsin solution until the cells are completely removed. S5. Place the clean sheepskin after removing cells in a freeze dryer until the sheepskin is completely dry. Then place the freeze-dried sheepskin in an ultra-clean workbench and irradiate both sides of the freeze-dried sheepskin with ultraviolet light for 3 hours each to obtain a cell-reinforced sheep acellular dermal biological dressing.

2. A cell-compounded sheep acellular dermis biological dressing, the preparation method of the cell-compounded sheep acellular dermis biological dressing according to claim 1, characterized in that: The cell-compoundable sheep acellular dermal biological dressing is prepared from sheepskin through depilation, splitting, disinfection, decellularization, freeze drying, sterilization and co-culturing with cells.

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