Acellular matrix biological dressing and preparation method thereof

Abstract

The invention discloses an extracellular matrix biological dressing and a preparation method thereof, and the preparation method of the extracellular matrix biological dressing comprises the following steps: removing cells and immunogenicity of pretreated organs and vascularized tissues by using a detergent until the DNA content is less than 100 ng / mg dry weight and the alpha-Gal clearance rate is greater than 90%; putting into a solution containing growth factors for treatment; the treated organ and the vascularized tissue are cross-linked by adopting a chemical reagent, after cleaning, the organ and the vascularized tissue are treated by adopting glycerol and an end-capping reagent, moisture is removed, after sterilization treatment, the acellular matrix biological dressing is obtained, the volume ratio of the organ after moisture removal to the organ before moisture removal is 0.5: 1-1: 1, and the pore size of the organ and the vascularized tissue is 15-500 microns.

Description

Technical Field

[0001] The present invention relates to the technical field of medical devices, and in particular to a decellularized extracellular matrix biological dressing and a preparation method thereof. Background Art

[0002] Skin is the first line of defense of the human body and is vulnerable to various external injuries, such as burns, mechanical trauma, and chronic diseases. Usually, mild and small-scale skin injuries can heal by themselves. When the skin wound is too large or the local environment of the wound is disordered, the wound cannot heal itself and needs to be solved by wound dressing.

[0003] Common dressings on the market can be divided into traditional gauze dressings, synthetic dressings, biological dressings, etc. Traditional medical dressings can no longer meet the treatment needs of chronic wounds and complex wounds, and the biocompatibility of synthetic dressings needs to be improved. For wound management, more efficient and effective care methods are needed.

[0004] Wound healing in diabetic foot is an extremely complex physiological process. The reasons why wounds in diabetic foot patients are difficult to heal involve the following points: 1) Weakened defense function of diabetic patients: In a high-sugar environment, leukocyte chemotaxis decreases, phagocytosis weakens, and lethality decreases, resulting in impaired immune function and low cell-mediated immunity in diabetic patients; 2) Impaired tissue repair function in diabetic patients: Due to abnormal insulin secretion and regulation in the body, diabetic patients affect the body's sugar metabolism and reduce sugar utilization. The healing of incision tissue requires protein and other tissue factors from the growth of granulation tissue to the formation of collagen fibers, but diabetic patients have abnormal sugar utilization and cannot effectively convert glucose into protein and other substances. Tissue hyperglycemia leads to hyperosmotic tissue cells, which is not conducive to cell survival. All of these lead to impaired tissue regeneration and repair functions, resulting in delayed wound healing; 3) Neurovascular disorders in diabetic patients: The blood sugar level of diabetic patients increases, which promotes the deposition of glucose oxides in tissue collagen. Oxidative stress can easily cause tissue damage and cell death, leading to the occurrence of diabetic complications, causing microvascular lesions, and damaging blood vessels and nerves; 4) Hyperglycemia can easily lead to wound infection: There are glycosylated residues in diabetic wounds. Combined with the symptoms of hyperglycemia, the ability of macrophages in the wound to transform from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype is weakened, resulting in the enrichment of M1 macrophages and the strengthening of a harsh microenvironment (long-term inflammation, intense proteolysis, and excessive oxidative stress).

[0005] The treatment principles for wound healing in diabetic foot are: 1) Wound debridement, removal of necrotic tissue in ulcerated wounds, and reduction of the possibility of further spread of the wound; 2) Wound anti-infection treatment. If the infected surface is not effectively controlled, it will rapidly develop and cause amputation and serious life-threatening; 3) Improve blood microcirculation in the wound, increase blood supply to body tissues, reduce hypoxia, promote nerve repair and capillary regeneration, and thus improve blood circulation in the lower limbs of diabetic foot; 4) Tissue regeneration. The use of dressings or "target" drug administration allows the pathological fluid in the wound to be continuously drawn out, the physiological body fluid to be continuously renewed, the wound granulation tissue to be rapidly produced, and the tissue to regenerate, thereby achieving the effect of wound healing.

[0006] Among the above-mentioned treatment principles for diabetic foot, traditional dressings and common biomaterial dressings on the market can basically achieve the effect of anti-infection of wounds, but improving blood microcirculation in the wound and accelerating tissue regeneration are clinical problems that biological dressings have not yet solved.

[0007] Biological dressings can be divided into two categories: artificial biological dressings and biological allogeneic tissue wound coverings. Common artificial biological dressings are made of materials such as collagen, chitosan, and hyaluronic acid. They have hemostatic and coagulation effects and can induce proliferation and differentiation of multiple cells. Their disadvantages are poor stability and weak ability to absorb exudate. The tissue of biological dressings is similar to that of human collagen and has good compatibility. Implantation or covering of wounds does not cause adverse reactions with the body. It can treat or replace defective tissues in the body and promote wound healing and repair. Biological allogeneic tissue wound coverings include pig skin, frog skin, fish skin, etc., such as glutaraldehyde cross-linked pig skin and irradiated pig skin. However, the traditional glutaraldehyde fixation and chrome tanning treatment technology does not specifically remove antigens, has residual aldehyde toxicity, incomplete antigen removal, weak rejection reaction, poor dry softness, and enlarged pores after drying, poor bacterial isolation, and unsatisfactory performance and treatment effects.

[0008] In addition, low-temperature large-area burns, bedsores, poor healing of surgical incisions, external trauma, etc. are known as "difficult-to-heal wounds" and are in urgent need of new dressings for clinically effective body repair.

[0009] Therefore, in view of the blank area of ​​the prior art, the purpose of the present invention is to provide a method for preparing a biological dressing. Summary of the invention

[0010] The main purpose of the present invention is to provide a decellularized extracellular matrix biological dressing and a preparation method thereof, aiming to solve the above problems.

[0011] To achieve the above object, the present invention provides a method for preparing a decellularized extracellular matrix biological dressing, the method comprising the following steps: The pretreated organs and vascularized tissues were treated with a solution containing growth factors after removing cells and immunogenicity using detergents until the DNA content was <100 ng / mg (dry weight) and the α-Gal clearance rate was >90%. The organ treated with growth factors and the vascularized tissue are cross-linked with chemical reagents, treated with glycerol and a capping agent after washing and moisture is removed, and sterilized to obtain a decellularized extracellular matrix biological dressing, wherein the volume ratio after moisture removal to that before moisture removal is 0.5:1~1:1, and the pores between the organ and the vascularized tissue are 15~500um.

[0012] Preferably, in the method for preparing the decellularized extracellular matrix biological dressing, the steps of cross-linking the organ treated with growth factors and the vascularized tissue using chemical reagents, treating with glycerol and a capping agent after washing and removing moisture, and sterilizing to obtain the decellularized extracellular matrix biological dressing include: The organ treated with the growth factor is cross-linked with the vascularized tissue by using a chemical reagent, and after washing, it is treated with glycerol and a capping agent and moisture is removed under a first condition, and sterilized to obtain a decellularized extracellular matrix biological dressing; the first condition includes a temperature of -80° to 40°C and normal pressure or below atmospheric pressure; The water content of organs and vascularized tissues treated with glycerol and capping agents ranged from 0.5% to 50%; The sterilization method adopts one or more combinations of irradiation sterilization, ethylene oxide sterilization, and chemical liquid sterilization.

[0013] Preferably, in the method for preparing the acellular extracellular matrix biological dressing, in the step of treating the pretreated organ and vascularized tissue with a solution containing growth factors after removing cells and immunogenicity using a detergent to reduce the DNA content to less than 100 ng / mg dry weight and the α-Gal clearance rate to more than 90%, the growth factor is one or more of αFGF, βFGF, EGF, TGFα, TGFβ, and PDGF; The solution containing the growth factor is prepared by mixing one or more of bovine serum, PBS, purified water and cell culture medium.

[0014] Preferably, in the preparation method of the decellularized extracellular matrix biological dressing, in the step of cross-linking the organ treated with growth factors and the vascularized tissue using chemical reagents, treating with glycerol and a capping agent after washing and removing moisture, and obtaining the decellularized extracellular matrix biological dressing after sterilization, the capping agent is one or more of aminosulfonates, sodium borohydride, cyanoborohydride, and sodium bisulfite.

[0015] Preferably, in the method for preparing the decellularized extracellular matrix biological dressing, the growth factors are cross-linked with the treated organs and vascularized tissues using chemical reagents, and after washing, they are treated with glycerol and a capping agent to remove moisture, and sterilized to obtain the decellularized extracellular matrix biological dressing, in the step, the chemical reagents include glutaraldehyde and epoxide.

[0016] Preferably, in the method for preparing the decellularized extracellular matrix biological dressing, the detergent comprises a mixture of at least three of anionic detergents, cationic detergents, zwitterionic detergents, and nonionic detergents; The anionic detergent includes one or more of sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecyl sulfonate, sodium taurocholate, water, sodium cholate, ursodeoxycholic acid, and sodium deoxycholate; The cationic detergent comprises one or more of cetyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, benzalkonium bromide and / or cetyltrimethylammonium bromide; The zwitterionic detergent comprises one or more of aminosulfonyl betaine, 3-sulfopropyl tetradecyl dimethyl ammonium, 3-sulfopropyl hexadecyl dimethyl ammonium, 3-[(3-cholamidopropyl)-dimethylammonium]-1-propanesulfonic acid inner salt, and n-tetradecyl-N,N-dimethyl-3-ammonium-1-propane sulfonate; The nonionic detergent includes one or more of 5-cyclohexylpentyl β-D-maltoside, 5-cyclohexylpentyl β-D-maltoside, decyl glucopyranoside, digitonin, poloxamer 188, N-decanoyl-N-methylglucamine, Triton, and Tween.

[0017] Preferably, in the method for preparing the acellular extracellular matrix biological dressing, in the step of removing cells and immunogenicity from the pretreated organ and vascularized tissue using a detergent until the DNA content is less than 100 ng / mg dry weight and the α-Gal clearance rate is greater than 90%, and then treating with a solution containing growth factors, the treatment conditions for removing cells include treatment temperature and treatment time; The treatment temperature is 20°C to 38°C; the treatment time is 3 to 72 hours; the chemical reagent is replaced every 4 to 24 hours during the decellularization process; Preferably, in the method for preparing the decellularized extracellular matrix biological dressing, after the pretreated organ and vascularized tissue are treated with a detergent to remove cells and immunogenicity until the DNA content is less than 100 ng / mg dry weight and the α-Gal clearance rate is greater than 90%, before the step of treating with a solution containing growth factors, the method for preparing the decellularized extracellular matrix biological dressing further comprises: Organs and vascularized tissues are removed from mammals, and foreign matter outside the organs and vascularized tissues is removed and washed.

[0018] Preferably, in the method for preparing the acellular extracellular matrix biological dressing, in the process of removing organs and vascularized tissues from the body of a mammal, removing impurities outside the organs and vascularized tissues and washing them, the removed organs include one or more of the heart, liver, kidney, spleen, pancreas, bladder, lung, large intestine, small intestine, stomach, brain, and lung; The removed vascularized tissue includes one or more of the heart, liver, kidney, spleen, pancreas, bladder, lung, large intestine, small intestine, stomach, brain, and lung of a mammal; The mammals include one or more of humans, cows, horses, sheep, pigs, rabbits, dogs, monkeys, cats, and rodents.

[0019] In order to achieve the above object, the present invention further provides a decellularized extracellular matrix biological dressing, which is prepared by the above-mentioned decellularized extracellular matrix biological dressing preparation method.

[0020] Preferably, in the acellular extracellular matrix biological dressing, the acellular extracellular matrix biological dressing comprises collagen or collagen and elastin; and also comprises a small amount of fibronectin, fibrin, laminin, extracellular matrix-related growth protein, and glycosaminoglycan; Among them, extracellular matrix-related growth proteins include growth factors and cytokines.

[0021] Preferably, in the acellular extracellular matrix bio-type dressing, the acellular extracellular matrix bio-type dressing is cut into blocks and porous sheets of different sizes for wound management, including deep wounds, diabetic foot ulcer wounds, traumatic wounds, surgical wounds, pressure sores, venous ulcers, and chronic vascular ulcers.

[0022] The present invention has at least the following beneficial effects: The invention provides a method for preparing a decellularized extracellular matrix biological dressing, comprising the following steps: using a detergent to remove cells and immunogenicity from pretreated organs and vascularized tissues until the DNA content is less than 100 ng / mg dry weight and the α-Gal clearance rate is greater than 90%, and then treating the pretreated organs and vascularized tissues with a solution containing growth factors; cross-linking the treated organs and vascularized tissues with chemical reagents, washing the treated organs and vascularized tissues with glycerol and a capping agent to remove moisture, and sterilizing the pretreated organs to obtain the decellularized extracellular matrix biological dressing, wherein the volume ratio after the moisture removal is to that before the moisture removal is 0.5:1-1:1, and the pores of the organs and vascularized tissues are 15-500 um. The decellularized extracellular matrix biological dressing obtained in this way has no immune response when implanted in vivo and has good biocompatibility.

[0023] Furthermore, the biological dressing provided by the present invention has excellent biocompatibility after being implanted in the body, has no immune rejection reaction, is anti-infective, can be recellularized, has excellent mechanical properties, and has a certain plasticity.

[0024] Furthermore, compared with other decellularization technologies, the decellularization method used in the present invention retains more original microstructures of the ECM of organs or tissues (such as visceral organs), including complete blood vessels and / or microvascular systems; the perfused decellularized ECM from visceral organs retains collagen content, other binding and signaling factors, and vascular structure, thereby providing an environment for the re-endothelialization of introduced cells. The mild decellularization method removes cells without changing the extracellular matrix and the structure of visceral organs, so that the structure and integrity of visceral organs are not affected, and the immunogenicity of blood vessels after decellularization is reduced. Finally, the water in the viscera is removed without changing the structural collapse and protein of the visceral organs, which is conducive to the re-cellularization of visceral organs after implantation in the body. This method has a short preparation cycle and low cost, and the product can be used in multiple damaged parts in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 A schematic diagram of a process of one embodiment of a method for preparing acellular extracellular matrix biological dressing provided by the present invention; Figure 2a This is a diagram showing the detection results of the biological dressing in Example 1 of the present invention when DAPI staining analysis is performed; Figure 2b This is a diagram showing the test results of the biological dressing in Example 1 of the present invention when performing H&E staining histological analysis; Figure 3a This is a product picture of the biological dressing of different sizes after cutting in Example 1 of the present invention; Figure 3b This is a product picture of a sterilized dressing packaged in Example 1 of the present invention; Figure 4a This is a test result diagram of the biological dressing in Comparative Example 1 of the present invention when DAPI staining analysis is performed; Figure 4b This is a graph showing the test results of the biological dressing in Comparative Example 1 of the present invention when performing H&E staining histological analysis; Figure 5 This is a physical picture of the biological dressing after cutting in Comparative Example 1 of the present invention; Figure 6 This is a photo of the biological dressing after cutting in Example 2 of the present invention; Figure 7a , Figure 7b , Figure 7c , Figure 7d The diabetic mice in the control group that did not use dressings had their backs shaved before surgery, 1 / 2 of the back skin removed, the affected area fixed with sterile gauze after surgery, and the wound healing status 21 days after surgery; Figure 8a , Figure 8b , Figure 8c , Figure 8d In the present invention, the back of the diabetic mice in the experimental group was shaved after anesthesia, 1 / 2 of the back area was peeled, and the biological dressing was used for fixation and repair. About 90% of the back area of ​​the diabetic mice healed 21 days after the operation. Fig. 9 Comparison of wound healing rates between diabetic mice in the control group and diabetic mice in the experimental group; Fig.10 Comparison of the content of M1 and M2 macrophages in the wound tissue of mice in the control group after surgery; Fig.11 Comparison of the content of M1 and M2 macrophages in the wound tissue of diabetic mice in the postoperative experimental group.

[0027] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0028] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0029] Figure 1 The present invention provides a schematic flow chart of an embodiment of the method for preparing the decellularized extracellular matrix biological dressing. Figure 1 As shown, Figure 1In step S100, organs and vascularized tissues are removed from the mammal, and impurities outside the organs and vascularized tissues are removed and washed.

[0030] The organs and vascularized tissues are removed from the body of a mammal, and foreign matter outside the organs and vascularized tissues is removed and cleaned. The organs removed include one or more of the heart, liver, kidney, spleen, pancreas, bladder, lung, large intestine, small intestine, stomach, brain, and lung; the vascularized tissues removed include one or more of the heart, liver, kidney, spleen, pancreas, bladder, lung, large intestine, small intestine, stomach, brain, and lung of a mammal; and the mammal includes one or more of humans, cattle, horses, sheep, pigs, rabbits, dogs, monkeys, cats, and rodents.

[0031] It should be noted that the sources of the above raw materials must comply with the national animal breeding standards for animal-derived medical devices.

[0032] In step S200, the pretreated organs and vascularized tissues are treated with a solution containing growth factors after using a detergent to remove cells and immunogenicity until the DNA content is less than 100 ng / mg (dry weight) and the α-Gal clearance rate is greater than 90%.

[0033] The treatment conditions for removing cells include treatment temperature and treatment time. The treatment temperature is 20°C to 38°C (in other embodiments, the treatment temperature can also be 25°C, 30°C, and 35°C); the treatment time is 3 to 72 hours (in other embodiments, the treatment time can also be 10 hours, 20 hours, 30 hours, 40 hours, or 50 hours); during the decellularization process, the chemical reagent is replaced every 4 to 24 hours (in other embodiments, the interval time can also be 10 hours, 15 hours, or 20 hours).

[0034] The detergent comprises at least three of anionic detergent, cationic detergent, zwitterionic detergent and nonionic detergent. The anionic detergent comprises one or more of sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecyl sulfonate, sodium taurocholate, water and sodium cholate, ursodeoxycholic acid and sodium deoxycholate.

[0035] The cationic detergent includes one or more of hexadecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, benzalkonium bromide and / or hexadecyltrimethylammonium bromide. The zwitterionic detergent includes one or more of aminosulfobetaine, 3-sulfopropyl tetradecyldimethylammonium, 3-sulfopropyl hexadecyldimethylammonium, 3-[(3-cholamidopropyl)-dimethylammonium]-1-propanesulfonic acid inner salt, n-tetradecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate.

[0036] The nonionic detergent includes one or more of 5-cyclohexylpentyl β-D-maltoside, 5-cyclohexylpentyl β-D-maltoside, decyl glucopyranoside, digitonin, poloxamer 188, N-decanoyl-N-methylglucamine, Triton, and Tween.

[0037] The growth factor is one or more of αFGF, βFGF, EGF, TGFα, TGFβ, and PDGF. The solvent for preparing the solution containing the growth factor is a mixture of one or more of bovine serum, PBS, purified water, and cell culture medium.

[0038] In step S300, the organ treated with the growth factor and the vascularized tissue are cross-linked with a chemical reagent, washed, treated with glycerol and a capping agent, and dehydrated, and sterilized to obtain a decellularized extracellular matrix biological dressing, wherein the volume ratio after dehydration to that before dehydration is 0.5:1 to 1:1, and the pores of the organ and the vascularized tissue are 15 to 500 um. Preferably, the chemical reagent includes glutaraldehyde and epoxide.

[0039] More specifically, the treated organ and vascularized tissue are cross-linked with chemical reagents, treated with glycerol and a capping agent after washing, and water is removed under a first condition, and sterilized to obtain a decellularized extracellular matrix biological dressing; the first condition includes a temperature of -80° to 40°C and normal pressure or below atmospheric pressure; the moisture content of the organ and vascularized tissue treated with glycerol and a capping agent is 0.5% to 50%; The sterilization method adopts one or more combinations of irradiation sterilization, ethylene oxide sterilization, and chemical liquid sterilization.

[0040] The volume ratio after water removal to that before water removal can also be 0.6:0.8, or 0.7:1. The pores of the organs and vascularized tissues can also be 50um, 100um, or 150um. The water content of the organs and vascularized tissues treated with glycerol and a capping agent can also be 1%, 10%, 20%, 30%, or 40%. The temperature in the first condition can also be -70°C, -50°C, -10°C, 20°C, or 30°C.

[0041] In addition, the end-capping agent is one or more of aminosulfonate, sodium borohydride, cyanoborohydride, and sodium bisulfite.

[0042] The acellular extracellular matrix biological dressing is cut into blocks and porous sheets of different sizes for wound management, including deep wounds (diabetic ulcer wounds), pressure sores, venous ulcers, chronic vascular ulcers, diabetic foot ulcer wounds, traumatic wounds (abrasions, lacerations, second-degree burns, skin tears), surgical wounds, pressure sores, venous ulcers, and chronic vascular ulcers.

[0043] The components of biological dressings include collagen or collagen and elastin; they also include small amounts of fibronectin, fibrin, laminin, extracellular matrix-related growth proteins (including growth factors and cytokines), glycosaminoglycans, matrix substances, reticular fibers and thrombospondin.

[0044] In order to verify the beneficial effects of the method for preparing the acellular extracellular matrix biological dressing provided by the present invention, examples and comparative examples are provided for comparison and explanation.

[0045] Example 1

[0046] S1, Obtain livers from healthy pigs weighing 200 kg from a slaughterhouse. Use medical scissors to carefully remove fat and connective tissue outside the liver without damaging the liver membrane; S2, the liver was placed in 40mmol / L ursodeoxycholic acid, 150mmol / L ammonium bromide, 75mmol / L 3-sulfopropyl hexadecyl dimethyl ammonium solution, and 10mmol / L Triton solution at 25℃ for 72h, and the solution was changed every 12h until the DNA content was <100ng / mg dry weight and the α-Gal clearance rate was >90%; the liver was treated with 100ng / ml βFGF solution prepared with bovine serum for 15h; S3, cross-linked with 0.625% glutaraldehyde at 25°C, washed with PBS for 48 hours; the liver soaked in growth factors was soaked in glycerol and 5% sodium borohydride solution for 4 hours, the liver was inverted and the surface moisture was dried, pre-frozen at -20°C for 3 hours, and freeze-dried at -60°C for 72 hours.

[0047] S4, Histological analysis of liver samples ( Figure 2a The figure shows the test result of the biological dressing in Example 1 of the present invention when DAPI staining analysis is performed; Figure 2b The results of the H&E staining histological analysis of the biological dressing in Example 1 of the present invention are shown in Figure 1). There are no residual nuclei in the DAPI staining and HE staining images, the residual DNA is 32.1±0.62ng / mg, and the a-gal clearance rate is 95.97%. The physical properties of the freeze-dried tissue were tested, and the axial tensile strength was 2.34±0.12N; after tissue freeze-drying: freeze-dried precursor 0.93:1, SEM characterization of the dressing surface pores is 15~100um, and the water content is 3.13±0.08%.

[0048] S5, cut the freeze-dried liver into blocks of varying volumes of 2*2cm~2*10cm according to clinical use requirements ( Figure 3a The product diagram of the bio-type dressing of different sizes after cutting in Example 1 of the present invention is shown), packaged in a sealed bag, and sterilized by irradiation to obtain the bio-type dressing ( Figure 3b The figure shows the sterilized dressing product of Example 1 of the present invention after packaging, which is used for the treatment of difficult-to-heal wounds such as diabetic foot and surgical wounds.

[0049] Example 2

[0050] S1, obtain the kidney of a healthy cow weighing 300 kg from a slaughterhouse. Use medical scissors to carefully remove the fat and connective tissue outside the kidney without damaging the liver membrane; S2, the kidneys were placed in a solution of 40 mmol / L tetradecyltrimethylammonium bromide, 320 mmol / L 3-[(3-cholamidopropyl)-dimethylammonium]-1-propanesulfonic acid inner salt, and 150 mmol / L decylpyranoglucoside at 37°C for 12 h, and the solution was changed every 6 h until the DNA content was <100 ng / mg dry weight and the α-Gal clearance rate was >90%; the kidneys were placed in a solution of 650 ng / ml PDGF at 37°C for 12 h; S3, the cross-linked kidney was washed with PBS for 4 hours, and then cross-linked with 10% 1,4-butanediol diglycidyl ether solution at 25°C for 24 hours; the treated kidney tissue was treated with glycerol and 8% borohydride for 5 hours, pre-frozen at -20°C for 3 hours, and freeze-dried at -60°C for 48 hours; S4, the residual DNA content of the kidney sample was 45.72±0.56ng / mg, and the a-gal clearance rate was 96.21%; the volume ratio of the kidney tissue before and after freeze-drying was 0.84:1, and SEM characterized the surface pores of the dressing as 45-90um, the water content was 2.42±0.12%, and the axial tensile strength was 1.86±0.24N.

[0051] S5, cut the freeze-dried kidney into 2*2cm~2*10cm blocks, seal and package them, and sterilize them with ethylene oxide to obtain biological dressings for the repair of diabetic foot, deep wounds, and chronic vascular ulcers.

[0052] Example 3

[0053] S1, Obtain the pancreas of a healthy sheep weighing 100 kg from a slaughterhouse. Use medical scissors to carefully remove the fat and connective tissue outside the pancreas without damaging the pancreatic outer membrane; S2, the pancreas was treated with 300mmol / L sodium taurocholate, 100mmol / L 3-sulfopropyl tetradecyl dimethyl ammonium, and 40mmol / L 5-cyclohexylpentyl β-D-maltoside solution at 25°C for 36h, and the solution was changed every 6h until the DNA content was <100ng / mg dry weight and the α-Gal clearance rate was >90%. The pancreas was placed in 5% TGFα solution and treated at 37°C for 24h; S3, cross-linked with 3.5% ethylene glycol diglycerol ether at 37°C for 24h, the cross-linked pancreas was washed with PBS for 4h; the treated pancreas was treated with glycerol and 10% sodium borohydride solution for 6h; and dried at 20°C for 72h; S4, the residual DNA content of the pancreatic sample was 21.41±0.86ng / mg, the a-gal clearance rate was 94.17%, the volume ratio of the pancreas before and after water removal was 0.79:1, SEM characterization of the dressing surface pores was 20-150um, the water content was 3.14±0.35%, and the axial tensile strength was 2.31±0.23N.

[0054] S5, the pancreas is cut after drying and sterilized with chemical liquid before being used for pressure sores, traumatic wounds, and surgical wounds.

[0055] Comparative Example 1 S1, Obtain liver from a 200 kg pig from a slaughterhouse, healthy and quarantine-qualified. Carefully remove fat and connective tissue outside the pancreas using medical scissors without damaging the liver membrane; S2, the liver was immersed in 30mmol / L taurocholic acid and 100mmol / L Tween solution at 25℃ for 48h, and the solution was changed every 24h; 0.625% glutaraldehyde was used for cross-linking at 25℃ for 48h; S3, after washing with 0.9% NaCl solution, the liver was placed in a 0.5 mg / ml αFGF growth factor solution prepared in normal saline and treated at 37°C for 10 h; the treated liver was treated with glycerol and 3% sodium bisulfite solution for 12 h, and dried under negative pressure at 25°C for 10 h; S4, the residual DNA content in liver tissue was 178.21±1.65ng / mg, the a-gal clearance rate was 75.64%, and DAPI staining ( Figure 4a The results of DAPI staining analysis of the biological dressing in Comparative Example 1 of the present invention are shown in FIG), H&E staining ( Figure 4b The test results of the biological dressing in comparative example 1 of the present invention when H&E staining histological analysis are performed are shown in Figure 1). A large number of cell nuclei remain in the liver tissue, the pores of the liver tissue are 1-10um, the volume of the liver after water removal: the volume before water removal is 0.59:1, the tissue water content is 10.14±0.46%, the axial tensile strength is 0.51±0.13N, and the cut tissue is as follows Figure 5 As shown, the tissue surface is denser and has fewer pores. It is compacted during the drying process in the height direction and has a higher density.

[0056] Comparative Example 2 S1, small intestinal mucosa of pigs weighing 200 kg was obtained from a slaughterhouse, and the small intestinal outer membrane and other tissues were removed, and the small intestinal submucosal layer was separated; S2, the submucosa of the porcine small intestine was treated in a solution of 100 mmol / L sodium deoxycholate and 50 mmol / L benzalkonium bromide at 37°C for 48 h, with the solution replaced every 12 g, and cross-linked with 1,4-butanediol diglycidyl ether at 25°C for 24 h; S3, after washing with purified water, the small intestinal submucosal layer was treated with 1% TGFα solution at 37°C for 12 h; the treated small intestinal mucosa was treated with glycerol and 3% sodium borohydride for 3 h, and dried at 20°C under normal pressure for 48 h; S4, the residual DNA content of the small intestinal mucosa was 216.63±1.25ng / mg, the a-gal clearance rate was 69.48%, the volume ratio of the small intestinal mucosa before and after water removal was 0.46:1, the pore size of the small intestinal mucosa surface was 5-17um, the water content was 17.12±0.63%, and the axial tensile strength was 0.42±0.04N.

[0057] The test methods for the various effects of the biological dressings provided in the above embodiments and comparative examples are as follows: (1) DAPI staining Take 5 vm of the tissue obtained in the embodiment and the comparative example, place at room temperature for 10 minutes; fix with PFA for 5 minutes, wash with PBS; add DAPI staining solution for 5 minutes; wash with PBS, and then seal with sealing agent; Observe under fluorescence microscope.

[0058] (2) Axial tensile mechanical properties The test was carried out according to the method in standard YY / T 1511-2017, with a dressing width of 1 cm, a clamp spacing of 3 cm, a tensile rate of 5 mm / min, and an axial tensile strength of not less than 0.6 N.

[0059] (3) SEM sample preparation method The biological dressing was washed three times with saline and fixed in glutaraldehyde solution for 3 hours; Wash three times with 0.1mol PBS buffer, 10-15min each time; Osmic acid fixation for 1 hour; wash with buffer 3 times, 10-15 minutes each time; Rinse with 30% tert-butanol (alcohol solvent) for 10 min, rinse with 50% tert-butanol (alcohol solvent) for 10 min, rinse with 70% tert-butanol (alcohol solvent) for 10 min, rinse with 80% tert-butanol (alcohol solvent) for 10 min, rinse with 90% tert-butanol (alcohol solvent) for 10 min, rinse with 100% tert-butanol (alcohol solvent) 3 times, 10 min each time, and store at 4°C for later use.

[0060] The volume ratio in Table 1 is the volume ratio after water removal to that before water removal; the pores are the pores of organs and vascularized tissues.

[0061] Table 1 Comparison of effects of embodiments and comparative examples

[0062] Furthermore, in order to further verify the beneficial effects of the present invention, the following experiment is used: (1) Refer to the standard test model for performance evaluation of contact wound dressings (YY / T 1476-2020) to evaluate the healing effect of biological dressings on type 2 diabetic mice; (2) 6-8 week old db / db mice with blood glucose ≥ 18 mmol / L were selected; mice were given 10% water and chloral at 0.3-0.4 mg / kg body weight, anesthetized and hair removed from the back, and iodine disinfected and 2*2 cm skin was removed from 1 / 2 of the back. The experimental group used the 2*2 cm (2*2 cm) skin from Example 2. Figure 6 ) Biological dressings promoted the repair of the affected area. The control group did not use dressings, and sterile gauze was used to fix the dressings and the affected area. The gauze was changed every 2 days. There were 10 diabetic mice in the control group and 10 diabetic mice in the experimental group. (3) Photos were taken on the 1st, 3rd, 7th, 14th, and 21st days after surgery, and the wound area was calculated using the image area software. The wound healing rate R was used to evaluate the healing rate of the biological dressing. The content of anti-inflammatory M1 macrophages and pro-repair M2 macrophages was compared to evaluate the in vivo conditions of the blank control group and the experimental group using dressings for repair after surgery. R=(A0-At) / A0, where A0 is the initial wound area and At is the wound area at time t.

[0063] The backs of diabetic mice in the control group were shaved before surgery without using dressings. Figure 7a )、1 / 2 of the back area peeled ( Figure 7b ), postoperative sterile gauze fixation of the affected area ( Figure 7c ), 21 days after surgery, only about 10% of the back of the control group healed ( Figure 7d ); The backs of diabetic mice in the experimental group using biological dressings were shaved after anesthesia ( Figure 8a )、1 / 2 of the back area peeled ( Figure 8b ), fix and repair the affected area with biological dressing ( Figure 8c ), about 90% of the back area of ​​the diabetic mice in the experimental group healed 21 days after surgery ( Figure 8d ); The healing rate at different time points is as follows Fig. 9 The wound healing rates of the experimental group were 15%±3%, 36%±4%, 68%±2%, and 92%±5% at 3, 7, 14, and 21 days after surgery, respectively. The healing rates showed a trend of gradual increase with time. The numbers of M1 and M2 macrophages in the control group and the experimental group were significantly higher than those in the control group ( Fig.10 , Fig.11), experiments have shown that biological dressings have a significant effect in promoting the repair of difficult-to-heal wounds.

[0064] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A method for preparing a cell-free extracellular matrix biological dressing, characterized in that: The preparation method of the decellularized extracellular matrix biological dressing comprises the following steps: The pretreated organs and vascularized tissues were treated with a solution containing growth factors after removing cells and immunogenicity using detergents to reduce DNA content to <100 ng / mg dry weight and α-Gal clearance to >90%. The organ treated with growth factors and the vascularized tissue are cross-linked with chemical reagents, treated with glycerol and a capping agent after washing and moisture is removed, and sterilized to obtain a decellularized extracellular matrix biological dressing, wherein the volume ratio after moisture removal to that before moisture removal is 0.5:1~1:1, and the pores between the organ and the vascularized tissue are 15~500um.

2. The method for preparing the acellular extracellular matrix biological dressing according to claim 1, characterized in that: The steps of cross-linking the organ treated with growth factors with the vascularized tissue using chemical reagents, treating with glycerol and a capping agent after washing and removing moisture, and sterilizing to obtain the decellularized extracellular matrix biological dressing include: The treated organ and vascularized tissue are cross-linked with chemical reagents, washed, treated with glycerol and a capping agent, and dehydrated under a first condition, and sterilized to obtain a decellularized extracellular matrix biological dressing; the first condition includes a temperature of -80° to 40°C and normal pressure or below atmospheric pressure; The water content of organs and vascularized tissues treated with glycerol and capping agents ranged from 0.5% to 50%; The sterilization method adopts one or more combinations of irradiation sterilization, ethylene oxide sterilization, and chemical liquid sterilization.

3. The method for preparing the acellular extracellular matrix biological dressing according to claim 1, characterized in that: In the step of using a detergent to remove cells and immunogenicity from the pretreated organ and vascularized tissue until the DNA content is less than 100 ng / mg dry weight and the α-Gal clearance rate is greater than 90%, and then treating with a solution containing growth factors, the growth factor is one or more of αFGF, βFGF, EGF, TGFα, TGFβ, and PDGF; The solution containing the growth factor is prepared by mixing one or more of bovine serum, PBS, purified water and cell culture medium.

4. The method for preparing the acellular extracellular matrix biological dressing according to claim 1, characterized in that: In the step of cross-linking the organ treated with growth factors with the vascularized tissue using chemical reagents, treating with glycerol and a blocking agent after washing and removing moisture, and sterilizing to obtain the decellularized extracellular matrix biological dressing, the blocking agent is one or more of aminosulfonate, sodium borohydride, cyanoborohydride, and sodium bisulfite.

5. The method for preparing the acellular extracellular matrix biological dressing according to claim 1, characterized in that: In the step of cross-linking the organ treated with growth factors with the vascularized tissue using chemical reagents, treating with glycerol and a capping agent after washing and removing moisture, and sterilizing to obtain the decellularized extracellular matrix biological dressing, the chemical reagents include glutaraldehyde and epoxide.

6. The method for preparing the acellular extracellular matrix biological dressing according to claim 5, characterized in that: The pretreated organs and vascularized tissues are treated with a solution containing growth factors after removing cells and immunogenicity with a detergent until the DNA content is less than 100 ng / mg dry weight and the α-Gal clearance rate is greater than 90%, wherein the detergent comprises a mixture of at least three of anionic detergents, cationic detergents, zwitterionic detergents, and non-ionic detergents; The anionic detergent includes one or more of sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecyl sulfonate, sodium taurocholate, water, sodium cholate, ursodeoxycholic acid, and sodium deoxycholate; The cationic detergent comprises one or more of cetyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, benzalkonium bromide and / or cetyltrimethylammonium bromide; The zwitterionic detergent comprises one or more of aminosulfonyl betaine, 3-sulfopropyl tetradecyl dimethyl ammonium, 3-sulfopropyl hexadecyl dimethyl ammonium, 3-[(3-cholamidopropyl)-dimethylammonium]-1-propanesulfonic acid inner salt, and n-tetradecyl-N,N-dimethyl-3-ammonium-1-propane sulfonate; The nonionic detergent includes one or more of 5-cyclohexylpentyl β-D-maltoside, 5-cyclohexylpentyl β-D-maltoside, decyl glucopyranoside, digitonin, poloxamer 188, N-decanoyl-N-methylglucamine, Triton, and Tween.

7. The method for preparing the acellular extracellular matrix biological dressing according to claim 5, characterized in that: In the step of using a detergent to remove cells and immunogenicity from the pretreated organ and vascularized tissue until the DNA content is less than 100 ng / mg dry weight and the α-Gal clearance rate is greater than 90%, and then treating with a solution containing growth factors, the treatment conditions for removing cells include treatment temperature and treatment time; The treatment temperature is 20°C to 38°C; the treatment time is 3 to 72 hours; and the chemical reagent is replaced every 4 to 24 hours during the decellularization process.

8. The method for preparing the acellular extracellular matrix biological dressing according to claim 1, characterized in that: After the pretreated organ and vascularized tissue are treated with a detergent to remove cells and immunogenicity until the DNA content is less than 100 ng / mg dry weight and the α-Gal clearance rate is greater than 90%, before the step of treating with a solution containing growth factors, the method for preparing the decellularized extracellular matrix biological dressing also includes: Organs and vascularized tissues are removed from mammals, and foreign matter outside the organs and vascularized tissues is removed and washed.

9. The method for preparing the acellular extracellular matrix biological dressing according to claim 8, characterized in that: The organs and vascularized tissues are removed from the mammal, and foreign matter outside the organs and vascularized tissues is removed and cleaned, wherein the removed organs include one or more of the heart, liver, kidney, spleen, pancreas, bladder, lung, large intestine, small intestine, stomach, brain, and lung; The removed vascularized tissue includes one or more of the heart, liver, kidney, spleen, pancreas, bladder, lung, large intestine, small intestine, stomach, brain, and lung of a mammal; The mammals include one or more of humans, cows, horses, sheep, pigs, rabbits, dogs, monkeys, cats, and rodents.

10. A decellularized extracellular matrix biological dressing, characterized in that: The decellularized extracellular matrix biological dressing is prepared by the preparation method of the decellularized extracellular matrix biological dressing according to any one of claims 1 to 9.

11. The acellular extracellular matrix biological dressing according to claim 10, characterized in that: The acellular extracellular matrix biological dressing contains collagen or collagen and elastin; it also contains a small amount of fibronectin, fibrin, laminin, extracellular matrix-related growth protein, and glycosaminoglycan; Among them, extracellular matrix-related growth proteins include growth factors and cytokines.

12. The acellular extracellular matrix biological dressing according to claim 11, characterized in that: The acellular extracellular matrix biological dressing is cut into blocks and porous sheets of different sizes for wound management, including deep wounds, diabetic foot ulcer wounds, traumatic wounds, surgical wounds, pressure sores, venous ulcers, and chronic vascular ulcers.

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