CN116019982A - Acellular dermal matrix, and preparation method and application thereof - Google Patents
Acellular dermal matrix, and preparation method and application thereof Download PDFInfo
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Abstract
The present application provides an acellular dermal matrix; wherein the collagen content of the acellular dermal matrix is 92-98wt%; the decellularized dermal matrix is derived from animal embryo skin; the animal skin is at least one selected from foetus cow skin, foetus pig skin and foetus sheep skin; preferably, the animal skin is selected from the group consisting of cow leather. The acellular dermal matrix also comprises inorganic ions, wherein the content of the inorganic ions is 0.01-0.2wt%; the inorganic ion is selected from Mg 2+ 、Zn 2+ 、Cu 2+ 、Ag + At least one of them is preferably Cu 2+ . Decellularized dermis prepared using the methods of the present applicationThe matrix has good biocompatibility and vascularization promoting effect, can reduce inflammatory cytokine secretion, can effectively improve the healing rate of chronic difficult-to-heal wound surface, and provides a solution for the treatment of clinical chronic difficult-to-heal wound surface.
Description
Technical Field
The application relates to the technical field of biomedical materials, in particular to a decellularized dermis matrix, a preparation method and application thereof.
Background
The chronic wound difficult to heal is a wound which can not achieve complete functions and anatomy through self repair after normal skin or tissue defects of organisms caused by various internal and external factors and conventional therapeutic intervention for more than 4 weeks. Chronic difficult-to-heal wounds are commonly known as ulcers, with diabetic skin ulcers (diabetic cutaneous ulcers, DCU) being one of the major conditions of chronic difficult-to-heal wounds. DCU is a common complication of diabetes, the main etiology of which includes skin injury, peripheral neuropathy, and vascular disease. Basic research and clinical practice prove that endogenous changes such as nerves, blood vessels, immunity, metabolism and the like and exogenous factors such as infection, wound, pressure and the like jointly cause difficult healing of the diabetic wound surface, and the interaction among pathogenic factors forms a complex pathophysiological process of the diabetic wound surface. The continuous high sugar environment in DCU patients can cause metabolic and endothelial cell dysfunction, lead to nitric oxide release and neovascular decrease, influence vasodilation function and appear lower limb/nerve ischemia. The neuropathy and the insufficient local blood flow can cause the damage of leucocytes and immune functions of DCU patients, so that the inflammatory reaction of wound tissues is insufficient, and the epithelialization and vascularization capacity are damaged, thereby causing the slow healing of the wounds, easily causing infection and even amputation of serious patients. At present, the basic principle of DCU treatment is to adopt anti-infection, debridement, wound decompression, proper blood circulation reconstruction, optimal wound dressing selection, negative pressure therapy, oxygen therapy and the like to promote wound healing according to the condition of a patient on the basis of maintaining stable blood sugar, blood pressure and blood fat.
The acellular dermal matrix (Acellular dermal matrix, ADM) removes the cell components with immunogenicity in epidermis and skin by a series of physicochemical and biological methods, retains the extracellular matrix components and structures such as collagen, and the like, thus obtaining the collagen matrix material with a three-dimensional space structure. Numerous studies have demonstrated that ADM has a micro-environment that induces tissue regeneration, provides cells with survival and motility, promotes cell growth and proliferation, helps promote neovascularization and epithelialization, and allows tissues to complete the healing process. ADM is reported to be beneficial to the healing of chronic difficult-to-heal wounds, but the wound healing rate is still not satisfactory, so that a new treatment strategy is required to be found clinically so as to improve the healing rate of chronic difficult-to-heal wounds.
Disclosure of Invention
The application aims to provide a decellularized dermal matrix for treating chronic wound surfaces difficult to heal. The specific technical scheme is as follows:
the first aspect of the present application provides an acellular dermal matrix; wherein the collagen content of the acellular dermal matrix is 92-98wt%; the decellularized dermal matrix is derived from animal fetuses; the animal skin is at least one selected from cow skin, pig skin and sheep skin; preferably, the animal skin is selected from the group consisting of cow leather.
In a second aspect, the present application provides a method for preparing a decellularized dermal matrix comprising the steps of:
pretreatment: washing animal skin with purified water for 3-8 times, placing the animal skin in 0.1-1wt% sodium chloride solution, oscillating for 5-13h, wringing out water, and removing epidermis layer and subcutaneous tissue layer to obtain animal dermis tablet;
degreasing: according to the mass volume ratio of 1g: mixing the animal dermis slices with degreasing agent in 5-20mL, and oscillating for 30-120min; wherein the degreasing agent is at least one selected from 40-80wt% ethanol solution, 0.5-2wt% sodium hydroxide solution and 50-100wt% isopropanol solution;
and (3) disinfection: according to the mass volume ratio of 1g: mixing the animal dermis slices with disinfectant in 10-30mL, and oscillating for 30-120min; wherein the disinfectant is at least one selected from 0.01-0.2wt% of peracetic acid solution, 10-50wt% of ethanol solution and 0.01-0.1wt% of benzalkonium bromide solution;
decellularization: according to the mass volume ratio of 1g: mixing the animal dermis slices with a decellularization reagent in 5-20mL, and oscillating for 30-120min; wherein the decellularization reagent is at least one selected from 0.5-2wt% sodium hydroxide solution and 0.2-2wt% trypsin-ethylenediamine tetraacetic acid (EDTA) solution;
α -Gal antigen removal: according to the mass volume ratio of 1g:3-20mL of the animal dermis tablet is mixed with 0.2-5U/mL of alpha-galactosidase solution, and the mixture is oscillated for 15-30h at 24-37 ℃;
and (3) sterilization: sterilizing by irradiation sterilization; wherein the irradiation sterilization is cobalt 60 irradiation sterilization, and the irradiation dose is 15-25kGy.
A third aspect of the present application provides an application of the acellular dermal matrix according to the first aspect of the present application or the acellular dermal matrix prepared by the preparation method according to the second aspect of the present application in the treatment of chronic difficult-to-heal wound surfaces; preferably, the chronic refractory wound is a diabetic skin ulcer.
The preparation method of the acellular dermal matrix provided by the application combines the specific pretreatment, degreasing, disinfection, acellular, alpha-Gal antigen removal and sterilization methods, can effectively remove animal tissue antigenic substances, can maximally retain the integrity of bioactive components and structures in the materials, shows excellent vascularization promoting effects, provides necessary conditions for nutrient substance transportation, and further accelerates the wound healing process. In addition, by adopting the method for adding inorganic ions, the inorganic ions are effectively compounded in the preparation process, on one hand, the activity of vascular endothelial cells and the high-efficiency expression of vascularization growth factors can be regulated, so that the excellent vascularization promoting effect is shown, necessary conditions are provided for the transportation of nutrient substances, and the wound healing process is further accelerated; on the other hand, the preparation can also regulate the expression of inflammatory factors of immune cells, and has anti-inflammatory effect. Furthermore, the preparation condition of the preparation method is simple, the quality is controllable, and the cost is low.
The acellular dermal matrix prepared by the preparation method has good biocompatibility and vascularization promoting effect, can reduce secretion of inflammatory cytokines, effectively improves the healing rate of chronic difficult-to-heal wound surfaces, and provides a solution for treating clinical chronic difficult-to-heal wound surfaces.
Of course, not all of the above-described advantages need be achieved simultaneously in practicing any one of the products or methods of the present application.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following description will briefly introduce the drawings that are required to be used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are only some embodiments of the present application, and other embodiments may also be obtained according to these drawings to those skilled in the art.
FIG. 1 is a hematoxylin-eosin staining chart (HE staining chart) of wound healing of a diabetic skin ulcer model rat filled with the decellularized dermal matrix of example 1;
FIG. 2 is a plot of HE staining for wound healing in a diabetic skin ulcer model rat filled with the decellularized dermal matrix of example 2;
FIG. 3 is a plot of HE staining of wound healing in a diabetic skin ulcer model rat filled with the decellularized dermal matrix of example 3;
FIG. 4 is a plot of HE staining of wound healing in a diabetic skin ulcer model rat filled with the decellularized dermal matrix of example 4;
FIG. 5 is a plot of HE staining of wound healing in a diabetic skin ulcer model rat filled with the decellularized dermal matrix of example 5;
FIG. 6 is a plot of HE staining of wound healing in a diabetic skin ulcer model rat filled with the decellularized dermal matrix of example 6.
Detailed Description
The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all, of the embodiments of the present application. Based on the embodiments herein, a person of ordinary skill in the art would be able to obtain all other embodiments based on the disclosure herein, which are within the scope of the disclosure herein.
The first aspect of the present application provides an acellular dermal matrix; wherein the collagen content of the acellular dermal matrix is 92-98wt%; the decellularized dermal matrix is derived from animal fetuses; the animal skin is at least one selected from cow skin, pig skin and sheep skin; preferably, the animal skin is selected from the group consisting of cow leather.
The inventor finds that the extracellular matrix structure of animal embryo skin contains a large amount of proteins for promoting the growth of tissue cells, the antigenic components are easier to remove, and the matrix components are easier to be absorbed by the tissue cells of the organism as nutrient substances. Preferably, the animal skin is selected from the group consisting of cow leather, which is more similar in structure to human dermis and is a natural biological scaffold material.
In some embodiments of the first aspect of the present application, the acellular dermal matrix further comprises inorganic ions in an amount of 0.01-0.2wt%; the inorganic ion is selected from Mg 2+ 、Zn 2+ 、Cu 2+ 、Ag + At least one of them is preferably Cu 2+ 。
The inventor finds that the acellular dermal matrix has less immunogenic substances and biological active ingredients; the inorganic ions with the specific content have excellent vascularization promoting effect, provide necessary conditions for the transportation of nutrient substances, further accelerate the wound healing process and play an anti-inflammatory role.
In some embodiments of the first aspect of the present application, the acellular dermal matrix is in the form of a powder, a floe or a patch, preferably a floe.
In this application, decellularized dermis matrix is powdered, flocculent or diaphragm form, because its wound depth and shape of chronic difficult healing wound surface position are inhomogeneous, adopts powdered, flocculent or diaphragm form product to fill, and the laminating wound that can be better accelerates wound healing process.
In some embodiments of the first aspect of the present application, the decellularized dermal matrix further comprises deferoxamine mesylate in an amount of 0.01 to 0.1wt%.
The inventor finds that the content of the deferoxamine mesylate is controlled within the scope of the application in the research, and the effective component of the deferoxamine mesylate in the decellularized dermal matrix can better realize the therapeutic effect, so that the effect of the decellularized dermal matrix in repairing chronic wound surfaces difficult to heal is enhanced. In the present application, the mode of adding deferoxamine mesylate is not limited, and the object of the present application can be achieved, for example, according to the mass ratio of the acellular dermal matrix to deferoxamine mesylate powder of 1mg:1g of deferoxamine mesylate powder is weighed and dissolved in pure water to form deferoxamine mesylate solution, the decellularized dermal matrix is placed in the deferoxamine mesylate solution to oscillate for 5-10h, and the product is obtained through freeze-drying treatment.
In a second aspect, the present application provides a method for preparing a decellularized dermal matrix comprising the steps of:
pretreatment: washing animal skin with purified water for 3-8 times, placing the animal skin in 0.1-1wt% sodium chloride solution, oscillating for 5-13h, wringing out water, and removing epidermis layer and subcutaneous tissue layer to obtain animal dermis tablet;
degreasing: according to the mass volume ratio of 1g: mixing the animal dermis slices with degreasing agent in 5-20mL, and oscillating for 30-120min; wherein the degreasing agent is at least one selected from 40-80wt% ethanol solution, 0.5-2wt% sodium hydroxide solution and 50-100wt% isopropanol solution;
and (3) disinfection: according to the mass volume ratio of 1g: mixing the animal dermis slices with disinfectant in 10-30mL, and oscillating for 30-120min; wherein the disinfectant is at least one selected from 0.01-0.2wt% of peracetic acid solution, 10-50wt% of ethanol solution and 0.01-0.1wt% of benzalkonium bromide solution;
decellularization: according to the mass volume ratio of 1g: mixing the animal dermis slices with a decellularization reagent in 5-20mL, and oscillating for 30-120min; wherein the decellularization reagent is at least one selected from 0.5-2wt% sodium hydroxide solution and 0.2-2wt% trypsin-EDTA solution;
α -Gal antigen removal: according to the mass volume ratio of 1g:3-20mL of the animal dermis tablet is mixed with 0.2-5U/mL of alpha-galactosidase solution, and the mixture is oscillated for 15-30h at 24-37 ℃;
and (3) sterilization: sterilizing by irradiation sterilization; wherein the irradiation sterilization is cobalt 60 irradiation sterilization, and the irradiation dose is 15-25kGy.
The inventor finds that the fetal animals are in a growth and development stage, the hair follicles of the skin are imperfect, the antigenic components are easier to remove, and the matrix components are easier to be absorbed by organism tissue cells as nutrient substances.
In the present application, the manner of removing the epidermis layer and the subcutaneous tissue layer is not limited, and for example, the epidermis layer containing hair and hair follicle may be removed by physical scraping, and the subcutaneous tissue layer may be peeled off using an electric skin remover.
The inventors found in the study that the decellularized dermal matrix prepared using the degreasing method and degreasing agent of the present application has a low fat content of 0.1-1wt%.
The inventor finds in the research that the acellular dermal matrix prepared by the acellular method and the acellular reagent basically maintains the complete structure, the matrix components are basically unaffected, and further, the content of collagen can reach 92-98wt% by adopting the acellular method.
The inventor finds that the method for removing the alpha-Gal antigen has low alpha-Gal antigen in the acellular dermal matrix and obvious effect of removing the alpha-Gal antigen.
In the application, the specific pretreatment, degreasing, disinfection, decellularization, alpha-Gal antigen removal and sterilization methods are combined, so that animal tissue antigenic substances can be effectively removed, meanwhile, bioactive components and structural integrity in the material can be reserved to the maximum extent, an excellent vascularization promoting effect is shown, necessary conditions are provided for nutrient substance transportation, and the wound healing process is further accelerated.
In the preparation method of the present application, after the pretreatment is completed, the steps of degreasing, sterilizing, decellularizing and removing the α -Gal antigen are not represented in order, and may be performed in different orders according to need. Illustratively, the preparation process of the present application may be performed in any order of: 1) Pretreatment, degreasing, disinfection, decellularization, alpha-Gal antigen removal and sterilization; 2) Pretreatment, disinfection, degreasing, alpha-Gal antigen removal, decellularization and sterilization; 3) Pretreatment, decellularization, disinfection, degreasing, alpha-Gal antigen removal and sterilization; 4) Pretreatment, decellularization, disinfection, removal of alpha-Gal antigen, degreasing and sterilization.
In some embodiments of the second aspect of the present application, further comprising:
inorganic ion addition: according to the mass volume ratio of 1g: mixing 20-60mL of the animal dermis tablet with 0.01-0.2wt% of inorganic ion solution, and oscillating for 60-180min; wherein the inorganic ion solution is at least one selected from copper chloride, zinc chloride, magnesium chloride and silver nitrate solution.
The inventor finds that by adopting the method for adding inorganic ions, inorganic ions can be effectively compounded in acellular dermal matrixes, on one hand, the activity of vascular endothelial cells and the high-efficiency expression of vascularized growth factors can be regulated, so that excellent vascularization promoting effect is shown, necessary conditions are provided for the transportation of nutrient substances, and the wound healing process is further accelerated; on the other hand, the preparation can also regulate the expression of inflammatory factors of immune cells, and has anti-inflammatory effect.
The inorganic ion addition is performed after the completion of each step of pretreatment, degreasing, sterilization, decellularization and α -Gal antigen removal, and before sterilization. Illustratively, the preparation process of the present application may be performed in the following order: pretreatment, degreasing, disinfection, decellularization, alpha-Gal antigen removal, inorganic ion addition and sterilization.
In some embodiments of the second aspect of the present application, the inorganic ion solution is selected from copper chloride solutions; the concentration of the inorganic ion solution is 0.01-0.1wt%.
The inventor finds that the concentration of the inorganic ion solution is controlled within the range of the application in the research, so that the inorganic ion in the acellular dermal matrix can better realize the therapeutic effect, thereby enhancing the effect of the acellular dermal matrix in the repair of chronic wound surfaces difficult to heal.
In some embodiments of the second aspect of the present application, the animal skin is selected from at least one of fetal bovine skin, fetal porcine skin, or fetal ovine skin; preferably, the animal skin is selected from the group consisting of cow leather.
The inventor finds that the extracellular matrix structure of animal embryo skin contains a large amount of proteins for promoting the growth of tissue cells, the antigenic components are easier to remove, and the matrix components are easier to be absorbed by the tissue cells of the organism as nutrient substances. Preferably, the fetal calf skin structure is more similar to human dermis, being a natural biological scaffold material.
In some embodiments of the second aspect of the present application, each of the degreasing, sterilizing, decellularizing and α -Gal antigen removal steps further comprises rinsing with a rinsing agent; the cleaning agent is at least one selected from 0.1-1wt% sodium chloride solution and 0.1-2M (mol/L) Phosphate (PBS) buffer solution.
In some embodiments of the second aspect of the present application, the washing step comprises, in terms of a feed liquid mass ratio of 1g: adding cleaning agent into 5-20mL, oscillating for 10-60min, and repeating cleaning for 3-20 times.
In this application, the oscillation is a conventional operation in the art, and the present application is not limited thereto, and the object of the present application can be achieved, for example, an air bath shaker may be used for oscillation, and the oscillation frequency is 100-200rpm.
The decellularized dermal matrix prepared using the methods of the present application can be dried by lyophilization, which is a routine procedure in the art, and is not limited herein. The lyophilization step may be completed prior to the sterilization process of the present application. For example, the method of the present application may include the steps of: pretreatment, degreasing, disinfection, decellularization, removal of alpha-Gal antigen, inorganic ion addition, freeze-drying and sterilization.
A third aspect of the present application provides an application of the acellular dermal matrix according to the first aspect of the present application or the acellular dermal matrix prepared by the preparation method according to the second aspect of the present application in the treatment of chronic difficult-to-heal wound surfaces; preferably, the chronic refractory wound is a diabetic skin ulcer.
The technical scheme of the application is further explained by specific examples.
The experimental materials and methods used in the examples below are conventional materials and methods unless otherwise specified.
Example 1
(1) Pretreatment: washing the animal embryo skin with purified water for 5 times, placing the animal embryo skin in 0.9wt% sodium chloride solution, oscillating for 8h, wringing out water, removing the epidermis layer containing hair and hair follicle by using a physical scraping mode, and stripping subcutaneous tissue layer by using an electric skin removing machine to obtain the cow dermis sheet;
(2) Degreasing: according to the mass volume ratio of 1g: mixing the cattle dermis slices with 75wt% ethanol solution by 10mL, oscillating for 60min, discarding the ethanol solution, adding 0.9wt% sodium chloride solution, and oscillating and cleaning for 3 times each for 15min;
(3) And (3) disinfection: according to the mass volume ratio of 1g:10mL of the defatted bovine dermis slices are mixed with a mixed solution of 0.1 weight percent of peroxyacetic acid and 50 weight percent of ethanol, the mixed solution of the peroxyacetic acid and the ethanol is removed after 60 minutes of shaking, and then 0.9 weight percent of sodium chloride solution is added for 3 times of shaking and cleaning for 15 minutes each time;
(4) Decellularization: according to the mass volume ratio of 1g: mixing the sterilized bovine dermis slices with 1wt% sodium hydroxide solution by 10mL, oscillating for 60min, discarding the sodium hydroxide solution, adding 0.1M PBS solution, and oscillating for 10 times each for 10min;
(5) α -Gal antigen removal: according to the mass volume ratio of 1g: mixing the decellularized animal dermis slices with 4.5U/mL alpha-galactosidase solution in 10mL, oscillating for 30h at 37 ℃, discarding the alpha-galactosidase solution, adding 0.1M PBS solution, and oscillating and cleaning for 3 times for 15min each time;
(6) Inorganic ion addition: according to the mass volume ratio of 1g: mixing the bovine dermis slices after removing the alpha-Gal antigen with 0.1wt% copper chloride solution in 20mL, and oscillating for 60min to obtain a decellularized dermis matrix of composite copper ions;
(7) And (3) freeze-drying: spreading the acellular dermal matrix of the composite copper ions in a steel plate mould, and freeze-drying;
(8) And (3) sterilization: sterilizing the acellular dermal matrix of the composite copper ions by adopting radiation sterilization; wherein the irradiation sterilization is cobalt 60 irradiation sterilization, and the irradiation dose is 20kGy.
Example 2
Example 1 was repeated except that the 0.1wt% copper chloride solution in step (6) was replaced with the 0.2wt% copper chloride solution.
Example 3
Example 1 was repeated except that the 0.1wt% copper chloride solution in step (6) was replaced with the 0.1wt% zinc chloride solution.
Example 4
Example 1 was repeated except that the 0.1wt% copper chloride solution in step (6) was replaced with the 0.1wt% magnesium chloride solution.
Example 5
Example 1 was repeated except that the 0.1wt% copper chloride solution in step (6) was replaced with a 0.1wt% silver nitrate solution.
Example 6
The procedure of example 1 was followed except that the step (6) was deleted to obtain a acellular dermal matrix.
Effect measurement
Analysis of biochemical Components, fat content
Fat content: the acid hydrolysis treatment specified in the second method of GB/T5009.6-2016 was carried out.
Collagen content: YY/T1453-2016 appendix B hydroxyproline content determination.
TABLE 1
| Total collagen (wt%) | Collagen III (wt%) | Inorganic ions (wt%) | Fat content (wt%) | |
| Example 1 | 95.95±1.15 | 1.64±0.45 | 0.1 | 0.64±0.22 |
| Example 2 | 94.63±2.04 | 1.76±0.23 | 0.2 | 0.59±0.27 |
| Example 3 | 93.79±1.73 | 1.27±0.17 | 0.1 | 0.62±0.13 |
| Example 4 | 96.25±1.18 | 1.53±0.39 | 0.1 | 0.46±0.35 |
| Example 5 | 93.65±1.07 | 1.23±0.46 | 0.1 | 0.57±0.39 |
| Example 6 | 94.29±1.21 | 1.25±0.22 | 0 | 0.53±0.32 |
Collagen is an important component constituting extracellular skeleton, and has good biocompatibility; collagen also has the degradable biological property, and can be decomposed into small molecules and short peptides which are easy to absorb in a certain period of time in vivo; the collagen and other bioactive components are used together, so that the collagen has a synergistic effect, and is easy for cells to adhere to the surface of the collagen material and proliferate and grow, so that collagen molecules are important components in the scaffold material for tissue repair, and have the advantage that other components cannot be replaced. As can be seen from the results of Table 1, the total collagen content of the acellular dermal matrix obtained by the preparation methods of examples 1 to 6 of the present application was as high as 95.95.+ -. 1.15wt%, 94.63.+ -. 2.04wt%, 93.79.+ -. 1.73wt%, 96.25.+ -. 1.18wt%, 93.65.+ -. 1.07wt%, 94.29.+ -. 1.21wt%, wherein the collagen III content was 1.64.+ -. 0.45wt%, 1.76.+ -. 0.23wt%, 1.27.+ -. 0.17wt%, 1.53.+ -. 0.39wt%, 1.23.+ -. 0.46wt%, 1.25.+ -. 0.22wt%, respectively. The preparation method has little damage to the collagen in the extracellular matrix, and the retention of the collagen III enhances the vascular promotion capability of the acellular dermal matrix, thereby being beneficial to the regeneration and repair of tissues.
It can also be seen from Table 1 that the decellularized dermal matrix obtained by the preparation methods of examples 1-6 of the present application has low fat content, indicating that the decellularized dermal matrix of the present application has good biosafety.
Experimental animal
20 SD rats, SPF grade, male, with a body weight of 150-200g, were healthy at 5 to 6 weeks of age and were offered by Peking Vitolihua laboratory animal technologies Inc. Raising in the environment with the temperature of 23+/-2 ℃ and the relative humidity of 55% -65%.
Experimental treatment
After the rats were adaptively bred for 7 days, the rats with normal blood sugar measured by blood sampling were used for the subsequent experiments, all rats were induced by diabetes, and skin excision was performed only at the same site to make wound surfaces.
Establishment of diabetic skin ulcer rat model
Rats were fasted for more than 12 hours and were intraperitoneally injected with Streptozotocin (STZ) solution at 60-80 mg/kg (calculated as STZ solute). Blood glucose was continuously monitored with a glucometer within 7 days after injection of STZ solution, and fasting blood glucose was greater than or equal to 11.1mmol/L, or postprandial blood glucose was greater than 16.7mmol/L, indicating success in inducing diabetes in rats. After successful induction, the diabetic rats should be closely tested for various physiological indexes. Blood sugar was measured periodically, and the color, weight, intake and ingestion of the hair were recorded, with or without abdominal distension and ascites formation, and with or without cataract formation.
After molding for 2-3 weeks, binding the rats in prone position, shaving the backs of diabetic rats, and sterilizing with iodine and alcohol after shaving. Each rat was pre-operatively intramuscular injected with 8-10 ten thousand units of penicillin for prophylactic treatment. The left side and the right side of the right center of the back of the rat are symmetrically called 1X 1cm full skin defect wound surfaces. According to each set of protocols, the corresponding materials are added and sampled at the appropriate time.
Wound morphology observation and healing rate detection
The acellular dermal matrix in the example 1 is filled in the back wound surface of a diabetic rat, and the acellular dermal matrix is externally applied with a wet dressing to maintain the necessary wet environment for wound healing, so as to be used as an experimental group; the control group was not added with any material. The recovery condition of the wound surface of the rats after each group of operations for 1 week, 2 weeks and 3 weeks is recorded, and the rats are completely covered by the epithelium to be used as the basis of wound surface healing. The area of the ulcer wound was measured using Image-Plus 6.0 Image analysis software, and the healing rate was calculated weekly. Wound healing rate = [ (original wound area-unhealed wound area)/original wound area ] ×100%. The results are shown in Table 2.
TABLE 2
As can be seen from Table 2, the wound healing rates of the control group and the experimental group of diabetic rats are increased along with the extension of time, but the wound healing rate of the experimental group is far higher than that of the control group, especially more obvious after 3 weeks of operation, the wound healing rate of the experimental group after 3 weeks of operation is as high as (94.17+/-1.12), and the control group is only (58.33+/-1.42), which indicates that the acellular dermal matrix prepared by the application has remarkable promoting effect on the wound healing of the diabetic skin ulcer model rats.
Histological analysis
The materials prepared in examples 1-6 are respectively filled in the back wound surface of a diabetic rat, the external wet dressing is applied, the necessary wet environment for wound healing is maintained, HE staining analysis is carried out 3 weeks after operation, and the results are shown in figures 1-6.
In fig. 1-4, the number of inflammatory cells at the material filling part is small, and the growth of blood cells and microvascular tissues to the center of the wound is obviously observed, so that the supply recovery of blood accelerates the vascularization of the wound surface and promotes the wound healing; fig. 5 shows that the material filling part has fewer inflammatory cells, more new micro-blood vessels and better epithelialization, and the wound surface has no bacterial infection phenomenon due to the unique antibacterial property of silver ions; FIG. 6 shows that the material filling part has moderate inflammatory cell number, more newly generated micro-blood vessels and better epithelialization. The acellular dermal matrix prepared by the application can regulate the activity of vascular endothelial cells and the high-efficiency expression of vascularization growth factors, so that the acellular dermal matrix has excellent vascularization promoting effect, provides necessary conditions for the transportation of nutrients, accelerates the wound healing process and improves the healing effect of the wound of a diabetic skin ulcer model rat.
The preparation method of the acellular dermal matrix provided by the application combines the specific pretreatment, degreasing, disinfection, acellular, alpha-Gal antigen removal and sterilization methods, can effectively remove animal tissue antigenic substances, can maximally retain the integrity of bioactive components and structures in the materials, shows excellent vascularization promoting effects, provides necessary conditions for nutrient substance transportation, and further accelerates the wound healing process. In addition, by adopting the method for adding inorganic ions, the inorganic ions are effectively compounded in the preparation process, on one hand, the activity of vascular endothelial cells and the high-efficiency expression of vascularization growth factors can be regulated, so that the excellent vascularization promoting effect is shown, necessary conditions are provided for the transportation of nutrient substances, and the wound healing process is further accelerated; on the other hand, the preparation can also regulate the expression of inflammatory factors of immune cells, and has anti-inflammatory effect. Furthermore, the preparation condition of the preparation method is simple, the quality is controllable, and the cost is low. The acellular dermal matrix prepared by the preparation method has good biocompatibility and vascularization promoting effect, and can also effectively improve the healing rate of chronic difficult-to-heal wound surfaces, thereby providing reference for the treatment of clinical chronic difficult-to-heal wound surfaces.
The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the scope of the present application. Any modifications, equivalent substitutions, improvements, etc. that are within the spirit and principles of the present application are intended to be included within the scope of the present application.
Claims (10)
1. Acellular dermal matrix; wherein the collagen content of the acellular dermal matrix is 92-98wt%; the decellularized dermal matrix is derived from animal fetuses; the animal skin is at least one selected from cow skin, pig skin and sheep skin; preferably, the animal skin is selected from the group consisting of cow leather.
2. The acellular dermal matrix of claim 1, wherein the acellular dermal matrix further comprises inorganic ions in an amount of 0.01-0.2wt%; the inorganic ion is selected from Mg 2+ 、Zn 2+ 、Cu 2+ 、Ag + At least one of (a)The seed is preferably Cu 2+ 。
3. The acellular dermal matrix according to any one of claims 1-2, wherein the acellular dermal matrix is in the form of a powder, a floc or a patch, preferably a floc.
4. The acellular dermal matrix according to any one of claims 1-2, wherein the acellular dermal matrix further comprises deferoxamine mesylate in an amount of 0.01-0.1wt%.
5. A method of preparing an acellular dermal matrix comprising the steps of:
pretreatment: washing animal skin with purified water for 3-8 times, placing the animal skin in 0.1-1wt% sodium chloride solution, oscillating for 5-13h, wringing out water, and removing epidermis layer and subcutaneous tissue layer to obtain animal dermis tablet;
degreasing: according to the mass volume ratio of 1g: mixing the animal dermis slices with degreasing agent in 5-20mL, and oscillating for 30-120min; wherein the degreasing agent is at least one selected from 40-80wt% ethanol solution, 0.5-2wt% sodium hydroxide solution and 50-100wt% isopropanol solution;
and (3) disinfection: according to the mass volume ratio of 1g: mixing the animal dermis slices with disinfectant in 10-30mL, and oscillating for 30-120min; wherein the disinfectant is at least one selected from 0.01-0.2wt% of peracetic acid solution, 10-50wt% of ethanol solution and 0.01-0.1wt% of benzalkonium bromide solution;
decellularization: according to the mass volume ratio of 1g: mixing the animal dermis slices with a decellularization reagent in 5-20mL, and oscillating for 30-120min; wherein the decellularization reagent is at least one selected from 0.5-2wt% sodium hydroxide solution and 0.2-2wt% trypsin-EDTA solution;
α -Gal antigen removal: according to the mass volume ratio of 1g:3-20mL of the animal dermis tablet is mixed with 0.2-5U/mL of alpha-galactosidase solution, and the mixture is oscillated for 15-30h at 24-37 ℃;
and (3) sterilization: sterilizing by irradiation sterilization; wherein the irradiation sterilization is cobalt 60 irradiation sterilization, and the irradiation dose is 15-25kGy.
6. The preparation method according to claim 5, further comprising:
inorganic ion addition: according to the mass volume ratio of 1g: mixing 20-60mL of the animal dermis tablet with 0.01-0.2wt% of inorganic ion solution, and oscillating for 60-180min; wherein the inorganic ion solution is at least one selected from copper chloride, zinc chloride, magnesium chloride and silver nitrate solution.
7. The preparation method according to claim 6, wherein the inorganic ion solution is selected from copper chloride solutions; the concentration of the inorganic ion solution is 0.01-0.1wt%.
8. The method according to claim 5, wherein the animal skin is at least one selected from the group consisting of fetal bovine skin, fetal porcine skin and fetal ovine skin; preferably, the animal skin is selected from the group consisting of cow leather.
9. The method of claim 5, wherein each of the degreasing, sterilizing, decellularizing and α -Gal antigen removal steps further comprises washing with a detergent; the cleaning agent is at least one selected from 0.1-1wt% sodium chloride solution and 0.1-2M PBS buffer solution; preferably, the cleaning step comprises the following steps of: adding cleaning agent into 5-20mL, oscillating for 10-60min, and repeating cleaning for 3-20 times.
10. Use of the acellular dermal matrix according to any one of claims 1-4 or prepared by the method of preparation according to any one of claims 5-9 for the treatment of chronic refractory wounds; preferably, the chronic refractory wound is a diabetic skin ulcer.
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| CN116077739A (en) * | 2023-03-23 | 2023-05-09 | 杭州华迈医疗科技有限公司 | Acellular pericardium and preparation method and application thereof |
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