CN116421789A - Preparation method of acellular dermal matrix composite scaffold - Google Patents
Preparation method of acellular dermal matrix composite scaffold Download PDFInfo
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- CN116421789A CN116421789A CN202310440822.0A CN202310440822A CN116421789A CN 116421789 A CN116421789 A CN 116421789A CN 202310440822 A CN202310440822 A CN 202310440822A CN 116421789 A CN116421789 A CN 116421789A
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- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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Abstract
The invention discloses a preparation method of a decellularized dermis matrix composite scaffold, which comprises the following steps: s1, providing animal source skin tissue, cleaning the surface of the source skin tissue, soaking and cleaning to obtain dermis tissue; s2, sequentially placing the dermal tissue into a PMSF solution and an EDTA solution, stirring, taking out the dermal tissue after stirring, cleaning, repeating the stirring and cleaning processes for a plurality of times, and freeze-drying to obtain a decellularized dermal matrix; s3, grinding the acellular dermal matrix to obtain acellular dermal matrix powder; s4, taking the acellular dermal matrix, putting the acellular dermal matrix into an acetic acid solution, and stirring until the acellular dermal matrix is completely dissolved to obtain an acellular dermal matrix solution; s5, adding the decellularized dermal matrix powder into the decellularized dermal matrix solution, uniformly stirring, pouring into a mold, and freeze-drying to obtain a preformed decellularized dermal matrix composite scaffold; s6, soaking the preformed acellular dermal matrix composite scaffold in a cross-linking agent for cross-linking, washing after cross-linking, and freeze-drying to obtain the acellular dermal matrix composite scaffold.
Description
Technical Field
The invention relates to the technical field of biological tissue engineering scaffold materials, in particular to a preparation method of a decellularized dermis matrix composite scaffold.
Background
The acellular dermal matrix is an extracellular matrix subjected to acellular treatment, contains biological components such as glycosaminoglycan, collagen, proteoglycan, cell growth factors and the like, has excellent biocompatibility, and is widely applied to the field of tissue engineering. The acellular dermal matrix is usually prepared in the form of a two-dimensional structure sheet, and needs to be processed to form a three-dimensional scaffold structure. Chinese patent CN114377207a discloses a preparation method of a pig acellular dermal matrix scaffold, which performs multi-loop treatments such as acetic acid gelation, chemical crosslinking, freeze drying and the like on the acellular dermal matrix, but the three-dimensional scaffold prepared by the method has a sheet-like structure, compact microstructure, uneven pore distribution and no contribution to cell growth and proliferation.
The traditional preparation method of the acellular dermal matrix scaffold comprises the following steps: the acellular dermal matrix is subjected to proteinase dissolution or acid dissolution and then freeze-dried for molding. The enzymolysis-freeze-drying method uses protease, which can destroy part of self growth factors and beneficial proteins of the dermal matrix, and reduce the biological performance of the acellular dermal matrix. The acid dissolution-freeze-drying method uses acid to dissolve the acellular dermal matrix, and the method realizes the three-dimensional structure of the acellular dermal matrix, but the prepared scaffold has compact structure and low porosity, so that cells are difficult to grow in the scaffold, and meanwhile, the mechanical strength is low, so that the mechanical property requirement of the tissue filling material cannot be met. Therefore, there is a need to develop a new method for decellularized dermal matrix scaffold, which can enhance the mechanical properties of the scaffold and improve the pore structure of the scaffold to meet the wider tissue filling requirements under the condition that the bioactivity of the decellularized dermal matrix itself is not damaged as much as possible.
Disclosure of Invention
The invention aims at: aiming at the problems of low mechanical strength and low porosity of the acellular dermal matrix scaffold prepared by the prior art, the invention develops a novel preparation method of the acellular dermal matrix composite scaffold, and the acellular dermal matrix composite scaffold with high mechanical strength and porous structure is prepared by the method without adding additives, so that the problems of low mechanical strength and low porosity of the existing acellular dermal matrix scaffold are solved.
The invention is realized by the following technical scheme:
the preparation method of the acellular dermal matrix composite scaffold is characterized by comprising the following steps of:
s1, preparation of dermis tissues: providing animal source skin tissue, cleaning the surface of the source skin tissue, soaking and cleaning to obtain dermis tissue;
s2, acellular treatment of the dermal matrix: sequentially placing the dermis tissues into PMSF (phenylmethanesulfonyl fluoride) solution and EDTA (ethylene diamine tetraacetic acid) solution, stirring, taking out the dermis tissues after stirring, cleaning, repeating the stirring and cleaning processes for a plurality of times, and freeze-drying to obtain acellular dermis matrixes;
s3, preparing acellular dermal matrix powder: grinding the acellular dermal matrix to obtain acellular dermal matrix powder;
s4, preparing a acellular dermal matrix solution: putting the acellular dermal matrix into acetic acid solution, and stirring until the acellular dermal matrix is completely dissolved to obtain acellular dermal matrix solution;
s5, preforming the acellular dermal matrix composite scaffold: adding the acellular dermal matrix powder into the acellular dermal matrix solution, stirring uniformly, pouring into a mold, and freeze-drying to obtain a preformed acellular dermal matrix composite scaffold;
s6, chemical crosslinking: and (3) immersing the preformed acellular dermal matrix composite scaffold in a cross-linking agent for cross-linking, washing after cross-linking, and freeze-drying to obtain the acellular dermal matrix composite scaffold.
Further, a preparation method of the acellular dermal matrix composite scaffold comprises the following steps: s1, preparation of dermis tissues: providing animal source skin tissue, then cleaning the source skin tissue with deionized water to remove surface impurities, removing surface animal hair, removing surface fat, soaking in normal saline for 10-24 hours to remove epidermis, and washing with deionized water to obtain dermis tissue.
Further, a preparation method of the acellular dermal matrix composite scaffold comprises the following steps: the animal-derived skin tissue described in step S1 is selected from the skin tissue of swine or bovine. Preferably porcine skin tissue.
Further, a preparation method of the acellular dermal matrix composite scaffold comprises the following steps: s2, acellular treatment of the dermal matrix: sequentially placing the dermis tissue into a PMSF solution with the concentration of 1.0-5.0 mg/ml and an EDTA solution with the concentration of 0.5-2.5 mg/ml, stirring at the speed of 200-500 rpm for 5-10 minutes, taking out the dermis tissue after stirring, flushing the dermis tissue with deionized water, repeating the stirring and cleaning processes for 2-5 times, and then placing the dermis tissue into a freeze dryer for freeze drying to obtain the acellular dermis matrix.
Further, a preparation method of the acellular dermal matrix composite scaffold comprises the following steps: the PMSF solution in the step S2 takes deionized water as a solvent; the EDTA solution takes PBS buffer solution as a solvent.
Further, a preparation method of the acellular dermal matrix composite scaffold comprises the following steps: s3, preparing acellular dermal matrix powder: and (3) putting the acellular dermal matrix into a grinder, grinding for 3-5 minutes, and repeating grinding for 3-6 times to obtain acellular dermal matrix powder.
Further, a preparation method of the acellular dermal matrix composite scaffold comprises the following steps: s4, preparing a acellular dermal matrix solution: the acellular dermal matrix is taken and put into acetic acid solution, and stirred at the speed of 200-500 rpm until the acellular dermal matrix is completely dissolved, so that acellular dermal matrix solution with the concentration of 0.1-1.0g/ml is obtained.
Further, a preparation method of the acellular dermal matrix composite scaffold comprises the following steps: s5, preforming the acellular dermal matrix composite scaffold: adding the acellular dermal matrix powder into the acellular dermal matrix solution, uniformly stirring, pouring into a mold, and then placing into a freeze dryer for freeze drying to obtain a preformed acellular dermal matrix composite scaffold; wherein: the mass volume ratio of the acellular dermal matrix powder to the acellular dermal matrix solution is 0.1-1.0g/ml.
Further, a preparation method of the acellular dermal matrix composite scaffold comprises the following steps: s6, chemical crosslinking: immersing the preformed acellular dermal matrix composite stent in a cross-linking agent with the concentration of 0.05-0.25mg/ml for cross-linking for 1-3 hours, washing with deionized water after cross-linking, and then freeze-drying at the temperature of minus 30 ℃ to minus 15 ℃ for 10-24 hours to obtain the acellular dermal matrix composite stent.
Further, a preparation method of the acellular dermal matrix composite scaffold comprises the following steps: the cross-linking agent in the step S6 is selected from one of aldehyde solution, genipin solution or carbodiimide solution.
The invention has the beneficial effects that:
(1) The acellular dermal matrix selected in the preparation method of the acellular dermal matrix composite scaffold provided by the invention is used as a substrate material, and the acellular dermal matrix has no immunogenicity and good biocompatibility.
(2) In the preparation method of the acellular dermal matrix composite scaffold, the acellular dermal matrix is subjected to acellular by adopting the PMSF solution and the EDTA solution, and the acellular effect is thorough.
(3) The preparation method of the acellular dermal matrix composite scaffold provided by the invention adopts an acid dissolution method to replace an enzymolysis method to prepare the acellular dermal matrix solution, reduces damage to extracellular matrix components such as collagen, hyaluronic acid, growth factors and the like in the acellular dermal matrix, and maintains the microscopic three-dimensional structure of the acellular dermal matrix by using the acellular dermal matrix powder prepared by a grinding method.
(4) The mechanical strength of the acellular dermal matrix composite scaffold prepared by the method is higher than that of a single scaffold prepared by an enzymolysis solution, and the acellular dermal matrix composite scaffold has elasticity, so that the acellular dermal matrix composite scaffold is more convenient for implantation operation.
(5) The acellular dermal matrix composite scaffold prepared by the method has a porous three-dimensional structure, and the internal pores are communicated, so that cells can grow in the scaffold.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the compressive strength of the stents of comparative example 1 and examples 1 to 3 according to the present invention at a compressive strain of 60%;
FIG. 2 is a graph showing the results of the porosity of scaffolds prepared in comparative example 1 and examples 1-3 of the present invention;
FIG. 3 is an SEM image of scaffolds prepared according to comparative examples 1 and 2 of the present invention; wherein: fig. 3 a is an SEM image of the decellularized dermal matrix scaffold prepared in comparative example 1, and fig. 3B is an SEM image of the decellularized dermal matrix composite scaffold prepared in example 2.
Detailed Description
The following description of the embodiments of the present invention 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 embodiments of the invention. The following description of at least one exemplary embodiment is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The preparation method of the acellular dermal matrix composite stent is characterized by comprising the following specific steps:
s1, preparation of dermis tissues: providing animal skin tissue (selected from pig skin tissue), cleaning the pig skin tissue with deionized water to remove surface impurities, removing surface hair, removing surface fat, soaking in physiological saline for 24 hr, removing epidermis, and repeatedly washing with deionized water to obtain dermis tissue;
s2, acellular treatment of the dermal matrix: sequentially placing the obtained dermal tissue into a PMSF solution with the concentration of 3.0mg/ml and an EDTA solution with the concentration of 1.5mg/ml, stirring at the speed of 300rpm for 5 minutes, taking out the dermal tissue after stirring, repeatedly washing with deionized water, repeating the stirring and washing processes for 2 times, and then placing into a freeze dryer for freeze drying to obtain the acellular dermal matrix;
s3, preparing acellular dermal matrix powder: grinding the acellular dermal matrix in a grinder for 5 minutes, and repeating grinding for 3 times to obtain acellular dermal matrix powder;
s4, preparing a acellular dermal matrix solution: putting the acellular dermal matrix into acetic acid solution, and stirring at 300rpm until the acellular dermal matrix is completely dissolved to obtain acellular dermal matrix solution with the concentration of 0.9 g/ml; wherein: the acetic acid solution is acetic acid solution with the volume fraction of 5%, and the solvent is deionized water;
s5, preforming the acellular dermal matrix composite scaffold: adding 1.0g of the acellular dermal matrix powder into 10ml of the 0.9g/ml acellular dermal matrix solution, stirring uniformly, pouring into a mould, and then placing into a freeze dryer for freeze drying for 24 hours to obtain a preformed acellular dermal matrix composite scaffold; the amount of the decellularized dermal matrix powder incorporated in this step was 10% of the total amount of the decellularized dermal matrix;
s6, chemical crosslinking: and (3) immersing the preformed acellular dermal matrix composite scaffold in genipin solution with the concentration of 0.1mg/ml for crosslinking for 2 hours, repeatedly flushing with deionized water after crosslinking, and then freeze-drying at the temperature of minus 30 ℃ to minus 15 ℃ for 24 hours to obtain the acellular dermal matrix composite scaffold.
In example 1, the DNA remaining amount of the acellular dermal matrix was measured by using a DNA measuring kit to determine the effect of the acellular dermal matrix, and the result showed that the DNA remaining amount of the acellular dermal matrix was only 1% of the dermal matrix, indicating that the acellular dermal matrix prepared in this example was completely acellular.
Example 2
The preparation method of the acellular dermal matrix composite stent is characterized by comprising the following specific steps:
s1, preparation of dermis tissues: providing animal skin tissue (selected from pig skin tissue), cleaning the pig skin tissue with deionized water to remove surface impurities, removing surface hair, removing surface fat, soaking in physiological saline for 24 hr, removing epidermis, and repeatedly washing with deionized water to obtain dermis tissue;
s2, acellular treatment of the dermal matrix: sequentially placing the obtained dermal tissue into a PMSF solution with the concentration of 3.0mg/ml and an EDTA solution with the concentration of 1.5mg/ml, stirring at the speed of 300rpm for 5 minutes, taking out the dermal tissue after stirring, repeatedly washing with deionized water, repeating the stirring and washing processes for 2 times, and then placing into a freeze dryer for freeze drying to obtain the acellular dermal matrix;
s3, preparing acellular dermal matrix powder: grinding the acellular dermal matrix in a grinder for 5 minutes, and repeating grinding for 3 times to obtain acellular dermal matrix powder;
s4, preparing a acellular dermal matrix solution: putting the acellular dermal matrix into acetic acid solution, and stirring at 300rpm until the acellular dermal matrix is completely dissolved to obtain acellular dermal matrix solution with the concentration of 0.75 g/ml; wherein: the acetic acid solution is acetic acid solution with the volume fraction of 5%, and the solvent is deionized water;
s5, preforming the acellular dermal matrix composite scaffold: adding 2.5g of the acellular dermal matrix powder into 10ml of the 0.75g/ml acellular dermal matrix solution, uniformly stirring, pouring into a mould, and then placing into a freeze dryer for freeze drying for 24 hours to obtain a preformed acellular dermal matrix composite scaffold; the amount of the decellularized dermal matrix powder incorporated in this step is 25% of the total amount of the decellularized dermal matrix;
s6, chemical crosslinking: and (3) immersing the preformed acellular dermal matrix composite scaffold in genipin solution with the concentration of 0.1mg/ml for crosslinking for 2 hours, repeatedly flushing with deionized water after crosslinking, and then freeze-drying at the temperature of minus 30 ℃ to minus 15 ℃ for 24 hours to obtain the acellular dermal matrix composite scaffold.
Example 3
The preparation method of the acellular dermal matrix composite stent is characterized by comprising the following specific steps:
s1, preparation of dermis tissues: providing animal skin tissue (selected from pig skin tissue), cleaning the pig skin tissue with deionized water to remove surface impurities, removing surface hair, removing surface fat, soaking in physiological saline for 24 hr, removing epidermis, and repeatedly washing with deionized water to obtain dermis tissue;
s2, acellular treatment of the dermal matrix: sequentially placing the obtained dermal tissue into a PMSF solution with the concentration of 3.0mg/ml and an EDTA solution with the concentration of 1.5mg/ml, stirring at the speed of 300rpm for 5 minutes, taking out the dermal tissue after stirring, repeatedly washing with deionized water, repeating the stirring and washing processes for 2 times, and then placing into a freeze dryer for freeze drying to obtain the acellular dermal matrix;
s3, preparing acellular dermal matrix powder: grinding the acellular dermal matrix in a grinder for 5 minutes, and repeating grinding for 3 times to obtain acellular dermal matrix powder;
s4, preparing a acellular dermal matrix solution: putting the acellular dermal matrix into acetic acid solution, and stirring at 300rpm until the acellular dermal matrix is completely dissolved to obtain acellular dermal matrix solution with the concentration of 0.5 g/ml; wherein: the acetic acid solution is acetic acid solution with the volume fraction of 5%, and the solvent is deionized water;
s5, preforming the acellular dermal matrix composite scaffold: adding 5.0g of the acellular dermal matrix powder into 10ml of the 0.5g/ml acellular dermal matrix solution, uniformly stirring, pouring into a mould, and then placing into a freeze dryer for freeze drying for 24 hours to obtain a preformed acellular dermal matrix composite scaffold; the amount of decellularized dermal matrix powder incorporated in this step is 50% of the total decellularized dermal matrix;
s6, chemical crosslinking: and (3) immersing the preformed acellular dermal matrix composite scaffold in genipin solution with the concentration of 0.1mg/ml for crosslinking for 2 hours, repeatedly flushing with deionized water after crosslinking, and then freeze-drying at the temperature of minus 30 ℃ to minus 15 ℃ for 24 hours to obtain the acellular dermal matrix composite scaffold.
The above embodiments 1 to 3 differ in that: the mixing amount of the acellular dermal matrix powder is different, and the other preparation conditions are the same; specifically, the amount of the decellularized dermal matrix powder added in example 1 was 10% of the total amount of the decellularized dermal matrix; the amount of the decellularized dermal matrix powder incorporated in example 2 was 25% of the total amount of the decellularized dermal matrix; the amount of decellularized dermal matrix powder incorporated in example 3 was 50% of the total decellularized dermal matrix.
Comparative example 1
A method for preparing a decellularized dermal matrix scaffold is provided, and comparative example 1 differs from example 1 in that: the acellular dermal matrix powder was not incorporated in comparative example 1, but the acellular dermal matrix solution having a concentration of 0.9g/ml was directly poured into a mold for freeze-drying. That is, the blending amount of the acellular dermal matrix powder in comparative example 1 was 0.
And (3) testing:
(1) Compression mechanical property test:
the compressive mechanical strength of the acellular dermal matrix composite scaffolds prepared in comparative example 1 and examples 1 to 3 was measured using a universal tester, respectively: setting the operation compression rate of the universal testing machine to be 0.5mm/min, stopping compressing when the compression strain of the bracket reaches 60%, and recording the stress change of each bracket under the stress state when the bracket is wetted, wherein the result is shown in figure 1;
fig. 1 shows the compressive strength of the decellularized dermal matrix composite scaffold at a compressive strain of 60%, and as a result, as the content of the decellularized dermal matrix powder increases, the compressive strength of the composite scaffold increases and then decreases, and as the content of the decellularized dermal matrix powder increases, the mechanical strength of the composite scaffold prepared is highest.
(2) Porosity test:
the acellular dermal matrix composite scaffolds prepared in comparative example 1 and examples 1 to 3 were tested for porosity by an absolute ethanol weighing method: first, the weight of each rack in the dry state was weighed and recorded as W 0 The method comprises the steps of carrying out a first treatment on the surface of the Then the brackets are completely soaked in absolute ethyl alcohol for 10min, the brackets are taken out, the brackets are waited for no liquid to drop out, the weights of the brackets are weighed again and recorded, and the weight is recorded as W 1 The method comprises the steps of carrying out a first treatment on the surface of the The porosity of the scaffold was then calculated according to the following formula:
wherein: ρ is anhydrousDensity of ethanol (0.783 g/cm) 3 ) V is the volume of the scaffold.
The porosity results of the prepared acellular dermal matrix composite scaffold are shown in figure 2, and the greater the mixing amount of acellular dermal matrix powder, the higher the porosity of the obtained composite scaffold can be seen from figure 2.
(3) Microcosmic characterization:
the decellularized dermal matrix scaffolds prepared in comparative example 1 and example 2 were selected for morphology contrast analysis: fixing a bracket on a sample table by using conductive adhesive for metal spraying treatment, observing the appearance of a sample by using a Scanning Electron Microscope (SEM) under an acceleration voltage of 15.0kV, and measuring the aperture size of the bracket in a scanning electron microscope image, wherein the result is shown in figure 3;
as shown in fig. 3, it is observed under a scanning electron microscope that the decellularized dermal matrix scaffold prepared from the solution alone (i.e., the scaffold prepared in comparative example 1) has a sheet-like structure, a compact internal structure, and a small pore diameter, which is unfavorable for cells to grow into the scaffold; and the internal fibers of the composite scaffold prepared by mixing the powder and the solution (namely the composite scaffold prepared in the embodiment 2) are mutually crosslinked, the pore is uniformly distributed, and the average pore size is 106.9 mu m, so that the composite scaffold is more suitable for cell growth.
In summary, the invention provides a preparation method of a decellularized dermal matrix composite scaffold, which prepares the decellularized dermal matrix composite scaffold with high compression strength and porous structure by adjusting the mixing ratio of powder and solution on the premise of not changing the total amount of the decellularized dermal matrix, wherein the pore size of the composite scaffold meets the requirement of cell growth, and is beneficial to the cell growth into the scaffold.
The acellular dermal matrix composite scaffold prepared by the method not only improves the mechanical property of the scaffold, but also improves the pore structure of the scaffold, and is more beneficial to cell growth and proliferation.
The above-described preferred embodiments of the present invention are only for illustrating the present invention, and are not to be construed as limiting the present invention. Obvious changes and modifications of the invention, which are introduced by the technical solution of the present invention, are still within the scope of the present invention.
Claims (10)
1. The preparation method of the acellular dermal matrix composite scaffold is characterized by comprising the following steps of:
s1, preparation of dermis tissues: providing animal source skin tissue, cleaning the surface of the source skin tissue, soaking and cleaning to obtain dermis tissue;
s2, acellular treatment of the dermal matrix: sequentially placing the dermis tissues into a PMSF solution and an EDTA solution for stirring, taking out the dermis tissues for cleaning after stirring, and then repeating the stirring and cleaning processes for a plurality of times, and freeze-drying to obtain a acellular dermis matrix;
s3, preparing acellular dermal matrix powder: grinding the acellular dermal matrix to obtain acellular dermal matrix powder;
s4, preparing a acellular dermal matrix solution: putting the acellular dermal matrix into acetic acid solution, and stirring until the acellular dermal matrix is completely dissolved to obtain acellular dermal matrix solution;
s5, preforming the acellular dermal matrix composite scaffold: adding the acellular dermal matrix powder into the acellular dermal matrix solution, stirring uniformly, pouring into a mold, and freeze-drying to obtain a preformed acellular dermal matrix composite scaffold;
s6, chemical crosslinking: and (3) immersing the preformed acellular dermal matrix composite scaffold in a cross-linking agent for cross-linking, washing after cross-linking, and freeze-drying to obtain the acellular dermal matrix composite scaffold.
2. The method for preparing the acellular dermal matrix composite scaffold according to claim 1, wherein the steps of S1 and dermal tissue preparation are as follows: providing animal source skin tissue, then cleaning the source skin tissue with deionized water to remove surface impurities, removing surface animal hair, removing surface fat, soaking in normal saline for 10-24 hours to remove epidermis, and washing with deionized water to obtain dermis tissue.
3. The method for preparing a decellularized dermal matrix composite scaffold according to claim 1 or 2, wherein the animal-derived dermal tissue in step S1 is selected from the group consisting of porcine and bovine dermal tissue.
4. The method for preparing the acellular dermal matrix composite scaffold according to claim 1, wherein the step of S2 is performed by acellular treatment of dermal matrix: sequentially placing the dermis tissue into a PMSF solution with the concentration of 1.0-5.0 mg/ml and an EDTA solution with the concentration of 0.5-2.5 mg/ml, stirring at the speed of 200-500 rpm for 5-10 minutes, taking out the dermis tissue after stirring, flushing the dermis tissue with deionized water, repeating the stirring and cleaning processes for 2-5 times, and then placing the dermis tissue into a freeze dryer for freeze drying to obtain the acellular dermis matrix.
5. The method for preparing a decellularized dermal matrix composite scaffold according to claim 1 or 4, wherein the PMSF solution in step S2 is deionized water as a solvent; the EDTA solution takes PBS buffer solution as a solvent.
6. The method for preparing the acellular dermal matrix composite scaffold according to claim 1, wherein S3 is prepared from acellular dermal matrix powder: and (3) putting the acellular dermal matrix into a grinder, grinding for 3-5 minutes, and repeating grinding for 3-6 times to obtain acellular dermal matrix powder.
7. The method for preparing the acellular dermal matrix composite scaffold according to claim 1, wherein the preparation of the S4 acellular dermal matrix solution is as follows: the acellular dermal matrix is taken and put into acetic acid solution, and stirred at the speed of 200-500 rpm until the acellular dermal matrix is completely dissolved, so that acellular dermal matrix solution with the concentration of 0.1-1.0g/ml is obtained.
8. The method for preparing the acellular dermal matrix composite scaffold according to claim 1, wherein the step of S5 is performed by: adding the acellular dermal matrix powder into the acellular dermal matrix solution, uniformly stirring, pouring into a mold, and then placing into a freeze dryer for freeze drying to obtain a preformed acellular dermal matrix composite scaffold; wherein: the mass volume ratio of the acellular dermal matrix powder to the acellular dermal matrix solution is 0.1-1.0g/ml.
9. The method for preparing the acellular dermal matrix composite scaffold according to claim 1, wherein the steps of S6 and chemical crosslinking: immersing the preformed acellular dermal matrix composite stent in a cross-linking agent with the concentration of 0.05-0.25mg/ml for cross-linking for 1-3 hours, washing with deionized water after cross-linking, and then freeze-drying for 10-24 hours to obtain the acellular dermal matrix composite stent.
10. The method for preparing a decellularized dermal matrix composite scaffold according to claim 1 or 9, wherein the crosslinking agent in step S6 is one selected from an aldehyde solution, a genipin solution and a carbodiimide solution.
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