CN110960729B - Polysaccharide modified acellular matrix composite material, preparation and application - Google Patents
Polysaccharide modified acellular matrix composite material, preparation and application Download PDFInfo
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Abstract
The invention belongs to the technical field of acellular matrix, and particularly relates to a polysaccharide modified acellular matrix composite material, and preparation and application thereof. The preparation method comprises the following steps of degreasing, alkali treatment, enzyme treatment and freeze thawing of a fresh bladder matrix, compounding the fresh bladder matrix with polysaccharide, forming crosslinking by utilizing a plurality of amino groups on the polysaccharide and active groups on a cell-free bladder, improving the stability of a three-dimensional fiber network structure of the material, and grafting the polysaccharide to the cell-free bladder material, thereby endowing the cell-free matrix material with excellent biocompatibility. Wherein the polysaccharide comprises chitosan, hyaluronic acid and chondroitin sulfate. The invention adopts four-step methods of acellular treatment, polysaccharide compounding, non-toxic crosslinking and the like to prepare the modified acellular bladder matrix composite material, and can be used for constructing regenerative cornea materials, bladder materials, oral cavity repairing membranes and other tissue regenerative repairing materials.
Description
Technical Field
The invention belongs to the technical field of acellular matrix, and particularly relates to a polysaccharide modified acellular matrix composite material, and a preparation method and application of the composite material.
Background
The information in this background section is only for enhancement of understanding of the general background of the disclosure and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The acellular bladder matrix is made of heterogeneous organisms, has the advantages of wide source, low cost, low immunogenicity, similarity to bladder in function and the like, and is a hotspot material for bladder repair. However, the acellular porcine bladder matrix material prepared by the existing acellular method has the defects of poor mechanical property, urine seepage, uncontrollable degradation, poor biocompatibility and the like, and in addition, due to the use of chemical reagents in the acellular process, the original bioactive factors in the bladder are damaged, and the capacity of the acellular bladder matrix for inducing tissue regeneration is reduced.
The chitosan has excellent biocompatibility and biodegradability, and can stimulate immune cells to secrete cytokines and immune factors after being implanted into a body, accelerate cell proliferation and accelerate the fusion of the implanted scaffold material and tissues. Under the action of lysozyme in vivo, chitosan can be slowly degraded into oligosaccharide and monosaccharide, which can be absorbed by human body, and the porosity of matrix can be reduced by composite chitosan, thereby reducing the possibility of urine leakage during repair. The chondroitin sulfate is widely present on extracellular matrix and cell surface, has an anticoagulant effect, can cause the loss of the chondroitin sulfate in the acellular process, causes the performance reduction of the acellular bladder matrix, and can enhance the biocompatibility of the matrix by compounding the chondroitin sulfate on the matrix, thereby promoting the adhesion and proliferation of cells and inducing the tissue regeneration. Hyaluronic acid is widely present on tissues and organs, and has functions of protecting cells from being invaded by pathogenic bacteria, preventing infection, and accelerating wound recovery. The acellular bladder matrix can regulate the generation of collagen after being compounded with hyaluronic acid, and reduce the occurrence of graft contracture. In the previous research of the inventor, a method for modifying and modifying an acellular dermal matrix material by compounding water-soluble polysaccharide is provided, and the modified composite material can be used for preparing biological auxiliary materials, composite transplanting stents and the like and has good mechanical properties.
An ideal bladder repair material should have at least the following characteristics: 1. the preparation method has the advantages of good biocompatibility, no immunological rejection, 2. enough mechanical property is provided, the bladder is not broken when being filled with and emptied with urine, 3. the infiltration of the urine can be effectively prevented, the infiltration of the urine is avoided, the surrounding tissues are prevented from being eroded, 4. the neogenesis of blood vessels and tissues can be induced, and 5. the preparation method has a proper degradation rate. However, the acellular bladder matrix prepared by the existing method generally has the defects of urine seepage, low mechanical strength, easy degradation and the like, so a new idea and a new preparation process need to be developed to prepare the modified acellular bladder matrix tissue engineering repair material with excellent physical and chemical properties.
Disclosure of Invention
Against the background of the above research, the inventors believe that the mechanical strength of the acellular bladder matrix is improved by modification, so that the leakage rate of the acellular bladder material can be effectively reduced, and the acellular bladder material can be applied to bladder repair and the like. Aiming at the defects of the cell-free bladder matrix adopted at present, the invention treats the bladder by modification and crosslinking, can reduce the antigenicity, improve the degradation resistance and biocompatibility, and enhance the capability of inducing tissue regeneration, and has huge application prospect. Aiming at the research result, the invention provides the following technical scheme:
in a first aspect, a method for preparing a modified acellular bladder matrix composite is provided, the method comprising the steps of acellular treatment of bladder tissue and complexing the acellular treated bladder matrix with a water-soluble polysaccharide, the acellular treatment comprising treatment with a base solution, a surfactant and a protease.
Preferably, the cell-free treatment comprises the following specific steps:
adding alkali liquor and surfactant components into the bladder tissue without fat and fascia according to the feed-liquid ratio of 3-5: 1 for acting for a period of time; dealkalizing the bladder tissue by a saline solution and an acid solution; adding protease into the dealkalized pig bladder to remove residual cells, and freezing and thawing.
Further preferably, the alkali liquor is mixed with a surfactant.
In some specific embodiments, the surfactant is Trition X-100 or SDS and the base is NaOH.
In some specific embodiments, the concentration of the surfactant is 0.3% to 0.4%.
In some specific embodiments, the concentration of the NaOH solution is 0.2% to 1.0%.
In some embodiments, the action time of the alkali solution and the surfactant is 13-18 hours.
Further preferably, the dealkalization comprises the following specific steps: adding a saline solution into the bladder after alkali treatment according to the material-liquid ratio of 3-5: 1, and keeping the pH of the solution at 8-8.5 by using hydrochloric acid.
In some specific embodiments, the salt solution is NH with a concentration of 1.8-2.2%4And (4) Cl solution.
Further preferably, the protease is trypsin or neutral protease.
Preferably, the step of compounding the bladder matrix after the cell-free treatment by using the water-soluble polysaccharide specifically comprises the following steps:
sequentially adding chitosan, glutaraldehyde and chondroitin sulfate into the cell-free bladder matrix after freeze thawing for crosslinking, dehydrating to obtain a cell-free bladder matrix material compounded with chitosan and chondroitin sulfate, and coating a hyaluronic acid solution on the surface of the matrix material.
Preferably, a chitosan solution with the concentration of 0.1-0.4% is added into the freeze-thawed acellular bladder matrix according to the material-liquid ratio of 2-3: 1, the pH is adjusted to 5-6, reaction is carried out for 20-40 min, and then the pH is adjusted to 7.5-8.0, and reaction is carried out for 60-90 min.
Further preferably, the concentration of the glutaraldehyde solution is 0.01-0.04%, the feed-liquid ratio is 1.5-2.5:1, and the pH is adjusted to 8.0 in the reaction process.
Further preferably, the concentration of the chondroitin sulfate is 0.1% -0.4%, the feed-liquid ratio is 3-4:1, and the reaction time is 60-90 min.
Further preferably, the hyaluronic acid solution is prepared by the following method: dissolving hyaluronic acid in dilute acetic acid solution to prepare 0.8-1.2% w/v transparent solution.
Preferably, the preparation method further comprises the steps of washing, freeze-drying and sterilizing the matrix material after the hyaluronic acid is compounded.
Further preferably, the sterilization is performed by irradiation with cobalt 60.
In a second aspect, there is provided a modified acellular bladder matrix composite obtained by the method for preparing a modified acellular bladder matrix composite according to the first aspect.
In a third aspect, the modified acellular bladder matrix composite material of the second aspect is applied to bladder repair materials, regenerated cornea materials, oral repair materials and the like.
Compared with the prior art, the beneficial effect of this disclosure is:
1. aiming at the defects of the acellular bladder matrix in the prior art in the aspect of mechanical property, the invention provides a method for grafting polysaccharide to the acellular bladder matrixThe modified acellular bladder matrix composite material has sufficient space maintenance capacity, better compactness and stronger urine permeation resistance. Experiments show that after chitosan is compounded, the porosity index of the material is reduced, the seepage quantity is reduced, and the seepage quantity is 0.07-0.14 cc/cm2And the urine permeation can be resisted between h, so that the urine permeation is prevented during the restoration.
2. In addition, the capability of the modified acellular bladder matrix composite material for resisting enzymes is remarkably enhanced, experiments show that the concentration of glutaraldehyde has certain influence on the in-vitro degradability of the modified acellular bladder matrix composite material, and the capability of the material for resisting collagenase degradation is remarkably enhanced along with the increase of the concentration of the glutaraldehyde, so that the repair time of the material can be prolonged. The stable three-dimensional fiber network structure formed by modification and the adjustable pore size have wide application space, and technicians can construct tissue regeneration repair materials such as regenerative cornea materials, bladder materials, oral cavity repair films and the like according to different requirements.
3. The invention also optimizes the bladder cell-removing treatment method aiming at the composite material, so that the composite material is more suitable for the later grafting modification. The bladder matrix cells subjected to the decellularization treatment are removed more thoroughly, and the bladder matrix cells are lower in immunogenicity and better in biocompatibility.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the disclosure and not to limit the invention.
Figure 1 is a particle size plot (SEM 1500) of the modified acellular bladder matrix composite of example 1;
figure 2 is a cross-sectional view (SEM 2000) of the modified acellular bladder matrix composite of example 1;
FIG. 3 is a hematoxylin-eosin staining pattern of the modified acellular bladder matrix composite of example 1.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. The "concentrations" described in the following examples, unless otherwise specified, all represent mass fractions; "ratio of material to liquid" refers to the volume ratio of material to liquid.
As introduced by the background art, aiming at the defects in the prior art, the invention provides a polysaccharide modified acellular matrix composite material, a preparation method and application thereof.
In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific examples and comparative examples.
Example 1
The implementation provides a modified acellular bladder matrix composite material containing polysaccharide, which is prepared by the following steps:
(1) cutting off large fat, fascia and other tissues of the commercially available bladder, repeatedly washing the bladder by using a PBS (phosphate buffer solution) and weighing the bladder as a material measuring basis in the following working procedures;
(2) alkali treatment: adding NaOH with the mass concentration of 0.4% and Triton X-100 solution (the mass concentrations of the NaOH and the Triton X-100 in the solution are both 0.4%) at the temperature of 28 ℃ according to the ratio of the NaOH to the feed liquid of the pig bladder of 3:1, acting for 16h, fully removing the fibrous stroma and removing the cells; and adding distilled water according to the feed-liquid ratio of 1:4, and washing for three times, wherein each time lasts for 15 min.
(3) Eliminating alkali liquor: adding 2% NH at 30 deg.C and 3.0 mass/liquid ratio of pig bladder4CL solution. Controlling pH value at 8-8.5, acting for 1.5h, measuring pH value every half an hour, and adjusting pH value with dilute hydrochloric acid. And adding distilled water according to the feed-liquid ratio of 4.0 to wash for 15min twice.
(4) Enzyme treatment: adding trypsin with the mass concentration of 0.4% into the pig bladder feed liquid at the temperature of 40 ℃ according to the ratio of 4:1, allowing the trypsin to act for 30min, adding 1398 neutral protease to allow the mass concentration of the neutral protease in the solution to be 0.2%, allowing the neutral protease to act for 90min, and sufficiently removing residual cells; after the completion, distilled water is added according to the feed-liquid ratio of 4:1 to wash for two times, each time for 15 min.
(5) Freezing and thawing: freezing the enzyme-treated pig bladder in a refrigerator at-20 deg.C for 8 hr, and thawing at room temperature.
(6) And (3) composite chitosan: and (3) adding a chitosan solution with the mass concentration of 0.4% into the acellular bladder matrix obtained in the step (5), adjusting the material-liquid ratio to be 3.0, the temperature to be 30 ℃, adjusting the rotating speed of the rotary drum to react for 30min, adjusting the pH value to be 7.5-8.0, reacting for 60min, discharging waste liquid after the treatment is finished, adding distilled water with the volume being four times that of the treated material, and washing for 15min twice each time.
(7) And (3) glutaraldehyde crosslinking: adding glutaraldehyde solution into the material obtained in the step (6) according to the material-liquid ratio of 2.5:1, adjusting the temperature to 30 ℃, reacting for 90min under the condition of pH6.5, gradually adjusting the pH value to 8.0 in the process, and washing the material by using a large amount of distilled water until the pH value is about 7.0 after the reaction is finished.
(8) Compound chondroitin sulfate: adding 0.4% chondroitin sulfate solution at a ratio of material to liquid of 3:1, adjusting the temperature to 30-40 deg.C, reacting for 60-90min, and washing with distilled water twice at a ratio of material to liquid of 4:1 for 15min each time.
(9) Composite hyaluronic acid: fully dissolving hyaluronic acid in dilute acetic acid solution to prepare transparent solution with the concentration of 1.00 percent, centrifugally dehydrating the acellular bladder matrix material compounded with chitosan and chondroitin sulfate in the step (8) to ensure that the volume water content is 70-90 percent, and then spraying the hyaluronic acid solution on the surface of the acellular bladder matrix material by using a small spray bottle, wherein the spraying amount is 1g/m2。
(10) Washing with PBS buffer: adding PBS buffer solution according to the material-liquid ratio of 4:1, and washing twice, each time for 15 min.
(11) Freeze-drying: freezing in-20 deg.C refrigerator for 8 hr, adjusting freeze dryer temperature to-40 deg.C and air pressure to 4Pa, and vacuum freeze drying for 8 hr.
(12) And (3) sterilization: sterilizing by cobalt 60 irradiation.
Example 2
The implementation provides a modified acellular bladder matrix composite material containing polysaccharide, which is prepared by the following steps:
(1) cutting off large fat, fascia and other tissues of the commercially available bladder, repeatedly washing the bladder by using a PBS (phosphate buffer solution) and weighing the bladder as a material measuring basis in the following working procedures;
(2) alkali treatment: adding NaOH with the mass concentration of 0.3% and Triton X-100 solution (the mass concentrations of the NaOH and the Triton X-100 in the solution are both 0.3%) at the temperature of 28 ℃ according to the ratio of 5:1 to the feed liquid of the porcine bladder, acting for 13h, fully removing the fibrous stroma and removing the cells; after the completion, distilled water is added according to the feed-liquid ratio of 4:1 for washing for three times, and each time lasts for 15 min.
(3) Eliminating alkali liquor: adding 1.8% NH at 30 deg.C and 5:1 mass/liquid ratio of pig bladder4And (4) Cl solution. Controlling pH value at 8-8.5, acting for 1.5h, measuring pH value every half an hour, and adjusting pH value with dilute hydrochloric acid. After the completion, distilled water is added according to the feed-liquid ratio of 4:1 to wash for two times, each time for 15 min.
(4) Enzyme treatment: adding trypsin with the mass concentration of 0.4% into the pig bladder feed liquid according to the ratio of 4:1 at the temperature of 40 ℃ for acting for 30min, then adding 1398 neutral protease to enable the mass concentration of the neutral protease in the solution to be 0.2%, and acting for 90min to fully remove residual cells; after the completion, distilled water is added according to the feed-liquid ratio of 4:1 to wash for two times, each time for 15 min.
(5) Freezing and thawing: freezing the enzyme-treated pig bladder in a refrigerator at-20 deg.C for 8 hr, and thawing at room temperature.
(6) And (3) composite chitosan: adding a chitosan solution with the mass concentration of 0.1% into the acellular bladder matrix obtained in the step (5), adjusting the material-liquid ratio to be 3:1, the temperature to be 30 ℃, adjusting the rotating speed of the rotary drum to react for 30min, adjusting the pH value to be 7.5-8.0, reacting for 60min, discharging the waste liquid after the treatment is finished, adding distilled water with the volume being four times that of the treated material, and washing for 15min twice each time.
(7) And (3) glutaraldehyde crosslinking: adding glutaraldehyde solution into the material obtained in the step (6) according to the material-liquid ratio of 2.5:1, adjusting the temperature to 30 ℃, reacting for 90min under the condition of pH6.5, gradually adjusting the pH value to 8.0 in the process, and washing the material by using a large amount of distilled water until the pH value is about 7.0 after the reaction is finished.
(8) Compound chondroitin sulfate: adding 0.4% chondroitin sulfate solution at a ratio of material to liquid of 3:1, adjusting the temperature to 30-40 deg.C, reacting for 60-90min, and washing with distilled water twice at a ratio of material to liquid of 4:1 for 15min each time.
(9) Composite hyaluronic acid: fully dissolving hyaluronic acid in dilute acetic acid solution to prepare 0.8% transparent solution, centrifugally dehydrating the acellular bladder matrix material compounded with chitosan and chondroitin sulfate in the step (8) to ensure that the volume water content is 70-90%, and spraying the hyaluronic acid solution on the surface of the acellular bladder matrix material by using a small spray bottle, wherein the spraying amount is 1.1g/m2。
(10) Washing with PBS buffer: adding PBS buffer solution according to the feed-liquid ratio of 4.0, and washing twice for 15min each time.
(11) Freeze-drying: freezing in-20 deg.C refrigerator for 8 hr, adjusting freeze dryer temperature to-40 deg.C and air pressure to 4Pa, and vacuum freeze drying for 8 hr.
(12) And (3) sterilization: sterilizing by cobalt 60 irradiation.
Example 3
The implementation provides a modified acellular bladder matrix composite material containing polysaccharide, which is prepared by the following steps:
(1) cutting off large fat, fascia and other tissues of the commercially available bladder, repeatedly washing the bladder by using a PBS (phosphate buffer solution) and weighing the bladder as a material measuring basis in the following working procedures;
(2) alkali treatment: adding NaOH and Triton X-100 solutions with the mass concentration of 0.38% (the mass concentrations of NaOH and Triton X-100 in the solutions are both 0.38%) at the temperature of 28 ℃ according to the ratio of the NaOH to the feed liquid of the pig bladder of 3:1, acting for 18h, fully removing the fibrous interstitium and removing cells; and adding distilled water according to the feed-liquid ratio of 1:4, and washing for three times, wherein each time lasts for 15 min.
(3) Eliminating alkali liquor: adding NH with the concentration of 2.2 percent according to the mass ratio of the pig bladder to the feed liquid of 3:1 and the temperature of 30 DEG C4And (4) Cl solution. Controlling pH value at 8-8.5, acting for 1.5h, measuring pH value every half an hour, and adjusting pH value with dilute hydrochloric acid. After the completion, distilled water is added according to the feed-liquid ratio of 4:1 to wash for two times, each time for 15 min.
(4) Enzyme treatment: adding trypsin at 40 ℃ according to a ratio of 4:1 to the feed liquid of the pig bladder to enable the mass concentration of the trypsin to be 0.4% for 30min, then adding 1398 neutral protease to enable the mass concentration of the neutral protease in the solution to be 0.2%, and enabling the neutral protease to act for 90min to fully remove residual cells; after the completion, distilled water is added according to the feed-liquid ratio of 4:1 to wash for two times, each time for 15 min.
(5) Freezing and thawing: freezing the enzyme-treated pig bladder in a refrigerator at-20 deg.C for 8 hr, and thawing at room temperature.
(6) And (3) composite chitosan: adding a chitosan solution with the mass concentration of 0.4% into the acellular bladder matrix obtained in the step (5), adjusting the material-liquid ratio to be 3:1, the temperature to be 30 ℃, adjusting the rotating speed of the rotary drum to react for 30min, adjusting the pH value to be 7.5-8.0, reacting for 60min, discharging the waste liquid after the treatment is finished, adding distilled water with the volume being four times that of the treated material, and washing for 15min twice each time.
(7) And (3) glutaraldehyde crosslinking: adding glutaraldehyde solution into the material obtained in the step (6) according to the material-liquid ratio of 2.5:1, adjusting the temperature to 30 ℃, reacting for 90min under the condition of pH6.5, gradually adjusting the pH value to 8.0 in the process, and washing the material by using a large amount of distilled water until the pH value is about 7.0 after the reaction is finished.
(8) Compound chondroitin sulfate: adding 0.4% chondroitin sulfate solution at a ratio of material to liquid of 3:1, adjusting the temperature to 30-40 deg.C, reacting for 60-90min, and washing with distilled water twice at a ratio of material to liquid of 4:1 for 15min each time.
(9) Composite hyaluronic acid: fully dissolving hyaluronic acid in dilute acetic acid solution to obtain 1.00% transparent solution, and centrifuging and dehydrating the acellular bladder matrix material (8) compounded with chitosan and chondroitin sulfate to make the volume of the acellular bladder matrix materialThe water content is 70-90 percent, and then the hyaluronic acid solution is sprayed on the surface of the acellular bladder matrix material by using a small spray bottle, wherein the spraying amount is 0.8g/m2。
(10) Washing with PBS buffer: adding PBS buffer solution according to the material-liquid ratio of 4:1, and washing twice, each time for 15 min.
(11) Freeze-drying: freezing in-20 deg.C refrigerator for 8 hr, adjusting freeze dryer temperature to-40 deg.C and air pressure to 4Pa, and vacuum freeze drying for 8 hr.
(12) And (3) sterilization: sterilizing by cobalt 60 irradiation.
Through determination, the main technical indexes of the modified acellular bladder matrix composite prepared in the examples 1-3 are shown in the following table 1:
TABLE 1
| Item | Performance index |
| Porosity index (cc/cm 2 h) | ≤0.11 |
| Tensile Strength (MPa) | ≥8.15 |
| 48h degradation (%) | ≤66.7 |
| Hematoxylin-eosin staining | Cell residue is not seen |
| Observation by scanning electron microscope | Collagen is arranged orderly without cell residue |
| In vitro cytotoxicity | Cytotoxicity response is not more than grade 1 |
Example 4
The present example provides a modified acellular bladder matrix composite containing growth factors, prepared by the steps of:
(1) a modified acellular bladder matrix composite was prepared according to the procedure of example 1;
(2) compounding the material with 10 mu g/m25 μ g/m of vascular endothelial growth factor2The nerve growth factor of (1). The modified acellular bladder matrix composite prepared by the method can promote the generation of blood vessels and nerves and promote the recovery of tissues.
The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure, and various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.
Claims (8)
1. A preparation method of a modified acellular bladder matrix composite material is characterized by comprising the steps of carrying out acellular treatment on bladder tissues and compounding the acellular treated bladder matrix by using water-soluble polysaccharide, wherein the acellular treatment comprises treatment by using alkali liquor, a surfactant and protease;
the cell-free treatment comprises the following specific steps: adding alkali liquor and surfactant components into the bladder tissue without fat and fascia according to the feed-liquid ratio of 3-5: 1 for acting for a period of time; dealkalizing the bladder tissue by a saline solution and an acid solution; adding protease into the dealkalized pig bladder to remove residual cells, and freezing and thawing;
the alkali liquor is mixed with a surfactant; the surfactant is Trition X-100 or SDS, and the alkali is NaOH; the mass concentration of the surfactant is 0.3% -0.4%; the mass concentration of the NaOH solution is 0.2-1.0%; the action time of the alkali liquor and the surfactant is 13-18 h;
the step of compounding the bladder matrix after the cell-free treatment by adopting the water-soluble polysaccharide comprises the following specific steps:
sequentially adding chitosan, glutaraldehyde and chondroitin sulfate into the cell-free bladder matrix after freeze thawing for crosslinking, dehydrating to obtain a cell-free bladder matrix material compounded with chitosan and chondroitin sulfate, and coating a hyaluronic acid solution on the surface of the matrix material.
2. The method for preparing the modified acellular bladder matrix composite according to claim 1, wherein the dealkalization comprises the following specific steps: adding a salt solution into the bladder after alkali treatment according to the material-liquid ratio of 3-5: 1, and keeping the pH of the solution at 8-8.5 by using hydrochloric acid; or the salt solution is NH with the mass concentration of 2%4And (4) Cl solution.
3. The method of preparing the modified acellular bladder matrix composite of claim 1, wherein the protease is trypsin or neutral protease.
4. The method of preparing a modified acellular bladder matrix composite according to claim 1,
adding a chitosan solution with the mass concentration of 0.1-0.4% into the freeze-thawed acellular bladder matrix according to the material-liquid ratio of 2-3: 1, adjusting the pH to 5-6, reacting for 20-40 min, and adjusting the pH to 7.5-8.0, and reacting for 60-90 min;
or the mass concentration of the glutaraldehyde solution is 0.01-0.04%, and the material-liquid ratio is 1.5-2.5:1, adjusting the pH to 8.0 in the reaction process;
or the mass concentration of the chondroitin sulfate is 0.1-0.4%, and the material-liquid ratio is 3.0-4.0: 1, the reaction time is 60-90 min;
or the preparation method of the hyaluronic acid solution comprises the following steps: hyaluronic acid was dissolved in dilute acetic acid solution to make a 1.00% w/v clear solution.
5. The method of preparing a modified acellular bladder matrix composite according to claim 4, further comprising the steps of washing, lyophilizing and sterilizing the matrix material after complexing the hyaluronic acid.
6. The method of preparing the modified acellular bladder matrix composite of claim 5, wherein the sterilization is performed by cobalt 60 irradiation.
7. A modified acellular bladder matrix composite obtainable by the process for the preparation of a modified acellular bladder matrix composite according to any one of claims 1 to 6.
8. Use of the modified acellular bladder matrix composite material according to claim 7 as bladder repair material, regenerative corneal material, oral repair material.
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