CN109833119B - Crosslinking method for improving biocompatibility of acellular matrix - Google Patents
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Abstract
The invention discloses a crosslinking method for improving biocompatibility of an acellular matrix, wherein the acellular matrix needs to be cleaned before use, the method comprises the steps of soaking the cleaned acellular matrix in a glycidyl methacrylate solution, cleaning the acellular matrix soaked in the glycidyl methacrylate solution, crosslinking the acellular matrix soaked in the glycidyl methacrylate solution through a polymerization reaction, and cleaning the crosslinked acellular matrix; the biological tissue obtained by the crosslinking method has the advantages of improving the stability of elastin in the acellular matrix, improving the anti-calcification performance of the acellular matrix and improving the biocompatibility of the crosslinking acellular matrix material.
Description
Technical Field
The invention relates to a cross-linking treatment method of an acellular matrix, and the technical field of biological materials and medical instruments prepared by applying the cross-linking acellular matrix.
Background
As the population ages severely, the incidence of valvular heart disease is increasing. Valvular heart disease is a common valvular failure disease. Mainly manifested as stenosis at the heart valve or valvular insufficiency.
The primary treatment for valvular heart disease is heart valve replacement. The operation modes are mainly divided into an open chest valve replacement operation and an intervention valve replacement operation. The thoracotomy operation causes great trauma to patients, has high risk and slow recovery, needs extracorporeal circulation support, and can not be used for thoracotomy operations of many patients, especially the old patients. The interventional valve replacement surgery has small trauma to patients and low risk, and is the main development trend of the future valve replacement surgery.
Heart valves are largely divided into mechanical heart valves and biological heart valves. Patients who replace the mechanical valve need to take anticoagulant drugs for a long time to reduce the occurrence of blood coagulation at the mechanical valve due to poor biocompatibility of the mechanical valve. But at the same time, is also accompanied by a risk of bleeding. And replacement of the mechanical valve requires an open chest surgery, causing a great trauma to the patient. The biological valve is a very potential valve type due to the good biocompatibility and the development of an interventional replacement technology.
Biological heart valves currently in commercial use are typically prepared from bovine pericardium, porcine heart valves, and porcine pericardium by cross-linking glutaraldehyde. The glutaraldehyde crosslinking method has the advantages of simple process, low cost, stable collagen structure and better mechanical property. Glutaraldehyde cross-linked heart valves, however, present significant problems. The glutaraldehyde crosslinking can not protect the elastin, so that the mechanical property of the heart valve is easy to be reduced; the glutaraldehyde cross-linked heart valve is easy to calcify, which affects the service life; glutaraldehyde and residual aldehyde groups have strong toxicity, and are easy to cause inflammatory reaction, so that the valve fails.
Therefore, by changing the acellular matrix crosslinking method, on the basis of keeping the original crosslinking effect, the protection effect of elastin is improved, the calcification reaction is reduced, and the biocompatibility is improved, so that the method has great significance for the scientific research of biological heart valves and the development of related industrial applications.
Disclosure of Invention
The present invention aims to solve the above-mentioned deficiencies of the prior art and to provide a crosslinking method for improving the biocompatibility of an acellular matrix, which has the advantages of improving the stability of elastin in the acellular matrix, improving the anti-calcification performance of the acellular matrix and improving the biocompatibility of a crosslinked acellular matrix material.
The purpose of the invention is realized by the following technical scheme.
A crosslinking method for improving the biocompatibility of an acellular matrix specifically comprises the following steps:
1. obtaining fresh animal tissue material, and storing at low temperature of 4 deg.C;
2. decellularizing the animal tissue material to form a decellularized matrix;
3. placing the acellular matrix into a cleaning solution prepared by distilled water and alcohol according to a certain proportion, and cleaning the acellular matrix by ultrasonic cleaning at room temperature;
4. soaking the cleaned acellular matrix in 1-10% glycidyl methacrylate solution at the temperature of 20-45 ℃ and continuously reacting for 72-240 hours to combine the acellular matrix and the glycidyl methacrylate through chemical bonds;
5. cleaning the acellular matrix;
6. thermally initiating the double bonds connected into the acellular matrix for 12-48 hours at the temperature of 20-45 ℃ or polymerizing for 5-30 minutes at room temperature in a light initiation mode by an ultraviolet lamp light source through a thermally initiated polymerization initiator;
7. and cleaning the polymerized acellular matrix.
Further, the animal tissue in step 1 is derived from pig and cattle, and the site of the animal tissue comprises one or more of pericardium, valve, intestinal membrane, meninges, pulmonary membrane, blood vessel, small intestine, large intestine, skin and ligament.
Further, the main component of the acellular matrix in the step 2 is one or more of collagen, elastin, hyaluronic acid and gelatin.
Further, the acellular matrix in the step 4 is bonded with glycidyl methacrylate through one of a reaction of an amino group and an epoxy group, a reaction of a carboxyl group and an epoxy group, and a reaction of a hydroxyl group and an epoxy group.
Further, in the step 6, the thermal initiation polymerization initiator includes an organic peroxide initiator, an inorganic peroxide initiator and an azo initiator, the photo-initiated polymerization initiator includes 2-hydroxy-2-methyl-1-phenylpropanone, 1-hydroxycyclohexyl phenyl ketone, 2-methyl-2- (4-morpholinyl) -1- [4- (methylthio) phenyl ] -1-propanone, 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone, 2,4, 6-trimethylbenzoyl-diphenylphosphine oxide, and 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone.
The invention has the beneficial effects that:
1. the invention can improve the stability of elastin in the acellular matrix, potentially improve the mechanical property of the acellular matrix material and prolong the service life of the material.
2. The invention can improve the anti-calcification performance of the acellular matrix.
3. The invention can reduce the cytotoxicity and immunoreaction of the cross-linking acellular matrix and improve the biocompatibility of the cross-linking acellular matrix material.
Drawings
FIG. 1 shows a preparation process and a linkage manner of glycidyl methacrylate and acellular matrix according to the present invention;
fig. 2 is a schematic of the staining of elastin fibers with victoria blue.
FIG. 3 is a schematic representation of alizarin red staining for calcium.
FIG. 4 is a schematic representation of a Masson stain;
fig. 5 is a statistical data of fiber pocket density.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Furthermore, in all cases, the fresh animal tissue material was from a local slaughterhouse.
Example 1:
the treatment method of this example is as follows:
1. obtaining a fresh pig heart envelope, and storing the pig heart envelope in a wet state at 4 ℃;
2. decellularizing the animal tissue material to form a decellularized matrix;
3. cleaning the acellular matrix;
4. soaking the cleaned acellular matrix in 1% glycidyl methacrylate solution at the mass concentration for reaction for 72 hours at 25 ℃;
5. cleaning the acellular matrix;
6. polymerizing double bonds connected into the acellular matrix in a thermal initiation mode, specifically immersing the acellular matrix into 5mM potassium persulfate solution, and reacting for 24 hours at 40 ℃;
7. and cleaning the polymerized acellular matrix.
According to the treatment method provided by the embodiment, in the step 3, the mixed solution of distilled water and alcohol is used for ultrasonic cleaning for multiple times, and impurities introduced in the cell removing process are washed clean.
According to the treatment method provided by the embodiment, in the step 4, the acellular matrix needs to be vibrated and fully contacted with the glycidyl methacrylate solution, so that the epoxy group of the glycidyl methacrylate fully reacts with the residue in the acellular matrix, the content of the accessed double bond is increased, the crosslinking density is increased, and the crosslinked acellular matrix material is more stable.
According to the treatment method provided in this example, in step 5, the unreacted free glycidyl methacrylate introduced in step 4 is cleaned by ultrasonic cleaning for a plurality of times using a mixed solution of distilled water and alcohol.
According to the processing method provided in this example, in step 6, the initiator concentration, reaction temperature and reaction time are selected to be suitable for the polymerization reaction to occur, and the polymerization degree is large enough to increase the crosslinking density and the stability of the acellular matrix structure.
According to the treatment method provided by the present example, in step 7, distilled water is used for ultrasonic cleaning for multiple times, and impurities such as the initiator introduced in step 6 are cleaned.
According to the treatment method provided in this example, the prepared cross-linked acellular matrix protected elastin by hydrogen bonding between glycidyl methacrylate and elastin hydrophobic segment. In addition, the treatment method provided by the embodiment can improve the crosslinking density and promote the stabilization of the elastin.
According to the treatment method provided by the example, the prepared cross-linked acellular matrix does not introduce glutaraldehyde, and simultaneously shields most residues in the acellular matrix, so that the acellular matrix material has reduced capacity of binding calcium ions, and the calcification reaction is reduced.
According to the treatment method provided in this example, the prepared cross-linked acellular matrix does not incorporate a cross-linking agent, glutaraldehyde, which is a biologically toxic cross-linking agent, but instead a biocompatible cross-linking agent, thereby reducing the cytotoxicity and immunoreaction of the cross-linked acellular matrix material.
Example 2:
1. obtaining a fresh pig heart envelope, and storing the pig heart envelope in a wet state at 4 ℃;
2. decellularizing the animal tissue material to form a decellularized matrix;
3. cleaning the acellular matrix;
4. soaking the cleaned acellular matrix in a glycidyl methacrylate solution with the mass concentration of 3% to react for 120 hours at 25 ℃;
5. cleaning the acellular matrix;
6. polymerizing double bonds connected into the acellular matrix in a thermal initiation mode, specifically immersing the acellular matrix into 5mM potassium persulfate solution, and reacting for 24 hours at 40 ℃;
7. and cleaning the polymerized acellular matrix.
Example 3:
1. obtaining a fresh pig heart envelope, and storing the pig heart envelope in a wet state at 4 ℃;
2. decellularizing the animal tissue material to form a decellularized matrix;
3. cleaning the acellular matrix;
4. soaking the cleaned acellular matrix in a glycidyl methacrylate solution with the mass concentration of 5% to react for 240 hours at 25 ℃;
5. cleaning the acellular matrix;
6. polymerizing double bonds connected into the acellular matrix in a thermal initiation mode, specifically, immersing the acellular matrix into a 20mM potassium persulfate solution, and reacting for 24 hours at 40 ℃;
7. and cleaning the polymerized acellular matrix.
Example 4:
1. obtaining fresh porcine vascular tissue, and storing the porcine vascular tissue in a humid state at 4 ℃;
2. decellularizing the animal tissue material to form a decellularized matrix;
3. cleaning the acellular matrix;
4. soaking the cleaned acellular matrix in 5% glycidyl methacrylate solution at the mass concentration for reacting for 168 hours at 25 ℃;
5. cleaning the acellular matrix;
6. polymerizing double bonds connected into the acellular matrix in a thermal initiation mode, specifically, immersing the acellular matrix into a 20mM potassium persulfate solution, and reacting for 24 hours at 40 ℃;
7. and cleaning the polymerized acellular matrix.
Example 5:
1. obtaining a fresh pig heart envelope, and storing the pig heart envelope in a wet state at 4 ℃;
2. decellularizing the animal tissue material to form a decellularized matrix;
3. cleaning the acellular matrix;
4. soaking the cleaned acellular matrix in a glycidyl methacrylate solution with the mass concentration of 5% to react for 240 hours at 25 ℃;
5. cleaning the acellular matrix;
6. polymerizing double bonds connected into an acellular matrix in a photoinitiated mode, specifically, immersing the acellular matrix into 1 mass percent of Irgacure2959 solution, and immersing for 24 hours at 40 ℃;
7. placing the acellular matrix in an ultraviolet crosslinking box to irradiate for 10 minutes;
8. and cleaning the polymerized acellular matrix.
Examples of the experiments
As shown in FIG. 1, the preparation process and the linking mode of glycidyl methacrylate and acellular matrix are shown.
As shown in fig. 2, in the treatment method provided by the present invention, after the cross-linked acellular matrix and the traditional glutaraldehyde cross-linked acellular matrix are degraded by elastase, elastin in the acellular matrix can be effectively protected, wherein PGMA is the cross-linked acellular matrix prepared by the present invention, and GLUT is the glutaraldehyde cross-linked acellular matrix.
As shown in figure 3, the treatment method provided by the invention can effectively reduce calcification reaction after subcutaneous implantation of the cross-linked acellular matrix and the traditional glutaraldehyde cross-linked acellular matrix for 30 days, wherein PGMA is the cross-linked acellular matrix prepared by the invention, and GLUT is the glutaraldehyde cross-linked acellular matrix.
As shown in figure 4, the cross-linked acellular matrix and the traditional glutaraldehyde cross-linked acellular matrix are implanted subcutaneously for 30 days, so that the immunoreaction of the material after the material is implanted subcutaneously into animals can be effectively reduced, the asterisk in figure 4 is the material, and the other side of the red line is a fibrous capsule; in addition, the degree of fibrous capsule compaction in FIG. 4 may reflect the degree of immune response.
After the cross-linked acellular matrix prepared by the invention and the traditional glutaraldehyde cross-linked acellular matrix are implanted subcutaneously for 30 days, statistical data of the density of fibrous capsules in a Masson dyed section are shown in figure 5:
where the dotted line is PGMA and the solid line is GLUT.
The invention has the beneficial effects that:
1. the invention can improve the stability of elastin in the acellular matrix, potentially improve the mechanical property of the acellular matrix material and prolong the service life of the material.
2. The invention can improve the anti-calcification performance of the acellular matrix.
3. The invention can reduce the cytotoxicity and immunoreaction of the cross-linking acellular matrix and improve the biocompatibility of the cross-linking acellular matrix material.
The above are only typical examples of the present invention, and besides, the present invention may have other embodiments, and all the technical solutions formed by equivalent substitutions or equivalent changes are within the scope of the present invention as claimed.
Claims (5)
1. A crosslinking method for improving biocompatibility of an acellular matrix, which is characterized by improving the stability of elastin in the acellular matrix, comprises the following steps:
1. obtaining fresh animal tissue material, and storing at low temperature of 4 deg.C;
2. decellularizing the animal tissue material to form a decellularized matrix;
3. placing the acellular matrix into a cleaning solution prepared by distilled water and alcohol according to a certain proportion, and cleaning the acellular matrix by ultrasonic cleaning at room temperature;
4. soaking the cleaned acellular matrix in 1-10% glycidyl methacrylate solution at the temperature of 20-45 ℃ and continuously reacting for 72-240 hours to combine the acellular matrix and the glycidyl methacrylate through chemical bonds;
5. cleaning the acellular matrix;
6. thermally initiating the double bonds connected into the acellular matrix for 12-48 hours at the temperature of 20-45 ℃ or polymerizing for 5-30 minutes at room temperature in a light initiation mode by an ultraviolet lamp light source through a thermally initiated polymerization initiator;
7. and cleaning the polymerized acellular matrix.
2. The crosslinking method of claim 1, wherein the crosslinking method comprises the steps of: the animal tissue in step 1 is from pig and cattle, and the animal tissue site comprises one or more of pericardium, valve, intestinal membrane, meninges, pulmonary membrane, blood vessel, small intestine, large intestine, skin and ligament.
3. The crosslinking method of claim 1, wherein the crosslinking method comprises the steps of: the main components of the acellular matrix in the step 2 are one or more of collagen, elastin, hyaluronic acid and gelatin.
4. The crosslinking method of claim 1, wherein the crosslinking method comprises the steps of: the acellular matrix in the step 4 is combined with the glycidyl methacrylate through one of the reaction of an amino group and an epoxy group, the reaction of a carboxyl group and an epoxy group and the reaction of a hydroxyl group and an epoxy group.
5. The crosslinking method of claim 1, wherein the crosslinking method comprises the steps of: in the step 6, the thermal initiation polymerization initiator comprises an organic peroxide initiator, an inorganic peroxide initiator and an azo initiator, the photo-initiated polymerization initiator includes 2-hydroxy-2-methyl-1-phenylpropanone, 1-hydroxycyclohexyl phenyl ketone, 2-methyl-2- (4-morpholinyl) -1- [4- (methylthio) phenyl ] -1-propanone, 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone, 2,4, 6-trimethylbenzoyl-diphenylphosphine oxide, and 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone.
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Effective date of registration: 20210510 Address after: 310052 Room 311, 3/F, Building 88, Jiangling Road, Binjiang District, Hangzhou City, Zhejiang Province Patentee after: Hangzhou Qiming Medical Devices Co.,Ltd. Address before: 610000 No. 24 south part of Wuhou District first ring road, Chengdu, Sichuan. Patentee before: SICHUAN University |