CN115887770A - Biological valve material crosslinked by utilizing isocyano ethyl methacrylate, medical instrument, method and application thereof - Google Patents
Biological valve material crosslinked by utilizing isocyano ethyl methacrylate, medical instrument, method and application thereof Download PDFInfo
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- CN115887770A CN115887770A CN202111363858.0A CN202111363858A CN115887770A CN 115887770 A CN115887770 A CN 115887770A CN 202111363858 A CN202111363858 A CN 202111363858A CN 115887770 A CN115887770 A CN 115887770A
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Abstract
The invention discloses a biological valve material crosslinked by utilizing isocyano ethyl methacrylate and a method thereof, wherein the cleaned biological valve material is soaked in an isocyano ethyl methacrylate solution to obtain an in-situ modified biological valve of the isocyano ethyl methacrylate; and cleaning the biological valve, and soaking the biological valve in a free radical polymerization initiator to enable methacrylate ester groups on the biological valve to react with the free radical polymerization initiator to obtain the isocyano ethyl methacrylate cross-linked biological valve material. Solves a series of technical defects existing in the prior glutaraldehyde crosslinking method, can effectively improve the calcification resistance, enzyme degradation resistance and cell compatibility of biological materials such as biological heart valves and the like, and potentially prolongs the service life of the biological heart valves and the like.
Description
Technical Field
The invention relates to a biological valve material crosslinked by utilizing isocyano ethyl methacrylate, a medical instrument, a method and application thereof, in particular to a method for preparing a biological valve material by utilizing isocyano ethyl methacrylate, the biological valve material prepared by the method, the application of the biological valve material and a medical instrument containing the biological valve material, and belongs to the technical field of biomedical material medical instruments.
Background
Heart valve disease is a common valve failure disease. Anatomically manifested as narrowing of the blood access or incomplete valve closure. Treatment of heart valve disease includes open chest valve replacement surgery and percutaneous heart valve replacement surgery. The thoracotomy has the defects of large wound, high risk, slow recovery and need of extracorporeal circulation support, and is unacceptable for many patients. Percutaneous heart valve replacement surgery is a main trend of valve surgery in the future because of small trauma and low risk to patients.
The biological heart valve is a biomedical material and a device thereof for replacing a diseased heart valve of a human body. The biological heart valve leaflet is generally prepared by crosslinking a porcine pericardium, a bovine pericardium and the like through glutaraldehyde. The glutaraldehyde crosslinking treatment has the characteristics of simple operation, low cost and high collagen crosslinking degree. However, glutaraldehyde cross-linked bioprosthetic heart valves have certain problems of toxicity, material degradation, thrombosis, and calcification due to the presence of residual aldehyde groups on the valve. For damaged valves of young patients, the metabolic capacity of the patient population is vigorous, so that the biological heart valve is easier to calcify and has an effective service life of about 10 years. Therefore, although collagen in the material can be stabilized to a certain extent by the glutaraldehyde crosslinking method, certain cytotoxicity and calcification are caused by the existence of residual aldehyde groups, and the anticoagulation performance of the glutaraldehyde crosslinking method needs to be improved, so that the glutaraldehyde crosslinking biomaterial has certain technical defects.
The invention patent with publication number CN109833519A discloses a method for preparing a biological valve prosthesis, which can prepare a dry biological valve by compounding hydrogel and biological tissue, and during the preparation, the biological tissue is soaked in a hydrophilic polymer/hydrophilic monomer solution, then a cross-linking agent is added, the biological tissue is subjected to a cross-linking reaction in the presence of a catalyst/an initiator to form a three-dimensional network structure, so that a hydrogel compounded biological tissue is obtained, and then protein fibers in the biological tissue are subjected to cross-linking and dehydration, so that the dry biological valve is obtained.
Disclosure of Invention
The invention aims to provide a method for preparing a biological valve material by utilizing isocyano ethyl methacrylate crosslinking, which solves a series of technical defects existing in the existing glutaraldehyde crosslinking method, can effectively improve the calcification resistance, enzyme degradation resistance and cell compatibility of biological materials such as biological heart valves and the like, and potentially prolongs the service life of the biological heart valves.
The invention is realized by the following technical scheme: a method for preparing a biological valve material by utilizing isocyano ethyl methacrylate crosslinking comprises the following steps:
s1, soaking the cleaned biological material in an isocyano ethyl methacrylate solution to obtain an isocyano ethyl methacrylate in-situ modified biological valve;
s2, cleaning the biological valve in the step S1, and enabling methacrylate ester groups on the biological valve to react in the presence of a free radical polymerization initiator to obtain the isocyano ethyl methacrylate cross-linked biological valve material.
In the step S1, the in-situ modification of the biological material with the isocyano ethyl methacrylate means that the biological material is soaked in an ethanol solution of the isocyano ethyl methacrylate to enable an amino group in the biological valve material to react with an isocyanate group in the isocyano ethyl methacrylate to realize chemical bonding, so that a methacrylate group capable of free radical polymerization is modified on the biological valve material in situ.
In the step S1, the biological material is at least one selected from pericardium, valve, intestinal membrane, meninges, lung membrane, blood vessel, skin, and ligament.
In the step S1, when the biomaterial is washed, the pericardial tissue may be washed with distilled water under the oscillation condition by using soft friction and fluid pressure to remove the adhered non-pericardial and non-collagen tissues. Further, effective decellularization of pericardial tissue can be achieved by osmotic shock, with washing continuing until there is no visible adherent non-pericardial or non-collagenous tissue, such as: the washing was performed with distilled water for 2 hours under a condition of 4 degrees centigrade and 100RPM rotation speed oscillation.
In the step S1, the volume percentage concentration of the isocyanoethyl methacrylate solution is 0.1-20%.
In the step S1, the solvent in the isocyanoethyl methacrylate solution is at least one selected from ethanol, a mixed solution of tween and water, a mixed solution of ethanol and water, and isopropanol or a mixed solution of isopropanol and water.
For example: using 0.1-10% ethanol solution of isocyano ethyl methacrylate, treating for 0.5-24 hours at 0 deg.C.
In the step S2, the biological valve may be cleaned with distilled water to remove other impurities adhered to the surface of the biological valve material.
In the step S2, the radical polymerization initiator is at least one selected from ammonium persulfate-sodium bisulfite, potassium persulfate-sodium bisulfite, ammonium persulfate-potassium bisulfite, potassium persulfate-potassium bisulfite, ammonium persulfate-tetramethylethylenediamine, and potassium persulfate-tetramethylethylenediamine.
In the step S2, the concentration of the radical polymerization initiator is 1 to 100mM.
The technical scheme of the invention also comprises the biological valve material prepared by the method, and the application of the biological valve material, which is used for preparing a percutaneous intervention biological heart valve material or a biological valve material used in an open-chest valve replacement operation.
The technical scheme of the invention also comprises a medical device containing the biological valve material. The medical device comprises an interventional biological valve, an artificial blood vessel, an artificial skin, an artificial cardiac muscle patch and a dura mater.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention can realize various advantages in the aspects of improving the stability, the calcification-resisting performance and the like of the biological valve material by using the isocyano ethyl methacrylate for the cross-linking of the biological valve material. Wherein, the methacrylate group capable of free radical polymerization can form relatively stable carbon-carbon single bond under the action of the free radical polymerization initiator to realize the stable crosslinking of the biological valve material. Compared with reversible Schiff base bonds of the glutaraldehyde crosslinking agent in crosslinking, the reaction activity of isocyanate groups and amino groups in the biological valve material is higher, the formed urea bonds have higher stability, and the stability of collagen components in the biological valve material is favorably improved. In addition, compared with a glutaraldehyde crosslinking agent, the isocyano ethyl methacrylate crosslinking agent has no residual toxic aldehyde group, and can potentially avoid the problems of cytotoxicity, calcification and the like caused by the residual aldehyde group.
Drawings
FIG. 1 is a specific flow chart for preparing an isocyano ethyl methacrylate cross-linked biological heart valve material;
FIG. 2 is a schematic diagram of the present invention using isocyano ethyl methacrylate to crosslink a biological heart valve material;
FIG. 3 shows the enzyme degradation weight loss ratio of biological valve material treated by different cross-linking methods;
FIG. 4 is a graph of enzymatic degradation cytotoxicity of bioprosthetic valve material treated with different cross-linking methods;
fig. 5 is a graph of the enzyme-degraded endothelial cell compatibility of biological valve materials treated by different cross-linking methods.
Fig. 6 shows the calcification-resistant performance of the biological valve material treated by different cross-linking methods.
Detailed Description
The objects, technical solutions and advantageous effects of the present invention will be described in further detail below.
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention claimed, and unless otherwise specified, 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 invention belongs.
The biological valve material is an important medical material and an apparatus used in heart valve replacement surgery, the glutaraldehyde crosslinking method is a treatment method for crosslinking the biological heart valve through glutaraldehyde, and the method has the problems of certain toxicity, material degradation, thrombus and calcification due to the existence of residual aldehyde groups on the valve. Therefore, it is important to research and develop a new cross-linking method that can be used for the biological valve material.
The invention provides a biological valve material crosslinked by utilizing isocyano ethyl methacrylate, which is used for crosslinking the biological valve material for the first time, and the principle of the biological valve material is shown in figure 1. The isocyano ethyl methacrylate is a cross-linking agent with high reactivity with biological valve materials, and has a structure with high electrophilic isocyanate groups and free radical polymerizable methacrylate groups. Firstly, isocyanate groups react with nucleophilic groups (such as amino groups) on the biological valve material to form stable urea bonds, and free radical polymerizable methacrylate groups are covalently introduced onto the biological valve material; secondly, the methacrylate group capable of free radical polymerization can form relatively stable carbon-carbon single bond under the action of a free radical polymerization initiator so as to realize stable crosslinking of the biological valve material. Compared with reversible Schiff base bonds of the glutaraldehyde crosslinking agent in crosslinking, the reaction activity of isocyanate groups and amino groups in the biological valve material is higher, the formed urea bonds have higher stability, and the stability of collagen components in the biological valve material is favorably improved. In addition, compared with a glutaraldehyde crosslinking agent, the isocyano ethyl methacrylate crosslinking agent has no residual toxic aldehyde group, and can potentially avoid the problems of cytotoxicity, calcification and the like caused by the residual aldehyde group. Therefore, the isocyano ethyl methacrylate can be used for crosslinking of the biological valve material so as to improve the stability, the calcification resistance and the like of the material.
In the prior art, patent publication No. CN109833519A discloses that isocyanate compounds can be used as cross-linking agents for preparing cross-linked biological valves, and the reaction mechanism is to react isocyanate compounds with hydrophilic polymers, so as to cross-link protein fibers in biological tissues. However, in the method of the present invention, isocyanate in isocyano ethyl methacrylate as a cross-linking agent reacts with nucleophilic groups on the biological valve material to form relatively stable urea bonds, and methacrylate groups capable of free radical polymerization are introduced, and the method is characterized in that the biological valve material is soaked in an isocyanate solution and then methacrylate groups are introduced, so that the reaction mechanism of the two is different from the invention motivation.
In summary, the method of the invention adopts the isocyanuric ethyl methacrylate cross-linked biological valve material, the stable urea bond is formed by the reaction of the isocyanate group and the amino group, and the calcification of the biological valve material can be reduced while the stability of the biological heart valve is improved by the isocyanuric ethyl methacrylate cross-linking method, and no relevant report is found in the prior art. Therefore, the use of isocyanoethyl methacrylate to crosslink biological valve materials is of great significance to scientific research and development of related industrial fields.
The following examples are provided to illustrate specific embodiments of the present invention, and reference is made to the process flow shown in fig. 2, but the scope of the present invention is not limited to the following examples.
In all of the following embodiments, the fresh porcine/bovine pericardium is from a local slaughterhouse; isocyanoethyl methacrylate (2-Isocyanoethyl methacrylate, ICM), glutaraldehyde (GA), ammonium Persulfate (APS), potassium persulfate (KPS), sodium bisulfite (SHS), potassium bisulfite (KHS), tetramethylethylenediamine (N, N, N ', N' -Tetramethylethylenediamine, TEMED) are available from Sigma-Aldrich.
Example 1:
freshly collected pig hearts were washed with distilled water for 2 hours at 4 ℃ under 100RPM shaking. Then soaked in 3% isocyano ethyl methacrylate ethanol solution and treated at 4 ℃ for 24 hours. Then the pericardium is soaked in an ammonium persulfate-sodium bisulfite aqueous solution with the concentration of 40mM, and treated at 37 ℃ for 24 hours. And finally, washing the soaked pericardium by using distilled water, wherein the pericardium is soaked by using an ammonium persulfate-sodium bisulfite aqueous solution, and marking the obtained pig heart envelope material crosslinked by the isocyano ethyl methacrylate as ICM3-P.
Example 2:
freshly collected pig hearts were washed with distilled water for 2 hours at 4 ℃ under 100RPM shaking. Then soaked in 5% isocyano ethyl methacrylate ethanol solution and treated at 4 ℃ for 24 hours. Then the pericardium is soaked in an ammonium persulfate-sodium bisulfite aqueous solution with the concentration of 40mM, and treated at 37 ℃ for 24 hours. And finally, washing the pericardium soaked by the potassium persulfate-sodium bisulfite aqueous solution with distilled water. The obtained isocyano ethyl methacrylate cross-linked pig heart envelope material is marked as ICM5-P.
Example 3:
freshly collected pig hearts were washed with distilled water for 2 hours at 4 ℃ under 100RPM shaking. Then soaked in 7% isocyano ethyl methacrylate ethanol solution and treated at 4 ℃ for 24 hours. Then the pericardium is soaked in an ammonium persulfate-sodium bisulfite aqueous solution, and is treated for 24 hours at 37 ℃, and the concentration of the ammonium persulfate-sodium bisulfite aqueous solution is 40mM. And finally, washing the pericardium soaked by the potassium persulfate-sodium bisulfite aqueous solution with distilled water. The obtained isocyano ethyl methacrylate cross-linked pig heart envelope material is marked as ICM7-P.
Example 4:
freshly collected pig hearts were washed with distilled water for 2 hours at 4 ℃ under 100RPM shaking. Then soaked in 10% isocyano ethyl methacrylate ethanol solution and treated at 4 ℃ for 24 hours. Then the pericardium is soaked in an ammonium persulfate-sodium bisulfite aqueous solution, and is treated for 24 hours at 37 ℃, and the concentration of the ammonium persulfate-sodium bisulfite aqueous solution is 40mM. Finally, the pericardium soaked by the potassium persulfate-sodium bisulfite aqueous solution is washed by distilled water. The obtained isocyano ethyl methacrylate cross-linked pig heart envelope material is marked as ICM10-P.
Example 5:
the material obtained in example 1 is used for preparing a percutaneous interventional biological heart valve material, and the material also comprises other medical materials required by percutaneous interventional operations besides the required biological valve material.
Example 6:
the material obtained in example 2 is used for preparing a biological valve material for replacement of an open chest valve, and in addition to the required biological valve material, the material also comprises other medical materials required by the open chest valve replacement surgery.
Example 7:
a medical device comprising the material of example 3, which may be an interventional biovalve, a vascular prosthesis, a skin prosthesis, a myocardial patch or dura mater, is prepared to include other medically desirable materials in addition to the desired biovalve material.
In the process of treatment, the same comparison group is set: glutaraldehyde (GA) treated group. I.e. the pericardium is soaked in 0.625% glutaraldehyde for 24 hours.
When the materials obtained in examples 1 to 4 and the materials of the Glutaraldehyde (GA) treatment group were subjected to an enzyme degradation experiment, as shown in fig. 3, it was found that the weight loss rate of the pericardium material of isocyano ethyl methacrylate was not higher than that of the glutaraldehyde-crosslinked pericardium, wherein the weight loss rate gradually decreased with the increase of the concentration of isocyano ethyl methacrylate, and the weight loss rate was the lowest at a concentration of 10%.
Cytotoxicity tests were performed on the isocyano ethyl methacrylate-crosslinked pericardium ICM10-P obtained in example 4 and glutaraldehyde-crosslinked pericardium, and as shown in fig. 4, it was found that the cell viability of the ICM10-P group was higher, indicating that isocyano ethyl methacrylate-crosslinked pericardium was less cytotoxic than glutaraldehyde-crosslinked pericardium.
Endothelial cell compatibility tests were performed on the isocyano ethyl methacrylate-crosslinked pericardium ICM10-P obtained in example 4 and glutaraldehyde-crosslinked pericardium, and as shown in FIG. 5, it was found that the ICM10-P group had higher cell viability, indicating that the isocyano ethyl methacrylate-crosslinked pericardium had higher endothelial cell compatibility than the glutaraldehyde-crosslinked pericardium.
Rat subcutaneous calcification resistance test was performed on the isocyano ethyl methacrylate-crosslinked pericardium ICM10-P and glutaraldehyde-crosslinked pericardium obtained in example 4, and as shown in fig. 6, it was found that the calcification degree of ICM10-P group was lower, which indicates that isocyano ethyl methacrylate-crosslinked pericardium had higher calcification resistance than glutaraldehyde-crosslinked pericardium endothelial cells.
In conclusion, compared with the pericardium material crosslinked by glutaraldehyde, the biological valve material crosslinked by isocyano ethyl methacrylate provided by the invention not only solves the problem of cytotoxicity, but also can improve the calcification-resistant performance, the enzyme degradation-resistant performance and the cell compatibility of the material, thereby prolonging the service life of the material.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and all simple modifications and equivalent variations of the above embodiments according to the technical spirit of the present invention are included in the scope of the present invention.
Claims (10)
1. A method for preparing a biological valve material by utilizing isocyano ethyl methacrylate crosslinking is characterized by comprising the following steps: the method comprises the following steps:
s1, soaking the cleaned biological material in an isocyano ethyl methacrylate solution to obtain an isocyano ethyl methacrylate in-situ modified biological valve;
s2, cleaning the biological valve in the step S1, and enabling methacrylate ester groups on the biological valve to react in the presence of a free radical polymerization initiator to obtain the isocyano ethyl methacrylate cross-linked biological valve material.
2. The method of claim 1, wherein: in the step S1, the biological material is at least one selected from pericardium, valve, intestine, meninges, lung, blood vessel, skin or ligament.
3. The method of claim 1, further comprising: in the step S1, the volume percentage concentration of the isocyano ethyl methacrylate solution is 0.1-20%.
4. The method of claim 1, further comprising: in the step S1, the solvent in the isocyanoethyl methacrylate solution is at least one selected from ethanol, a mixed solution of tween and water, a mixed solution of ethanol and water, and isopropanol or a mixed solution of isopropanol and water.
5. The method of claim 1, further comprising: in the step S2, the radical polymerization initiator is at least one selected from ammonium persulfate-sodium bisulfite, potassium persulfate-sodium bisulfite, ammonium persulfate-potassium bisulfite, potassium persulfate-potassium bisulfite, ammonium persulfate-tetramethylethylenediamine, and potassium persulfate-tetramethylethylenediamine.
6. The method of claim 1, further comprising: in the step S2, the concentration of the radical polymerization initiator is 1 to 100mM.
7. A biological valve material prepared by the method of any one of claims 1 to 6.
8. Use of the biological valve material of claim 7, wherein: is used for preparing percutaneous intervention biological heart valve material or biological valve material for open valve placement.
9. A medical device comprising the bioprosthetic valve material of claim 7.
10. The medical device of claim 9, comprising: including biological valve, artificial blood vessel, artificial skin, artificial cardiac muscle patch and dura mater.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111363858.0A CN115887770A (en) | 2021-11-17 | 2021-11-17 | Biological valve material crosslinked by utilizing isocyano ethyl methacrylate, medical instrument, method and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111363858.0A CN115887770A (en) | 2021-11-17 | 2021-11-17 | Biological valve material crosslinked by utilizing isocyano ethyl methacrylate, medical instrument, method and application thereof |
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| CN115887770A true CN115887770A (en) | 2023-04-04 |
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| CN202111363858.0A Pending CN115887770A (en) | 2021-11-17 | 2021-11-17 | Biological valve material crosslinked by utilizing isocyano ethyl methacrylate, medical instrument, method and application thereof |
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| CN (1) | CN115887770A (en) |
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