CN109833118B - Method for improving stability of biological valve by adopting cross-linking treatment - Google Patents
Method for improving stability of biological valve by adopting cross-linking treatment Download PDFInfo
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- CN109833118B CN109833118B CN201811148262.7A CN201811148262A CN109833118B CN 109833118 B CN109833118 B CN 109833118B CN 201811148262 A CN201811148262 A CN 201811148262A CN 109833118 B CN109833118 B CN 109833118B
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Abstract
The invention discloses a method for improving the stability of a biological valve by adopting cross-linking treatment, which comprises the step of soaking a pericardium cross-linked by glutaraldehyde into a glycosaminoglycan and sodium trimetaphosphate aqueous solution to realize in-situ cross-linking of glycosaminoglycan on the pericardium. The method provided by the invention can improve the glycosaminoglycan content and the anti-calcification performance of the biological material, and potentially prolong the service life of the biological material.
Description
Technical Field
The invention relates to the technical field of biomedical materials and medical instruments, in particular to a method for improving the stability of a biological valve by adopting cross-linking treatment.
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 large trauma to patients, high risk, slow recovery and needs extracorporeal circulation support, which 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.
Biological heart valves refer to a class of biomedical materials used to replace diseased heart valves in humans. The biological heart valve is generally prepared by cross-linking porcine pericardium, bovine pericardium and the like through glutaraldehyde.
The glutaraldehyde crosslinking treatment has the characteristics of simple operation, low cost and high collagen crosslinking degree, and is the first choice in the chemical crosslinking industry of the existing biological heart valve. However, glutaraldehyde-crosslinked bioprosthetic heart valves suffer from the problems of easy degradation and calcification, resulting in a bioprosthetic heart valve that has an effective service life of only about 10 years. Glutaraldehyde can achieve stable crosslinking of collagen, but cannot crosslink glycosaminoglycan, resulting in certain technical limitations.
Glycosaminoglycans are useful for enhancing the hydrophilicity of biological heart valves and reducing shear stress as well as compressive stress. There are studies that suggest that loss of glycosaminoglycan from pericardial tissue will increase the risk of calcification in biological heart valves, whereas increasing glycosaminoglycan content can reduce calcification. Neomycin sulfate has been studied to inhibit degradation of glycosaminoglycan in pericardial tissue, but neomycin sulfate has limitations, as evidenced by limited reactive groups on pericardial tissue, resulting in limited neomycin sulfate that can be chemically bound.
Therefore, by optimizing the chemical crosslinking method of the biological heart valve, especially developing a novel material treatment method capable of improving the content and the structural stability of glycosaminoglycan, the overall structural stability and the anti-calcification performance of the biological heart valve can be improved, which has great significance for the development of scientific research and related industrial fields, and no good method exists at present, so that improvement is needed.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provide a method for improving the stability of a biological valve by adopting cross-linking treatment, which can improve the glycosaminoglycan content and the calcification-resisting performance of a biological material and potentially prolong the service life of the biological material.
The purpose of the invention is realized by the following technical scheme.
A method for improving the stability of a biological valve by adopting cross-linking treatment specifically comprises the following steps:
s1, obtaining the biological material, washing the biological material for 2 hours by using distilled water under the conditions of soft friction and fluid pressure oscillation at the rotating speed of 100RPM at 4 ℃ until no visible adhered non-pericardial or non-collagen tissue exists, and realizing effective decellularization of the pericardial tissue through osmotic shock;
s2, glutaraldehyde crosslinking, namely treating the biological material cleaned in the step S1 for 1 to 7 days by using an aqueous solution with glutaraldehyde concentration of 0.1 to 10 percent or PBS buffer solution;
s3, soaking the biological material cleaned in the step S2 in exogenous glycosaminoglycan, wherein the mass solubility of the glycosaminoglycan is 0.1-20%, and the exogenous glycosaminoglycan is ensured to be fully soaked;
s4, carrying out in-situ crosslinking of glycosaminoglycan and sodium trimetaphosphate from the biological material treated in the step S3, wherein the molar concentration of the sodium trimetaphosphate is 0.01-1M;
s5, finally soaking and cleaning the blank by using distilled water to remove unreacted glycosaminoglycan and sodium trimetaphosphate.
Further, in step S1, the biological material is animal tissue, including one or more of pericardium, valve, intestinal membrane, meninges, lung membrane, blood vessel, skin, or ligament.
Further, in step S4, the glycosaminoglycan comprises one or more of hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, and heparin.
The invention has the beneficial effects that: the method provided by the invention comprises the step of soaking the glutaraldehyde crosslinked pericardium into the glycosaminoglycan and sodium trimetaphosphate aqueous solution to realize in-situ crosslinking of glycosaminoglycan on the pericardium. The method provided by the invention can improve the glycosaminoglycan content and the anti-calcification performance of the biological material, and potentially prolong the service life of the biological material.
Drawings
To further clarify the above and other advantages and features of one or more of the present inventions, a more particular description of one or more of the present inventions will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
Fig. 1 is a detailed flow diagram of a prepared biological heart valve.
FIG. 2 is a schematic representation of the principle of increasing pericardium content and stable glycosaminoglycan content and stability using glycosaminoglycan and sodium trimetaphosphate cross-linked.
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.
Example 1:
this example provides a method for improving the stability of a biological valve using a cross-linking treatment, and in all embodiments, the fresh porcine pericardium is from a local slaughterhouse.
As shown in fig. 1, freshly harvested pig hearts were washed with distilled water for 2 hours at 4 degrees celsius with 100RPM shaking. And then soaked in 0.625% glutaraldehyde for 24 hours. The pericardium was then soaked in 1% hyaluronic acid for 24 hours. Finally, the pericardium is soaked in 0.1M sodium trimetaphosphate aqueous solution for 24 hours.
In the treatment process, the pericardium was soaked in 0.625% glutaraldehyde for 24 hours as a control.
Example 2:
this example provides a method for improving the stability of a biological valve using a cross-linking treatment, and in all embodiments, the fresh porcine pericardium is from a local slaughterhouse.
As shown in fig. 1, freshly harvested pig hearts were washed with distilled water for 2 hours at 4 degrees celsius with 100RPM shaking. And then soaked in 0.625% glutaraldehyde for 24 hours. The pericardium was then soaked in 5% hyaluronic acid for 24 hours. Finally, the pericardium is soaked in 0.1M sodium trimetaphosphate aqueous solution for 24 hours.
Example 3:
this example provides a method for improving the stability of a biological valve using a cross-linking treatment, and in all embodiments, the fresh porcine pericardium is from a local slaughterhouse.
As shown in fig. 1, freshly harvested pig hearts were washed with distilled water for 2 hours at 4 degrees celsius with 100RPM shaking. And then soaked in 0.625% glutaraldehyde for 24 hours. The pericardium was then soaked in 10% hyaluronic acid for 24 hours. Finally, the pericardium is soaked in 0.1M sodium trimetaphosphate aqueous solution for 24 hours.
Example 4:
this example provides a method for improving the stability of a biological valve using a cross-linking treatment, and in all embodiments, the fresh porcine pericardium is from a local slaughterhouse.
As shown in fig. 1, freshly harvested pig hearts were washed with distilled water for 2 hours at 4 degrees celsius with 100RPM shaking. And then soaked in 0.625% glutaraldehyde for 24 hours. The pericardium was then soaked in 1% chondroitin sulfate for 24 hours. Finally, the pericardium is soaked in 0.1M sodium trimetaphosphate aqueous solution for 24 hours.
Example 5:
this example provides a method for improving the stability of a biological valve using a cross-linking treatment, and in all embodiments, the fresh porcine pericardium is from a local slaughterhouse.
As shown in fig. 1, freshly harvested pig hearts were washed with distilled water for 2 hours at 4 degrees celsius with 100RPM shaking. And then soaked in 0.625% glutaraldehyde for 24 hours. The pericardium was then soaked in 10% chondroitin sulfate for 24 hours. Finally, the pericardium is soaked in 0.1M sodium trimetaphosphate aqueous solution for 24 hours.
Example 6:
this example provides a method for improving the stability of a biological valve using a cross-linking treatment, and in all embodiments, the fresh porcine pericardium is from a local slaughterhouse.
As shown in fig. 1, freshly harvested pig hearts were washed with distilled water for 2 hours at 4 degrees celsius with 100RPM shaking. And then soaked in 0.625% glutaraldehyde for 24 hours. The pericardium was then soaked in 20% chondroitin sulfate for 24 hours. Finally, the pericardium is soaked in 0.1M sodium trimetaphosphate aqueous solution for 24 hours.
Examples of the experiments
As shown in FIG. 2, the diagram is a schematic diagram of the principle of increasing pericardium content and stabilizing glycosaminoglycan content and stability by crosslinking glycosaminoglycan and sodium trimetaphosphate.
The results of analyzing the contents of the total glycosaminoglycans in the six examples and the glutaraldehyde control group are shown in Table 1, and the amount of calcium incorporation is shown in Table 2. And in the treatment process, the pericardium is soaked in 0.25% glutaraldehyde for 24 hours in the set control group.
| Mu.g Total Glycoaminopolysaccharide/10 mg tissue | |
| Glutaraldehyde pairPhoto group | 51.5±2.6 |
| Example 1 | 91.2±1.5 |
| Example 2 | 154.7±2.1 |
| Example 3 | 156.6±2.8 |
| Example 4 | 86.8±3.7 |
| Example 5 | 193.7±3.7 |
| Example 6 | 199.6±3.5 |
TABLE 1
| The calcium content is mu g/mg | |
| Glutaraldehyde control group | 60.90±1.63 |
| Example 1 | 37.21±1.23 |
| Example 2 | 12.56±0.61 |
| Example 3 | 11.45±0.43 |
| Example 4 | 35.21±1.12 |
| Example 5 | 15.32±1.09 |
| Example 6 | 13.26±0.86 |
TABLE 2
When the evaluation is carried out, the content of the glycosaminoglycan in the experimental group is increased, and the calcium content of the rats implanted subcutaneously for 30 days is reduced.
The invention has the beneficial effects that: the method provided by the invention comprises the step of soaking the glutaraldehyde crosslinked pericardium into the glycosaminoglycan and sodium trimetaphosphate aqueous solution to realize in-situ crosslinking of glycosaminoglycan on the pericardium. The method provided by the invention can improve the glycosaminoglycan content and the anti-calcification performance of the biological material, and potentially prolong the service life of the biological 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 (4)
1. A method for improving the stability of a biological valve by adopting cross-linking treatment is characterized in that a biological material cross-linked by glutaraldehyde is soaked in aqueous solutions of glycosaminoglycan and sodium trimetaphosphate in sequence to realize in-situ cross-linking of glycosaminoglycan on the biological material, so that the content and the structural stability of glycosaminoglycan in the biological valve are improved, and the overall structural stability and the anti-calcification performance of the biological heart valve are improved by enhancing the hydrophilicity of the biological valve and reducing shear stress and compressive stress, and specifically comprises the following steps:
s1, obtaining the biological material, washing the biological material for 2 hours by using distilled water under the conditions of soft friction and fluid pressure oscillation at the rotating speed of 100RPM at 4 ℃ until no visible adhered non-pericardial or non-collagen tissue exists, and realizing effective decellularization of the pericardial tissue through osmotic shock;
s2, glutaraldehyde crosslinking, namely treating the biological material cleaned in the step S1 for 1 to 7 days by using an aqueous solution with glutaraldehyde concentration of 0.1 to 10 percent or PBS buffer solution;
s3, soaking the biological material cleaned in the step S2 in exogenous glycosaminoglycan, wherein the mass concentration of the glycosaminoglycan is 0.1-20%, and the exogenous glycosaminoglycan is ensured to be fully soaked;
s4, carrying out in-situ crosslinking of glycosaminoglycan and sodium trimetaphosphate from the biological material treated in the step S3, wherein the molar concentration of the sodium trimetaphosphate is 0.01-1M;
s5, finally soaking and cleaning the blank by using distilled water to remove unreacted glycosaminoglycan and sodium trimetaphosphate.
2. The method of claim 1, wherein the cross-linking treatment is used to improve the stability of the biological valve, and the method comprises the following steps: in step S1, the biological material is animal tissue.
3. The method of claim 1, wherein the cross-linking treatment is used to improve the stability of the biological valve, and the method comprises the following steps: the biological material comprises one or more of pericardium, valves, gut membrane, meninges, lung membrane, blood vessels, skin, or ligaments.
4. The method of claim 1, wherein the cross-linking treatment is used to improve the stability of the biological valve, and the method comprises the following steps: in step S4, the glycosaminoglycan comprises one or more of hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, and heparin.
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| CN111420120A (en) * | 2020-05-28 | 2020-07-17 | 四川大学 | Biological valve with anticoagulation and anti-calcification functions and preparation method thereof |
| CN111658824B (en) * | 2020-06-15 | 2021-03-30 | 四川大学 | Valve membrane material with synergistic anticoagulation and anti-calcification functions and preparation method thereof |
| CN114681673B (en) * | 2020-12-31 | 2023-05-23 | 杭州启明医疗器械股份有限公司 | Crease-resistant dehydrated crosslinking biological material and preparation method and application thereof |
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| US20070218038A1 (en) * | 2006-03-17 | 2007-09-20 | Pegasus Biologics, Inc. | Stabilized, sterilized collagen scaffolds with active adjuncts attached |
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| CN1950114A (en) * | 2004-05-12 | 2007-04-18 | 苏尔莫迪克斯公司 | Natural biodegradable polysaccharide coatings for medical articles |
| CN104220104A (en) * | 2011-12-13 | 2014-12-17 | 波士顿科学医学有限公司 | Decalcifying heart valve |
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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 |