阅读说明：本技术 一种抗凝促细胞黏附生物瓣膜材料及其制备方法 (Anticoagulation cell adhesion-promoting biological valve material and preparation method thereof ) 是由 陈茂 庄伟华 李淑芬 冯沅 于 2021-10-22 设计创作，主要内容包括：本发明公开了一种抗凝促细胞黏附生物瓣膜材料及其制备方法。其制备方法为：对生物瓣膜材料进行戊二醛交联,并在戊二醛交联前和/或后采用改性溶液A和/或改性溶液B对生物瓣膜材料进行改性处理。本发明通过化学键作用、电荷相互作用将一种或多种改性材料结合在心包膜上,能够使人工生物瓣膜具有长效抗凝和促细胞黏附的性能,此外,还能提高人工生物瓣膜的力学性能和结构稳定性。(The invention discloses an anticoagulation and cell adhesion promotion biological valve material and a preparation method thereof. The preparation method comprises the following steps: and performing glutaraldehyde crosslinking on the biological valve material, and performing modification treatment on the biological valve material by using a modification solution A and/or a modification solution B before and/or after the glutaraldehyde crosslinking. The invention combines one or more modified materials on the pericardium through the chemical bond action and the charge interaction, can ensure that the artificial biological valve has the performances of long-acting anticoagulation and cell adhesion promotion, and can also improve the mechanical property and the structural stability of the artificial biological valve.)

1. A preparation method of an anticoagulation and cell adhesion promotion biological valve material is characterized in that a biological valve material is subjected to glutaraldehyde crosslinking, and a modification solution A and/or a modification solution B is/are adopted to carry out modification treatment on the biological valve material before and/or after the glutaraldehyde crosslinking.

2. The preparation method according to claim 1, wherein the modification treatment of the biological valve material by using the modification solution A or the modification solution B comprises the following steps:

soaking a biological valve material or a biological membrane material crosslinked by glutaraldehyde into a modified solution A or a modified solution B with the pH value of 0.5-12, and soaking for 2-96 h at 4-40 ℃ in a shaking manner.

3. The method according to claim 1 or 2, wherein the modifying solution A or B has a mass concentration of 0.001 to 20%.

4. The method according to claim 3, wherein the modified solution A is cysteine, penicillamine, cystamine, lysine, arginine, histidine, cysteamine, mercaptopropylamine, aminothiophenol, at least one of dopamine, 1-ethyl- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, diisopropylcarbodiimide, 4-dimethylaminopyridine, 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, 1-hydroxybenzotriazole and O- (benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate.

5. The method according to claim 3, wherein the modifying solution B is polyethyleneimine, maleimide-undecenoic acid succinimide ester, lysine, arginine, polylysine, 3-maleimidopropionic acid hydroxysuccinimide ester, chitosan, ammonium polyphosphate, polyhexamethylene bromide, polyhydroxyethyl cellulose ether quaternary ammonium salt, guar gum, gum arabic, choline, polyimidazolium salt, hyaluronic acid, 4-maleimidobutyric acid, 3-maleimidopropionic acid, polyacrylic acid, polysulfonic acid, polyacrylic acid and sodium salt thereof, KPVC EPpeptide, polymethacrylic acid and sodium salt thereof, polyvinylsulfonic acid and salt thereof, polystyrenesulfonic acid and salt thereof, (2-acrylamido-2-methylpropanesulfonic acid) polymer, 6-maleimidocaproic acid, 6- (maleimido) caproic acid succinimide ester, and, At least one of maleimidoacetic acid succinimidyl ester, polyarginine, N- (2-aminoethyl) maleimide, 5-maleimidovaleric acid, polyacrylamide, heparin, 5-maleimidovaleric acid NHS, sodium alginate, 4-maleimidobutyric acid-N-succinimidyl ester, and CRGD peptide.

6. The method according to claim 3, wherein the modifying solution A or the modifying solution B is prepared by:

and stirring and dissolving the weighed modified solution A or modified solution B at 4-60 ℃, and adjusting the pH value to 0.5-12.

7. The method according to claim 1, wherein the glutaraldehyde crosslinking is carried out by:

and soaking the biological valve material into a glutaraldehyde solution with the pH value of 0.5-12 and the temperature of 4-40 ℃, and oscillating and soaking for 2-96 hours.

8. The method of claim 1 or 2, wherein the biological valve material is a decellularized porcine pericardium or bovine pericardium.

9. An anticoagulation cell adhesion-promoting biological valve material, which is prepared by the method of any one of claims 1-8.

Technical Field

The invention belongs to the technical field of preparation of medical materials and medical instruments, and particularly relates to an anticoagulation cell adhesion-promoting biological valve material and a preparation method thereof.

Background

In recent years, cardiovascular diseases have become the biggest threat to human life health, wherein valvular heart diseases have become common heart diseases in our country. With the aging of the population becoming more serious, patients with valvular heart disease are increasing year by year. Heart valvular diseases are mainly manifested as stenosis or insufficiency. Once a heart valve is narrowed or incompletely closed, it can obstruct the normal flow of blood, severely increase the heart burden, cause damage to the heart function, and lead to heart failure. For severe heart valve disease, surgical valve replacement and transcatheter minimally invasive valve replacement are currently the most common treatment modalities. Bioprosthetic valves are the main components of traditional surgical open-chest valve replacement and emerging transcatheter valve replacement, and are the most value-added components. The artificial biological valve used clinically at present is mainly prepared by acellular and glutaraldehyde crosslinking treatment of pig pericardium or bovine pericardium. However, the biological valve prosthesis prepared by glutaraldehyde crosslinking has high toxicity, is difficult to realize cell adhesion, is easy to cause adverse clinical events such as thrombus and the like, and even worse, can also reduce the service life of the biological valve prosthesis and cause serious threat to the life health of patients.

The aldehyde group remained after the glutaraldehyde crosslinking is an important reason causing great toxicity of the artificial heart valve, and although part of free aldehyde group can be blocked by lysine, arginine or polycation and the like, the adhesion performance of the artificial biological valve cell is not obviously improved. In addition, grafting anticoagulant drugs by physical adsorption or chemical coupling and the like is a common method for improving the anticoagulant performance of the artificial biological valve, but the two strategies are respectively limited by short effective period and unsatisfactory anticoagulant effect. Therefore, effectively blocking free aldehyde groups on the glutaraldehyde cross-linked artificial biological valve and realizing long-acting anticoagulation of the artificial biological valve is expected to prolong the service life of the artificial biological valve, thereby improving the life of patients.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides an anticoagulation and cell adhesion promotion biological valve material and a preparation method thereof, aiming at endowing the anticoagulation and cell adhesion promotion capability to the artificial biological valve so as to prolong the service life of the artificial biological valve.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

a preparation method of an anticoagulant cell adhesion-promoting biological valve material comprises the following steps: and (3) carrying out glutaraldehyde crosslinking on the biological valve material, and modifying the biological valve material by using a modified solution A or a modified solution B before and/or after the glutaraldehyde crosslinking.

The method specifically comprises the following steps:

soaking the biological valve material in the modified solution A or the modified solution B, and then performing glutaraldehyde crosslinking;

or the biological valve material is firstly cross-linked by glutaraldehyde and then soaked in the modified solution A or the modified solution B;

or soaking the biological valve material in the modified solution A or the modified solution B, then carrying out glutaraldehyde crosslinking, and finally soaking the crosslinked biological valve material in the modified solution A or the modified solution B.

Further, the process of modifying the biological valve material by adopting the modified solution A or the modified solution B comprises the following steps:

soaking a biological valve material or a biological membrane material crosslinked by glutaraldehyde into a modified solution A or a modified solution B with the pH value of 0.5-12, and soaking for 2-96 h at 4-40 ℃ in a shaking manner.

Further, the mass concentration of the modification solution A or the modification solution B is 0.001-20%.

Further, the modified solution A is cysteine, penicillamine, cystamine, lysine, arginine, histidine, cysteamine, mercaptopropylamine and amino thiophenol, at least one of dopamine, 1-ethyl- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, diisopropylcarbodiimide, 4-dimethylaminopyridine, 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, 1-hydroxybenzotriazole and O- (benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate.

Further, the modifying solution B is polyethyleneimine, maleimide undecyl succinimide ester, lysine, arginine, polylysine, 3-maleimido propionic acid hydroxysuccinimide ester, chitosan, ammonium polyphosphate, polyhexamethylene dimethylammonium bromide, polyhydroxyethyl cellulose ether quaternary ammonium salt, guar gum, gum arabic, choline, polyimidazolium salt, hyaluronic acid, 4-maleimido butyric acid, 3-maleimido propionic acid, polyacrylic acid, polysulfonic acid, polyacrylic acid and sodium salt thereof, KPEPVC peptide, polymethacrylic acid and sodium salt thereof, polyvinylsulfonic acid and salt thereof, polystyrenesulfonic acid and salt thereof, (2-acrylamido-2-methylpropanesulfonic acid) polymer, 6-maleimido caproic acid, 6- (maleimido) caproic acid succinimide ester, poly (2-acrylamido) propionic acid, poly (2-maleimido) succinimide ester, poly (2-maleimido) caproic acid, poly (2-maleimido) copolymer, poly (2-maleimido) propionic acid, poly (2-ethylenephosphonic acid), poly (2-maleimido) succinimide ester, poly (2-ethylenephosphonic acid), poly (2-maleimido) amide), poly (2-ethylenephosphonic acid), poly (2-maleimido) amide), poly (2-bis (2-maleimido-one, poly (2-ethylenephosphonic acid), poly (2-maleimide), poly (ethylenephosphonic acid), poly (2-maleimide), poly (ethylenephosphonic acid), poly (, At least one of maleimidoacetic acid succinimidyl ester, polyarginine, N- (2-aminoethyl) maleimide, 5-maleimidovaleric acid, polyacrylamide, heparin, 5-maleimidovaleric acid NHS, sodium alginate, 4-maleimidobutyric acid-N-succinimidyl ester, and CRGD peptide.

Further, the preparation process of the modified solution A or the modified solution B comprises the following steps:

and stirring and dissolving the weighed modified solution A or modified solution B at 4-60 ℃, and adjusting the pH value to 0.5-12.

Further, the pH value of the modified solution A or the modified solution B is 5-8.

Further, the pH values of the modifying solution a and the modifying solution B were both 7.4.

Further, the pH values of the modifying solution a and the modifying solution B were both 5.5.

Further, the modification solution A comprises mercaptopropylamine, 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in a molar weight ratio of 1:1.5: 1.5.

Modification solution B: comprises a mixed solution, 5-maleimidovaleric acid, 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in a mass ratio of 2:0.1:0.12: 0.12; the mixed solution comprises polylysine and heparin, the mass concentration of the polylysine is 0.3%, the mass concentration of the heparin is 0.2%, and the volume ratio of the polylysine to the heparin is 1:1.

Further, the process of glutaraldehyde crosslinking is:

and soaking the biological valve material into a glutaraldehyde solution with the pH value of 0.5-12 and the temperature of 4-40 ℃, and oscillating and soaking for 2-96 hours.

Further, the biological valve material is acellular pericardium.

Further, decellularized pericardium includes, but is not limited to, porcine pericardium and bovine pericardium.

An anticoagulant cell adhesion-promoting biological valve material is prepared by the method.

The invention has the beneficial effects that:

1. the invention realizes the long-acting anticoagulation and cell adhesion promotion performance of the artificial biological valve by soaking the acellular pericardium in a modified solution and then crosslinking glutaraldehyde, or soaking the glutaraldehyde crosslinked pericardium in the modified solution, or soaking the acellular pericardium in the modified solution and crosslinking glutaraldehyde, and then further soaking the acellular pericardium in the modified solution, and combining one or more modified materials on the pericardium through the chemical bond action and charge interaction.

2. According to the invention, the acellular pericardium is soaked in the modified solution, so that the modified material is fully combined with the pericardium and is further crosslinked with glutaraldehyde, thereby increasing crosslinking sites, enabling the modified material to be stably combined with the pericardium, improving the functionality of the artificial biological valve, and improving the mechanical property and structural stability of the artificial biological valve.

3. Compared with the artificial biological valve which is cross-linked by simple glutaraldehyde, the artificial biological valve provided by the invention has the advantages of obviously improved anticoagulation performance and capability of promoting cell adhesion.

Drawings

FIG. 1 is a scanning electron microscope (A) of the adhesion of platelets and a scanning electron microscope (B) of the adhesion of platelets of a pure glutaraldehyde cross-linked membrane of a biological valve material prepared in example 2;

FIG. 2 is a fluorescent microscope picture (A) of endothelial cell adhesion and a fluorescent microscope picture (B) of pure glutaraldehyde cross-linked membrane cell adhesion of the biological valve material prepared in example 2.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.

Example 1

A preparation method of an anticoagulant cell adhesion-promoting biological valve material comprises the following specific processes:

modification solution A: 0.5 percent of dopamine by mass concentration and 7.4 of pH value;

modification solution B: mixed solution of polyethyleneimine and heparin with mass concentration of 0.3% and 0.2%, respectively, and pH value of 7.4.

Soaking the acellular bovine pericardium in the modified solution A at 4 ℃ for 24 hours under a slight oscillation condition; then, cleaning the membrane with clear water, soaking the membrane in the modified solution B for 24 hours at 4 ℃ under a slight oscillation condition; taking out the membrane, washing the membrane with clear water, then further treating the membrane with glutaraldehyde, wherein the concentration of the glutaraldehyde is 1 percent, the pH value of the glutaraldehyde is 7.4, and crosslinking the membrane for 48 hours at room temperature under the oscillation condition; and taking out the glutaraldehyde crosslinked membrane, washing the membrane with clear water, and further treating the membrane with a modified solution B for 24 hours to obtain the biological valve material with anticoagulation and cell adhesion promotion functions.

The tensile breaking stress of the artificial biological valve material prepared by the embodiment is slightly better than that of a membrane prepared by glutaraldehyde crosslinking. Platelet adhesion experiments show that the artificial biological valve material prepared by the embodiment has remarkably improved anti-platelet adhesion capability, a large amount of cell adhesion can be observed in the artificial biological valve prepared by the embodiment, and the cell adhesion phenomenon is not observed basically in the membrane prepared by glutaraldehyde crosslinking.

Example 2

A preparation method of an anticoagulant cell adhesion-promoting biological valve material comprises the following specific processes:

modification solution A: mercaptopropylamine, 1-ethyl- (3-dimethylaminopropyl) carbodiimide, and N-hydroxysuccinimide in a molar ratio of 1:1.5:1.5 were dissolved in distilled water at a pH of 5.5.

Modification solution B: comprises a mixed solution, 5-maleimidovaleric acid, 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in a mass ratio of 2:0.1:0.12: 0.12; the mixed solution comprises polylysine and heparin, the mass concentration of the polylysine is 0.3%, the mass concentration of the heparin is 0.2%, and the volume ratio of the polylysine to the heparin is 1:1. Then, the above components were dissolved in distilled water to have a pH of 5.5.

Soaking the acellular bovine pericardium in the modified solution A at 25 ℃ for 24 hours under the oscillation condition; then, cleaning the membrane with clear water, soaking the membrane in the modified solution B for 24 hours at 25 ℃ under an oscillation condition; after soaking, taking out the membrane, washing with clear water, further treating with glutaraldehyde, wherein the concentration of the glutaraldehyde is 1%, the pH value is 7.4, and crosslinking for 48 hours at 25 ℃ under an oscillation condition; and taking out the crosslinked membrane, washing the membrane with clear water, and further treating the membrane with a modified solution B for 24 hours to obtain the biological valve material with anticoagulation and cell adhesion promotion functions.

The tensile breaking stress of the artificial biological valve material prepared by the embodiment is equivalent to that of a membrane prepared by crosslinking glutaraldehyde. Platelet adhesion experiments show that the artificial biological valve material prepared by the embodiment has remarkably improved anti-platelet adhesion capability, a large amount of cell adhesion can be observed in the artificial biological valve prepared by the embodiment, and the cell adhesion phenomenon is not observed basically in the membrane prepared by glutaraldehyde crosslinking.

Example 3

A preparation method of an anticoagulant cell adhesion-promoting biological valve material comprises the following specific processes:

modification solution B: comprises a mixed solution, 5-maleimidovaleric acid, 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in a mass ratio of 3:0.1:0.12: 0.12; the mixed solution comprises polylysine and heparin, the mass concentration of the polylysine is 0.3%, the mass concentration of the heparin is 0.2%, and the volume ratio of the polylysine to the heparin is 1:1. Then, the above components were dissolved in distilled water to have a pH of 5.5.

Soaking the acellular bovine pericardium in the modified solution B at 25 ℃ for 24 hours under the oscillation condition; after soaking, taking out the membrane, washing with clear water, further treating with glutaraldehyde, wherein the concentration of the glutaraldehyde is 1%, the pH value is 7.4, and crosslinking for 48 hours at 25 ℃ under an oscillation condition; and taking out the crosslinked membrane, washing the membrane with clear water, and further treating the membrane with a modified solution B for 24 hours to obtain the biological valve material with anticoagulation and cell adhesion promotion functions.

The tensile breaking stress of the artificial biological valve material prepared by the embodiment is slightly better than that of a membrane prepared by glutaraldehyde crosslinking. Platelet adhesion experiments show that the artificial biological valve material prepared by the embodiment has remarkably improved anti-platelet adhesion capability, a large amount of cell adhesion can be observed in the artificial biological valve prepared by the embodiment, and the cell adhesion phenomenon is not observed basically in the membrane prepared by glutaraldehyde crosslinking.