CN113425910A - Composite cross-linked biological valve and preparation method thereof - Google Patents

Abstract

The application discloses a preparation method of a composite cross-linked biological valve, which comprises the following steps: 1) treating the decellularized biological valve with tannic acid; 2) treating the biological valve treated by the tannic acid by using neomycin; 3) treating the biological valve treated by neomycin by using 5-riboflavin monophosphate under the condition of ultraviolet irradiation; 4) rinsing to obtain the product. The present application also provides a composite crosslinked biological valve. The application provides a preparation method of compound crosslinked biological valve and compound crosslinked biological valve of preparation adopts riboflavin photooxidation, and in coordination with tannic acid and neomycin, carries out the crosslinking to the bovine pericardium that removes the cell to reach the purpose of the equal cross-linking of three kinds of principal ingredients of extracellular matrix, can obtain good extracellular matrix structure, outstanding mechanical effect and hot shrink temperature, and thickness is thinner, can have wider application range, the biological valve that anti collagen enzymolysis and elasticity enzymolysis ability are good.

Description

Composite cross-linked biological valve and preparation method thereof

Technical Field

The application relates to the technical field of biological materials, in particular to a composite cross-linked biological valve and a preparation method thereof.

Background

The heart of a human body is provided with four heart valves, and the four heart valves are normally opened and closed to cooperate with the contraction of cardiac muscle to circulate blood throughout the body and maintain normal life activities. However, due to congenital malformations and acquired factors such as rheumatic heart disease and heart degeneration, heart valve abnormalities can be found out, and the life of people is seriously threatened. Valve replacement is the primary treatment for advanced valvular lesions. Over 25 million valve replacement surgeries are performed worldwide each year, and over 50 million valve replacement surgeries are expected to occur each year by 2050.

Currently, there are two main options for prosthetic valves: mechanical valves and biological valves, the mechanical valves are mainly used for middle-aged and elderly patients because the mechanical valves require lifetime anticoagulation of patients, and a series of complications are brought, and teenagers select more biological valves which can bring better hemodynamic effects and do not need lifetime anticoagulation.

The biological valve on the market is mainly prepared by two tissues at present: (1) porcine pericardium and (2) bovine pericardium. The extracellular matrix of bovine pericardium and porcine pericardium mainly comprises three components: collagen fibers, elastic fibers and amino polysaccharide. The collagen fiber can provide tension resistance, the elastic fiber endows the tissue with deformation resistance, and the amino glycan can combine a large number of water molecules and can be used as a medium between the two to provide shearing resistance and endow the tissue with certain pressure resistance.

The biological valve needs to be subjected to decellularization treatment before use, and extracellular matrix in the tissue is damaged and lost to different degrees in the process, so that the mechanical property of the tissue after decellularization treatment is reduced, the tissue is easy to degrade and the like, and the service life is influenced. Therefore, the bioprosthetic valve needs to be cross-linked before use, so as to cross-link extracellular matrix components of the biological tissue, thereby improving mechanical properties of the tissue and resistance to in vivo enzymatic reaction. So that the implanted biological valve tissue remains in the body for long-term use without degeneration.

The mainstream treatment method in the market at present is to utilize glutaraldehyde to treat biological tissues, so that cross-linking is formed among collagen proteins, covalent bonds are formed among collagen fibers, the structural stability of the collagen fibers is improved, and meanwhile, antigens of heterogeneous organisms can be covered, and the collagen fibers are prevented from being attacked by a human immune system. But glutaraldehyde can only cross-link collagen fibers and cannot cross-link elastic fibers, so the fixed valve still has slight changes, such as change of the valve orifice area and reduction of extensibility. When biological valves are exposed to extracellular fluid with high calcium levels, glutaraldehyde residue groups can serve as binding sites for crystal nucleation and are one of the main factors of valve calcification. And the average life of the biological valve is only 12-15 years due to the toxicity of residual aldehyde groups, the degradation of elastin, the failure of endothelialization, the initiation of inflammatory reaction and other factors.

Disclosure of Invention

In order to solve the above technical problems, a first object of the present invention is to provide a method for preparing a composite crosslinked biological valve; a second object of the present invention is to provide a composite cross-linked bioprosthetic valve made by the above method; the application provides a preparation method of compound crosslinked biological valve and compound crosslinked biological valve of preparation, adopt riboflavin photooxidation, with tannic acid and neomycin in coordination, cross-link to removing cell bovine pericardium, in order to reach the purpose of the equal cross-linking of three kinds of principal ingredients of extracellular matrix, can obtain good extracellular matrix structure, outstanding mechanical effect and hot shrink temperature, and thickness is thinner, can have wider application range, the biological valve that anti collagen enzymolysis and elasticity enzymolysis ability are good, can effectively avoid using glutaraldehyde crosslinking simultaneously, the toxicity of residual aldehyde group and the calcification problem that arouses, compare with current glutaraldehyde crosslinking and possess better application space.

The technical scheme provided by the invention is as follows:

a method for preparing a composite cross-linked biological valve comprises the following steps:

1) treating the decellularized biological valve with tannic acid;

2) treating the biological valve treated by the tannic acid by using neomycin;

3) treating the biological valve treated by neomycin by using 5-riboflavin monophosphate under the condition of ultraviolet irradiation;

4) rinsing to obtain the product.

Preferably, in step 1), the tannic acid is specifically: 0.5 to 2 mass percent of tannic acid solution, and 70 percent of ethanol-water solution as solvent, and adjusting the pH of the tannic acid solution to 5 to 6.

Preferably, in the step 1), after the tannic acid is added, the mixture is shaken for 45 to 50 hours at a temperature of between 20 and 30 ℃.

Preferably, in step 2), the neomycin is specifically: 1-2mmol/L neomycin solution, 0.03-0.06 mmol/L morpholine ethanesulfonic acid as solvent, and adjusting pH of neomycin solution to 6-7.

Preferably, after neomycin is added, the mixture is shaken for 1 to 1.5 hours at normal temperature.

Preferably, in step 3), the riboflavin 5-monophosphate is specifically: 10-15 mmol/L5-riboflavin monophosphate solution, and the solvent is PBS buffer solution.

Preferably, in step 3), the wavelength of the ultraviolet light is 320-400 nm; and/or the presence of a gas in the gas,

after the 5-riboflavin monophosphate is added, ultraviolet light is used for irradiating for 12 to 14 hours under the stirring state.

Preferably, the biological valve is decellularized by the following steps: disinfecting with benzalkonium bromide, then treating with Trixon-100 solution by shaking, and then treating with DNase and RNase; and then sequentially rinsing with distilled water and PBS buffer solution to obtain the biological valve after cell removal.

Preferably, the biological valve is decellularized by the following steps:

soaking fresh biological material with benzalkonium bromide 0.1-0.2% for 30-40min, and stirring once every 10-15 min;

then, using Trixon-100 solution with the mass percentage concentration of 0.25-0.3%, oscillating the biological material at 35-37 ℃ for 45-50h, and changing the solution every 10-12 h;

then treating the biological material with the mixed solution by shaking at 35-37 deg.CChanging the liquid every 10-12h for 24-26 h; the mixture contains 3-4U/mL DNase and 0.03-0.05Mg/mL RNase, and contains 2-3mmol/L Mg²⁺0.1-0.2mmol/L Ca²⁺；

Then, oscillating and rinsing the biological material by using distilled water for 45-50h, and changing the liquid once every 8-10 h;

the biological material was then rinsed with PBS buffer for 24-26h, changed and then stored in PBS buffer.

A composite cross-linked biological valve prepared by the preparation method of any one of the above.

The application provides a preparation method of compound crosslinked biological valve, adopt riboflavin photooxidation, with tannic acid and neomycin are in coordination, cross-link to removing cell bovine pericardium, in order to reach the purpose of the equal cross-linking of three kinds of principal ingredients of extracellular matrix, can obtain good extracellular matrix structure, outstanding mechanical effect and hot shrinkage temperature, and the thickness is thinner, can have wider application range, biological valve that anti collagen enzymolysis and elasticity enzymolysis ability are good, can effectively avoid using glutaraldehyde crosslinking simultaneously, the toxicity of residual aldehyde group and the calcification problem that arouses, compare with current glutaraldehyde crosslinking and possess better application space.

Specifically, the method uses riboflavin to induce photooxidation, and carries out covalent crosslinking on tyrosine of collagen, so that the valve tissue mechanics and the enzymolysis resistance are improved, good mechanics and degradation resistance are achieved, the formation of calcium crystals caused by residual aldehyde groups of glutaraldehyde is avoided, and the problem of valve calcification is reduced. The riboflavin is used as a vitamin naturally contained in a human body, has the characteristics of no toxicity and low immunogenicity, is only used as a catalyst for a photo-oxidation reaction, does not introduce new substances into biological tissues, can reduce inflammatory reaction, and is favorable for attachment and endothelialization of new cells.

Tannin as a natural plant polyphenol compound can form various bonds with proline-rich protein (such as elastin and collagen), so that the enzymolysis resistance of elastin to elastase is improved, the effect of supplementing and crosslinking riboflavin to collagen is achieved, and the synergistic effect is achieved for the collagen fibers and the elastin fibers, so that good degradation resistance is achieved, and calcification induced by the degradation of elastin is reduced. The tannin has low cytotoxicity, and is also beneficial to cell attachment and endothelialization of acellular biological valves.

The neomycin serving as a hyaluronidase inhibitor can inhibit the enzymolysis of hyaluronic acid, so that the aminoglycan is stabilized, the anti-shearing force is provided for biological valves, and certain anti-compression performance is given to tissues.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a photograph at 10 times magnification of tissue after Masson staining and HE staining in an example of the present invention;

FIG. 2 is a statistical graph of Young's modulus in an example of the present invention;

FIG. 3 is a statistical chart of the maximum tensile strength in the example of the present invention;

FIG. 4 is a statistical chart of the thermal shrinkage temperature in an embodiment of the present invention;

FIG. 5 is a thickness histogram in accordance with an embodiment of the present invention;

FIG. 6 is a histogram of anti-collagenase ability in an example of the present invention;

FIG. 7 is a statistical chart of the resistance to elastase in the examples of the present invention.

Detailed Description

In order to make those skilled in the art better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The embodiments of the present application are written in a progressive manner.

Example 1 fresh bovine pericardium was decellularized

Soaking fresh bovine pericardium with 0.1% benzalkonium bromide by mass percent, sterilizing for 30min, and stirring once every 10 min;

placing the sterilized fresh bovine pericardium into 0.25 percent Trixon-100 solution by mass percentage, and oscillating for 48 hours at 37 ℃, and changing the solution once every 12 hours;

3U/mL DNase, 0.03mg/mL RNase, 2.5mmol/L MgCl were used₂0.1mmol/L of CaCl₂Preparing a mixed solution, placing the bovine pericardium treated by Trixon-100 in the mixed solution, and carrying out oscillation treatment at 37 ℃ for 24 hours, wherein the solution is changed once every 12 hours;

then, oscillating and rinsing for 48 hours by using distilled water, and changing the solution once every 8 hours;

the bovine pericardium was then rinsed with PBS buffer for 24h, changed, and then preserved with fresh PBS buffer.

Wherein, the PBS buffer solution formula is as follows: per 10XPBS 1L: NACL 80g, KCL 2g, KH₂PO₄ 2g，NA₂HPO₄*12H₂28.9g of O, and the deionized water is used for containing 1L.

Example 2 Cross-linking of decellularized bovine pericardium

1) Placing the decellularized bovine pericardium prepared in example 1 in a tannic acid solution (using 70% ethanol-water solution as a solvent, and adjusting the pH to 5 with hydrochloric acid) with a mass percentage concentration of 1%, and performing shaking treatment at normal temperature (25 ± 5 ℃) for 48 hours;

2) the tannic acid-decellularized bovine pericardium was put in a neomycin solution of 1mmol/L (using a morpholine ethanesulfonic acid solution of 0.05 mmol/L as a solvent, pH 6 was adjusted with hydrochloric acid), and treated with shaking at room temperature (25 ± 5 ℃) for 1 hour.

3) Placing tannin-neomycin-decellularized bovine pericardium into a 10 mmol/L5-riboflavin monophosphate solution (using PBS buffer solution as a solvent), and placing the solution under ultraviolet rays with the wavelength of 360nm for irradiation for 12 hours;

4) rinsed with PBS buffer and then stored with fresh PBS buffer.

Wherein, the PBS buffer solution formula is as follows: per 10XPBS 1L: NACL 80g, KCL 2g, KH₂PO₄ 2g，NA₂HPO₄*12H₂28.9g of O, and the deionized water is used for containing 1L.

Comparative example

0.625% glutaraldehyde (sigma) was prepared using PBS as a solvent, and the decellularized bovine pericardium prepared in example 1 was placed in a 0.625% glutaraldehyde solution, and after shaking treatment at room temperature for 2 days, the crosslinked bovine pericardium was placed in low-concentration (0.2%) glutaraldehyde and crosslinked for 7 days.

The cross-linked bovine pericardium prepared in example 2 was examined as follows:

1. masson staining, HE staining, and then observation under an optical microscope were performed, as shown in FIG. 1. Where 1.a is Masson stained tissue at 10 magnification, 1.b is HE stained tissue at 10 magnification.

From fig. 1, it can be seen that the bovine pericardium extracellular matrix treated by the invention is well preserved, and the collagen fiber structure is intact. HE staining is used for observing the overall structure and the nucleus structure of a sample, and can be used for observing the cell removing effect and the tissue crosslinking effect. The collagen structure can be observed by Masson staining, and the collagen crosslinking effect can be preliminarily judged.

The method comprises the following specific steps:

HE staining:

1. baking the tissue slices in a thermostat at 50-56 ℃ for 30 min;

2. dewaxing: soaking the tissue slices in turpentine for 10min 2 times;

3. hydration: soaking the slices in anhydrous ethanol, 95% ethanol, and 75% ethanol for 5min respectively, and washing with distilled water for 5min and 2 times;

4. staining sappan wood semen for 3 min;

4. slightly washing the sappan wood semen for 3s by running water;

5.1% hydrochloric acid ethanol differentiation for 3 s;

6. washing with running water or warm water for 20 min;

7. washing with distilled water for 2 s;

8.0.5% Irish solution for 1.5 min;

9. slightly washing with distilled water for 1-2 s;

10.95% ethanol for 3-5min

11. Absolute ethyl alcohol for 5-10 min;

12. and (5) sealing by using neutral gum.

Masson staining:

1. section deparaffinization hydration (reference HE staining);

2, dyeing by using Weiger's ferrohematoxylin for 5-10 min;

3. slightly washing with running water;

4.1% hydrochloric acid alcohol differentiation;

5. flushing for several minutes with running water;

6. dyeing the ponceau acid fuchsin liquid for 5 min;

7. slightly washing with distilled water;

treating with 8.1% phosphomolybdic acid water solution for about 5 min;

9. directly re-dyeing with aniline blue solution or green solution for 5min without washing;

treating with 10.1% glacial acetic acid for 1 min;

dehydrating with 11.95% ethanol for 3 min;

12. dehydrated by absolute alcohol, transparent xylene and sealed by neutral gum.

2, comparing the cross-linked bovine pericardium prepared in the example 2 with the bovine pericardium prepared in the comparative example in the aspects of mechanical property, thickness and collagen enzymolysis resistance.

2.1 Young's modulus

The cross-linked bovine pericardium prepared in example 2 and the comparative example was subjected to young's modulus measurement, and the result was obtained. The results are shown in FIG. 2.

Wherein, the Young modulus measurement specifically comprises: example 2VS comparative example: 292.56 + -127.55 MpaVS251.26 + -123.11 MPa

2.2 maximum tensile Strength

The cross-linked bovine pericardium prepared in example 2 and comparative example was subjected to the measurement of the maximum tensile strength, and the result obtained was the comparative example 2 VS: 19.55 plus or minus 8.40Mpa VS 17.89 plus or minus 10.07 Mpa. The results are shown in FIG. 3.

From the results of Young modulus and maximum tensile strength, the mechanical strength of the bovine pericardium treated by the method provided by the invention is superior to that of the bovine pericardium treated by glutaraldehyde.

The Young modulus and the maximum tensile strength are measured specifically as follows: the thickness and tensile length of each sample was measured and recorded using an electronic tensile tester (Instron, usa). Setting the stretching speed to be 50mm/min, obtaining a stress-strain curve chart of each sample, and then calculating the elastic modulus and the maximum tensile strength of the material.

2.3 temperature of thermal shrinkage

The thermal shrinkage temperature was measured for the cross-linked bovine pericardium prepared in example 2, comparative example, example 2VS comparative example: 82.62 + -3.27 deg.C VS82.76 + -3.51 deg.C. The results are shown in FIG. 4.

As can be seen from FIG. 4, the thermal shrinkage temperature of the treated bovine pericardium of the present invention is not statistically different from that of the glutaraldehyde group, which indicates that the thermal stability of the treated bovine pericardium of the present invention is not inferior to that of glutaraldehyde and the collagen crosslinking degree is not lower than that of glutaraldehyde.

2.4 thickness

The thickness of the cross-linked bovine pericardium prepared in example 2 and comparative example was measured, and example 2VS comparative example was 0.54 ± 0.079mmvs0.64 ± 0.039 mm. The results are shown in FIG. 5.

As can be seen from FIG. 5, the bovine pericardium treated by the present invention has a thinner thickness than the glutaraldehyde group, indicating that the plasticity of the biomaterial is better. The biological heart valve has the requirement of thickness, and if the thickness of the biological heart valve is 0.25-0.4mm, the thinner the biological heart valve is, the wider the application range is proved.

Wherein, the thickness measurement specifically is: and (3) paving the bovine pericardium, recording the thicknesses of two ends and the middle of each bovine pericardium by adopting a micrometer caliper, and taking the average value of three points as a final measurement value.

2.5 resistance to enzymatic hydrolysis

The collagen enzymolysis resistance and the elastase hydrolysis resistance of the fresh bovine pericardium before cell removal and the cross-linked bovine pericardium prepared in the example 2 and the comparative example are measured, and the collagen of the comparative example 2 VS: 98.03 ± 0.66% VS96.24 ± 0.82% spring force: 98.18 + -1.01% VS93.83 + -0.74%. The results are shown in FIGS. 6 to 7.

As can be seen in FIG. 6, the anti-collagen enzymatic ability of the bovine pericardium treated by the present invention is similar to that of the glutaraldehyde group.

As can be seen from FIG. 7, the anti-elastase ability of the bovine pericardium treated by the present invention is significantly higher than that of the glutaraldehyde group.

Wherein, the measurement of the anti-collagenase ability specifically comprises the following steps: bovine pericardium was sliced into 2 × 1cm size, vacuum freeze-dried and weighed W1, Tris 100mM +40mM CaCl₂+ collagenase 125U/ml, dissolved in PBS, adjusted pH 7.4 to make collagenase solution, freeze dried bovine pericardium placed in collagenase solution, at 37 degrees C reaction for 24 hours. The bovine pericardium after the reaction is lightly washed with deionized water for 3 times and freeze-dried for 24h again, and the measured mass is W2, and the weight loss percent is (W1-W2)/W1 is 100 percent, namely the resistance to enzymolysis, and the smaller the weight loss percent is, the stronger the resistance to enzymolysis is.

The elastic enzymolysis resistance experiment specifically comprises the following steps: preparing an elastase solution: tris 100mM +1mM CaCl₂pH 7.8+ elastase 30U/ml, dissolved in PBS and adjusted to pH 7.8. The remainder was subjected to anti-collagen enzymatic hydrolysis.

From the test results, the bovine pericardium treated by the ultraviolet crosslinking riboflavin-tannin composite crosslinking technology after cell treatment can obtain a good extracellular matrix structure, an excellent mechanical effect, a hot shrinkage temperature and a thin thickness, can have a wider application range, and has good collagen enzymolysis resistance and elastic enzymolysis resistance, and compared with the existing glutaraldehyde crosslinking, the bovine pericardium treated by the ultraviolet crosslinking riboflavin-tannin composite crosslinking technology has a better application space.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

1.A preparation method of a composite cross-linked biological valve is characterized by comprising the following steps:

1) treating the decellularized biological valve with tannic acid;

2) treating the biological valve treated by the tannic acid by using neomycin;

3) treating the biological valve treated by neomycin by using 5-riboflavin monophosphate under the condition of ultraviolet irradiation;

4) rinsing to obtain the product.

2. The method for preparing the composite cross-linked biological valve according to claim 1, wherein in the step 1), the tannic acid is: 0.5 to 2 mass percent of tannic acid solution, and 70 percent of ethanol-water solution as solvent, and adjusting the pH of the tannic acid solution to 5 to 6.

3. The method for preparing the composite cross-linked biological valve as claimed in any one of claims 1 to 2, wherein in the step 1), the vibration is performed for 45 to 50 hours at 20 to 30 ℃ after the tannin is added.

4. The method for preparing a composite cross-linked biological valve according to claim 1, wherein in step 2), the neomycin is specifically: 1-2mmol/L neomycin solution, 0.03-0.06 mmol/L morpholine ethanesulfonic acid as solvent, and adjusting pH of neomycin solution to 6-7.

5. The method for preparing the composite cross-linked biological valve according to any one of claims 1 or 4, wherein the neomycin is added and then the mixture is shaken for 1 to 1.5 hours at normal temperature.

6. The method for preparing a composite crosslinked biological valve according to claim 1, wherein in step 3), the riboflavin 5-monophosphate is in particular: 10-15 mmol/L5-riboflavin monophosphate solution, and the solvent is PBS buffer solution.

7. The method for preparing the composite cross-linked biological valve as claimed in any one of claims 1 or 6, wherein in the step 3), the wavelength of the ultraviolet light is 320-400 nm; and/or the presence of a gas in the gas,

after the 5-riboflavin monophosphate is added, ultraviolet light is used for irradiating for 12 to 14 hours under the stirring state.

8. The method for preparing a composite cross-linked biological valve according to claim 1, wherein the biological valve is decellularized by the steps of: disinfecting with benzalkonium bromide, then treating with Trixon-100 solution by shaking, and then treating with DNase and RNase; and then sequentially rinsing with distilled water and PBS buffer solution to obtain the biological valve after cell removal.

9. The method for preparing a composite cross-linked biological valve of claim 8, wherein the biological valve is decellularized by the steps of:

soaking fresh biological material with benzalkonium bromide 0.1-0.2% for 30-40min, and stirring once every 10-15 min;

then, using Trixon-100 solution with the mass percentage concentration of 0.25-0.3%, oscillating the biological material at 35-37 ℃ for 45-50h, and changing the solution every 10-12 h;

then using the mixed solution to perform oscillation treatment on the biological material at 35-37 ℃ for 24-26h, and changing the solution once every 10-12 h; the mixture contains 3-4U/mL DNase and 0.03-0.05Mg/mL RNase, and contains 2-3mmol/L Mg²⁺0.1-0.2mmol/L Ca²⁺；

Then, oscillating and rinsing the biological material by using distilled water for 45-50h, and changing the liquid once every 8-10 h;

the biological material was then rinsed with PBS buffer for 24-26h, changed and then stored in PBS buffer.

10. A composite cross-linked bioprosthetic valve made by the method of any one of claims 1-9.

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