CN109395164B

CN109395164B - Preparation method of dried animal extracellular matrix material

Info

Publication number: CN109395164B
Application number: CN201811441852.9A
Authority: CN
Inventors: 王云兵; 雷洋; 李高参
Original assignee: Sichuan University
Current assignee: Hangzhou Qiming Medical Devices Co.,Ltd.
Priority date: 2018-11-29
Filing date: 2018-11-29
Publication date: 2020-01-10
Anticipated expiration: 2038-11-29
Also published as: CN109395164A

Abstract

The invention discloses a preparation method of an anhydrated animal extracellular matrix material, which comprises the following steps: the animal pericardium, blood vessels and small intestines obtained from a slaughterhouse are subjected to multiple decellularization, protein cleaning, specific mold fixing appearance and specific solvent moderate dehydration, the obtained extracellular matrix material has good flatness and elasticity in a dry state, and a biological membrane obtained by cross-linking the pericardium after being stored for 3 months has equivalent maximum breaking tensile stress and elongation compared with the pericardium which is not subjected to drying direct cross-linking treatment; the method can greatly reduce the transportation and storage cost of the extracellular matrix material needing to be preserved by the wet aqueous solution at present, and provides a solution for preserving the extracellular matrix material for a long time.

Description

Preparation method of dried animal extracellular matrix material

Technical Field

The invention relates to the technical field of biomedical materials and medical instruments, in particular to a preparation method of a dried pericardium.

Background

Cardiovascular and cerebrovascular diseases have become the first killers threatening human health. Among them, coronary heart disease and heart valve diseases are the two most common diseases. Valvular heart disease refers to a disease in which the normal flow of blood is affected due to the occurrence of a pathological change in the valves of the body, thereby causing abnormal cardiac function. With the aging population, the incidence of valvular heart disease in China, particularly aortic stenosis disease in the elderly, is increasing year by year. According to incomplete statistics, only aortic valvular lesions are found in China, and about 20 ten thousand patients are newly added every year. For valves that produce severe lesions, replacement surgery is often required using prosthetic heart valves. Among them, artificial biological heart valves prepared from animal (porcine or bovine) pericardium are the mainstream products of valve replacement surgery. The current process flow of biological valves is to transport and store fresh pericardium obtained from slaughterhouses in an aqueous solution at low temperature (typically 4 ℃) and cold chain conditions and to cross-link as quickly as possible within a certain time (3-7 days). If the transport and storage are improper, for example, the fresh pericardium is not soaked in the water solution according to the specification, the pericardium can be air-dried and irreversibly deformed; or the fresh pericardium is not stored in a low-temperature environment according to the specification, so that the pericardium can be rotten; or stored in a low temperature environment of the aqueous solution for too long a time (more than 7 days or more), may also cause degradation damage of the fresh film.

Coronary heart disease is another common cardiac disease. Coronary artery bypass surgery is one of the important means for treating coronary heart disease at present, and the research of finding and developing an ideal artificial small vessel material is always the key point of research. The decellularized blood vessel stent has low immunogenicity and good mechanical properties, has mechanical properties similar to those of a recipient blood vessel, and is considered to be one of the most potential blood vessel substitute materials except for a self blood vessel. However, the current animal extracellular matrix materials for artificial small blood vessels generally require liquid storage, so that the animal extracellular matrix materials cannot be stored for a long time and transported over a long distance. The invention aims to optimize and improve the current process flow of the extracellular matrix material, prepare the dehydrated dried extracellular matrix material from the extracellular matrix material of the animal, greatly improve the convenience of the extracellular matrix material in the aspects of transportation and storage and reduce the cost of cold chain transportation and storage.

Disclosure of Invention

The present invention is directed to solving the above-mentioned deficiencies of the prior art and providing a method for preparing a pericardium that is directly cross-linked without drying and has a maximum tensile stress at break and an elongation that are comparable to those of a pericardium that is not dried.

The purpose of the invention is realized by the following technical scheme.

A preparation method of dried animal extracellular matrix material comprises the following specific steps:

a. multiple cell removal treatment: the combination of DNase, RNase, trypsin, sodium dodecyl sulfate, deoxycholic acid and polyethylene glycol octyl phenyl ether is adopted to carry out acellular treatment on the cardiac envelope and the blood vessel, and the acellular treatment time is from hours to days.

b. Protein cleaning: protein washing was performed using RIPA lysate.

c. Fixing the shape of a specific mold: including using a plexiglas plate mold to fix the shape.

d. Moderate dehydration of the specific solvent: comprises moderately dehydrating with a polyhydric alcohol solvent such as ethanol, glycerol, isopropanol, etc., for several hours to several days.

Further, the pericardium is a porcine pericardium or a bovine pericardium.

Further, the blood vessel is a small-caliber vein blood vessel of the animal.

Further, the concentration of the DNase for the multiple cell removal treatment in the step a is 10-1000U/ml, the concentration of the RNase is 2-200 mu g/ml, the concentration of the trypsin is 20-2000U/ml, the concentration of the SDS is 0.05-5%, the concentration of the deoxycholic acid is 0.01-1%, and the concentration of the Triton X-100 is 0.01-1%.

Further, the specific mold in the step c is one of a plastic glass plate mold or a plastic glass tube mold.

Further, the polyol solvent used in step d preferably comprises 75% ethanol, 100% ethanol, a mixture of 25% glycerol and 75% ethanol, a mixture of 100% glycerol, 25% glycerol with 50% ethanol and 25% isopropanol.

Furthermore, in the step d, after the dried pericardium which is properly dehydrated by the specific solvent is stored in a normal air environment at room temperature for 3 months, the pericardium is prepared by crosslinking.

The invention has the beneficial effects that: the extracellular matrix material prepared by the method has good flatness and elasticity in a dry state. Wherein the pericardium, which is cross-linked after long-term (at least 3 months) desiccation storage, has a comparable maximum tensile stress at break and elongation compared to a pericardium that is not cross-linked directly by desiccation.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not to be construed as limiting

b. Protein cleaning: protein washing was performed using RIPA lysate.

In this embodiment, the pericardium is porcine pericardium or bovine pericardium. The blood vessel is a small-bore venous blood vessel of an animal. The concentration of the DNA enzyme used in the multiple cell removing treatment in the step a is 10-1000U/ml, the concentration of the RNA enzyme used is 2-200 mu g/ml, the concentration of the trypsin used is 20-2000U/ml, the concentration of the SDS used is 0.05-5%, the concentration of the deoxycholic acid used is 0.01-1%, and the concentration of the Triton X-100 used is 0.01-1%. The specific mold in the step c is one of an organic glass plate mold or an organic glass tube mold. The polyol solvent used in step d preferably comprises a mixture of 75% ethanol, 100% ethanol, a mixture of 25% glycerol and 75% ethanol, 100% glycerol, 25% glycerol with 50% ethanol and 25% isopropanol. And d, storing the dried pericardium which is dehydrated properly by using a specific solvent for 3 months at room temperature in a common air environment, and crosslinking to prepare the obtained pericardium.

Five groups of implementations were performed on the pericardium according to the above:

example 1

Fresh pig heart envelopes were obtained from slaughterhouses and surface-adhering fat was removed. The whole complete pig heart envelope is fixed on a square organic glass plate mould, and the appearance is fixed by a rubber band. The mixture of 100U/ml DNase, 20. mu.g/ml RNase and 200U/ml trypsin was soaked for 4 hours. The RIPA lysate was soaked for 4 hours. 0.5% SDS was soaked for 24 hours. Soaking with 75% ethanol for 4 hr, soaking with 100% ethanol for 4 hr, and soaking with 25% glycerol, 50% glycerol and 25% isopropanol for 24 hr. Obtaining the dried pericardium.

Example 2

Fresh pig heart envelopes were obtained from slaughterhouses and surface-adhering fat was removed. The whole complete pig heart envelope is fixed on a square organic glass plate mould, and the appearance is fixed by a rubber band. The mixture of 100U/ml DNase, 20. mu.g/ml RNase and 200U/ml trypsin was soaked for 4 hours. The RIPA lysate was soaked for 4 hours. 0.5% SDS was soaked for 24 hours. 0.1 percent TritonX-100 is soaked for 24 hours. Soaking in 75% ethanol for 4 hr, soaking in 100% ethanol for 4 hr, and soaking in 25% glycerol, 50% glycerol and 25% isopropanol for 24 hr. Obtaining the dried pericardium.

Example 3

Fresh bovine pericardium was obtained from a slaughterhouse and surface-adhering fat was removed. The whole complete pig heart envelope is fixed on a square organic glass plate mould, and the appearance is fixed by a rubber band. The mixture of 100U/ml DNase, 20. mu.g/ml RNase and 200U/ml trypsin was soaked for 4 hours. The RIPA lysate was soaked for 4 hours. 0.5% SDS was soaked for 24 hours. Soaking with 75% ethanol for 4 hr, soaking with 100% ethanol for 4 hr, and soaking with 25% glycerol, 50% glycerol and 25% isopropanol for 24 hr. Obtaining the dried pericardium.

Example 4

Fresh porcine femoral vein vessels were obtained from the slaughterhouse and surface adhering fat was removed. The pig femoral vein blood vessel is fixed on a circular organic glass tube mold, and the shape is fixed by a rubber band. The mixture of 100U/ml DNase, 20. mu.g/ml RNase and 200U/ml trypsin was soaked for 4 hours. The RIPA lysate was soaked for 4 hours. 0.5% SDS was soaked for 24 hours. Soaking with 75% ethanol for 4 hr, soaking with 100% ethanol for 4 hr, and soaking with 25% glycerol, 50% glycerol and 25% isopropanol for 24 hr. Obtaining the dried porcine femoral vein blood vessel.

Example 5

Fresh porcine femoral vein vessels were obtained from the slaughterhouse and surface adhering fat was removed. The pig femoral vein blood vessel is fixed on a circular organic glass tube mold, and the shape is fixed by a rubber band. The mixture of 100U/ml DNase, 20. mu.g/ml RNase and 200U/ml trypsin was soaked for 4 hours. The RIPA lysate was soaked for 4 hours. 0.5% SDS was soaked for 24 hours. 0.1 percent TritonX-100 is soaked for 24 hours. Soaking in 75% ethanol for 4 hr, soaking in 100% ethanol for 4 hr, mixing with 25% glycerol, and soaking in 75% glycerol for 24 hr. Obtaining the dried porcine femoral vein blood vessel.

Examples of the experiments

To verify the maximum tensile stress and elongation at break and the long-term stability of the preparation of the dried pericardium, the dried animal extracellular matrix material prepared in examples 1-2 was therefore subjected to uniaxial tensile testing.

After the dried pericardium prepared in the embodiment is stored for 3 months in a normal air environment at room temperature, the dried pericardium is soaked in 1% glutaraldehyde solution for 24 hours and 0.25% glutaraldehyde for 24 hours, and the numbers of the prepared cross-linked biological membranes are respectively a dried membrane 1 and a dried membrane 2.

The glutaraldehyde control group is prepared by storing freshly collected pig heart envelope in normal saline at 4 degree of pH within 3-7 days, directly soaking with 1% glutaraldehyde solution for 24 hr, and soaking with 0.25% glutaraldehyde solution for 24 hr. Dumbbell-type tensile specimens with dimensions of 2cm x 35cm were prepared and specimen thickness was measured and recorded.

The test mode is as follows: and (4) adopting a universal mechanical experiment machine to carry out tensile test, wherein the tensile rate is 12mm/min, and the tensile test is carried out until the sample is broken.

The maximum tensile stress at break and the maximum elongation at break were recorded and calculated. Each set was tested in duplicate for 5 samples.

TABLE 1

TABLE 2

Table 1 maximum tensile stress at break calculation and statistics.

Table 2 maximum elongation at break calculation and statistics.

The results in tables 1 and 2 show that: the maximum tensile stress at break and the maximum elongation at break of the dried films 1 and 2 prepared in examples 1 and 2 are comparable to those of a control film which is not dried and directly crosslinked.

It is understood that the above are only exemplary embodiments of the present invention, and other embodiments of the present invention may be made by using equivalents or equivalent changes, which fall within the scope of the claims of the present invention.

Claims

1. A preparation method of dried animal tissue is characterized by comprising the following specific steps:

a. multiple cell removal treatment: adopting a combination of DNase, RNase, trypsin, sodium dodecyl sulfate, deoxycholic acid and polyethylene glycol octyl phenyl ether to carry out decellularization treatment on animal tissues, wherein the decellularization treatment time is from several hours to several days;

b. protein cleaning: protein cleaning is carried out by RIPA lysate;

c. fixing the shape by using a specific mold, wherein the specific mold is one of an organic glass plate mold or an organic glass tube mold;

d. moderate dehydration of the specific solvent: comprises moderate dehydration by using alcoholic solvent, wherein the alcoholic solvent is one or more of ethanol, glycerol and isopropanol, and the dehydration treatment time is several hours to several days.

2. The method for preparing the dried animal tissue according to claim 1, wherein the animal tissue is pericardium or blood vessel.

3. The method of claim 2, wherein the step of drying the animal tissue comprises: the pericardium is porcine pericardium or bovine pericardium.

4. The method of claim 2, wherein the step of drying the animal tissue comprises: the blood vessel is a small-bore venous blood vessel of an animal.

5. The method of claim 1 for preparing dried animal tissue, wherein the method comprises the steps of: the concentration of the DNA enzyme used in the multiple cell-removing treatment in the step a is 10-1000U/ml, the concentration of the RNA enzyme used is 2-200 mu g/ml, the concentration of the trypsin used is 20-2000U/ml, the concentration of the SDS used is 0.05-5%, the concentration of the deoxycholic acid used is 0.01-1%, and the concentration of the TritonX-100 used is 0.01-1%.

6. The method of claim 1 for preparing dried animal tissue, wherein the method comprises the steps of: the alcohol solvent used in step d comprises 75% ethanol, 100% ethanol, a mixture of 25% glycerol and 75% ethanol, a mixture of 100% glycerol, 25% glycerol with 50% ethanol and 25% isopropanol.

7. The method of claim 1 for preparing dried animal tissue, wherein the method comprises the steps of: and d, storing the dried pericardium which is dehydrated properly by using a specific solvent for 3 months at room temperature in a common air environment, and crosslinking to prepare the obtained pericardium.

8. An desiccated animal tissue prepared by the method of claim 1.