CN106561635A - Method for dry preservation of biological tissue - Google Patents

Abstract

The present invention relates to a method for dry preservation of biological tissue, the method comprises contacting three kinds of solutions containing organic solvent and glycerin with the biological tissue sequentially, and removing part of the solutions from the biological tissue treated by the solution, Sterilized, frozen and then stored dry. The size of the biological tissue treated by the above method is basically the same as that before dehydration, which completely eliminates the side effects of water and makes the dry tissue less susceptible to microorganisms, so that medical personnel can obtain it as close to the ready-to-use form as possible.

Description

A method for dry preservation of biological tissue

technical field

The invention relates to the field of implant materials, in particular to a method for dry preservation of biological tissues.

Background technique

As a kind of organic material, biological tissue material has become a research hotspot because of its good hemodynamic performance, less risk of embolism, less hemolysis, no need for anticoagulant therapy, convenient material acquisition, and low cost. But its main disadvantage is that it is more prone to tissue degeneration, calcification and rejection, which affects its durability.

Many scholars have engaged in a lot of research, trying to overcome these defects. It was not until 1968, when French scholars Carpentier et al. proposed the use of glutaraldehyde to treat and preserve biological tissues, that the durability of biological tissues was greatly improved, and biological tissues treated with glutaraldehyde were widely used clinically again.

However, long-term clinical practice has proved that the biological tissues treated with glutaraldehyde cross-linking have the following defects: 1) cytotoxicity, the long-term residual glutaraldehyde in the cross-linked tissue has toxic effects on cells. 2) The existence of aldehyde groups and other reasons lead to calcification of the tissue after cross-linking, which is one of the important reasons for the decline of biological tissues; 3) Incomplete elimination of immunogenicity is one of the reasons for calcification and also leads to inflammatory reactions One of the mechanisms; 4) Residual cells and debris are one of the main sites of tissue calcification and an important source of immunogenicity; 5) Tissues with residual reagents must be washed before implantation.

Therefore, how to overcome the defects caused by the treatment and preservation of biological tissues with glutaraldehyde has become an urgent problem to be solved.

CN101626682A discloses a biological tissue for surgical implantation and a treatment method thereof. The method for treating the biological tissue includes contacting the biological tissue with a non-aqueous treatment solution comprising polyhydric alcohols and C ₁ -C ₃ alcohols, thereby making the treated biological tissue The tissue will recover at least about 97% of its original hydrated size, while avoiding the drawbacks of glutaraldehyde treatment and preservation of biological tissue; however, the method requires a long processing time and a large volume of solution, which is not conducive to simple operation and reduce costs.

Contents of the invention

In order to solve the above-mentioned technical problems, the present invention provides a method for dry preservation of biological tissue, the size of the biological tissue treated by the above method is basically the same as that before dehydration, completely eliminates the side effects of water, and makes the dry tissue less susceptible to microorganisms impact, so that medical professionals can obtain as close to ready-to-use form as possible.

For reaching this purpose, the present invention adopts following technical scheme:

In a first aspect, the present invention provides a method for dry preservation of biological tissue, the method comprising:

(1) Preparation of solutions A, B and C comprising organic solvent and glycerin;

The solution A contains 5% to 50% by volume of organic solvent and 50% to 95% by volume of glycerol;

The solution B contains 5% to 30% by volume of organic solvent and 70% to 95% by volume of glycerol;

The solution C contains 1% to 28% by volume of organic solvent and 72% to 99% by volume of glycerin;

(2) Bringing the biological tissue into contact with the solutions A, B and C in the order of solutions A→B→C.

In the present invention, the solutions A, B and C are used to contact the biological tissue in the order of solution A → B → C, that is, the three solutions are combined and contacted with the biological tissue in a certain order; Compared with only one or two of the above solutions for treatment, it can make the size of the treated biological tissue closer to the size before dehydration, making the dried tissue less susceptible to microorganisms.

In the solution A of the present invention, the volume fraction of the organic solvent is 5% to 50%, such as 5%, 7%, 8%, 10%, 12%, 15%, 18%, 20%, 22%, 25%, 28%, 30%, 32%, 35%, 38%, 40%, 42%, 45%, 48% or 50%, and specific point values in between It is contemplated that the invention is not intended to be an exhaustive recitation of the specific point values encompassed by the stated ranges.

According to the present invention, the volume fraction of the organic solvent in the solution A is preferably 5%-25%, more preferably 25%.

In the solution A of the present invention, the volume fraction of glycerol is 50% to 95%, such as 50%, 52%, 55%, 58%, 60%, 62%, 65%, 68%, 70%, 75% %, 80%, 82%, 85%, 90%, 91%, 92%, 93%, 94% or 95%, as well as specific point values between the above-mentioned numerical values, limited by space and for the sake of simplicity, the present invention The specific point values encompassed by the stated ranges are not intended to be exhaustive.

According to the present invention, the volume fraction of glycerol in the solution A is preferably 75%-95%, more preferably 75%.

In the solution B of the present invention, the volume fraction of the organic solvent is 5% to 30%, such as 5%, 8%, 10%, 15%, 17%, 18%, 20%, 22%, 23%, 25%, 28% or 30%, as well as specific point values between the above values, are limited in space and for the sake of simplicity, the present invention will not exhaustively list the specific point values included in the range.

According to the present invention, the volume fraction of the organic solvent in the solution B is preferably 16%-20%, more preferably 20%.

In the solution B of the present invention, the volume fraction of glycerol is 70% to 95%, such as 70%, 72%, 75%, 77%, 78%, 80%, 82%, 85%, 88%, 90% %, 92% or 95%, and the specific point values between the above-mentioned numerical values, due to space limitation and for the sake of brevity, the present invention will not exhaustively list the specific point values included in the range.

According to the present invention, the volume fraction of glycerol in the solution B is preferably 80% to 84%, more preferably 80%.

In the solution C of the present invention, the volume fraction of the organic solvent is 1% to 28%, such as 1%, 2%, 5%, 10%, 12%, 15%, 18%, 20%, 22%, 23%, 25% or 28%, and specific point values between the above-mentioned numerical values, due to space limitation and for the sake of brevity, the present invention will not exhaustively list the specific point values included in the range.

According to the present invention, the volume fraction of the organic solvent in the solution C is preferably 5% to 15%, more preferably 15%.

In the solution C of the present invention, the volume fraction of glycerol is 72% to 99%, such as 72%, 75%, 77%, 78%, 80%, 82%, 85%, 88%, 90%, 92% %, 95% or 99%, and the specific point values between the above numerical values, due to space limitation and for the sake of brevity, the present invention will not exhaustively list the specific point values included in the range.

According to the present invention, the volume fraction of glycerol in the solution C is preferably 85%-90%, more preferably 85%.

According to the present invention, in step (1), the organic solvent is any one or a mixture of at least two of ethanol, n-propanol, 2-propanol, isopropanol or methanol, preferably ethanol, n-propanol or Any one or a mixture of at least two of 2-propanols, more preferably ethanol.

According to the present invention, in step (2), the biological tissue is a mammalian tissue.

Preferably, the mammalian tissue is any one or at least two of animal pericardium, aorta, mitral valve, tricuspid valve, pulmonary artery, ligament, skin, peritoneum, pleura, Achilles tendon or venous valved duct The mixture is more preferably bovine pericardium.

According to the present invention, in step (2), the contact time of the biological tissue with the solutions A, B and C is all greater than 40 minutes, preferably greater than 1 hour.

Preferably, the temperature at which the biological tissue is in contact with the solutions A, B and C is 0°C to 80°C, preferably 10°C to 30°C, more preferably 30°C.

According to the invention, the method further comprises removing parts of the solutions A, B and C from the solution-treated biological tissue.

In a second aspect, the present invention also provides a sterilized solution-treated biological tissue for surgical implantation into a human being prepared by a method comprising the steps of:

(1) Preparation of solutions A, B and C comprising organic solvent and glycerol;

The solution A contains 5% to 50% by volume of organic solvent and 50% to 95% by volume of glycerin;

The solution B contains 5% to 30% by volume of organic solvent and 70% to 95% by volume of glycerol;

The solution C contains 1% to 28% by volume of organic solvent and 72% to 99% by volume of glycerol;

(2) Bringing the biological tissue into contact with the solutions A, B and C in the order of solutions A→B→C.

The volume ratio of the organic solvent and glycerin in the above-mentioned solutions A, B and C in the present invention is the same as that in the first aspect, and will not be repeated here.

According to the present invention, the biological tissue is a mammalian tissue.

Preferably, the mammalian tissue is any one or at least two of animal pericardium, aorta, mitral valve, tricuspid valve, pulmonary artery, ligament, skin, peritoneum, pleura, Achilles tendon or venous valved duct The mixture is more preferably bovine pericardium.

The present invention uses different ratios of organic solvents and glycerol mixed solutions to contact biological tissues to obtain relatively dry dehydrated biological tissues. At this time, the biological tissue is reduced in size, followed by sterilization and packaging. When hydrated, biological tissue is approximately the same size as it was before dehydration

Compared with the prior art, the present invention has at least the following beneficial effects:

The size of the biological tissue treated by the above method is basically the same as the size before dehydration, which completely eliminates the side effects of water and makes the dry tissue less susceptible to microorganisms, so that medical personnel can obtain it as close to the ready-to-use form as possible; moreover, compared with separate Using solution A, B or C solution and using two kinds of combined treatment, the present invention can make the treated biological tissue contact with the solution A, B and C in the order of solution A→B→C. The size of the biological tissue is closer to the size before dehydration, making the dried tissue less susceptible to microorganisms.

detailed description

In order to facilitate understanding of the present invention, the present invention enumerates the following examples. It should be clear to those skilled in the art that the embodiments are only for helping to understand the present invention, and should not be regarded as specific limitations on the present invention.

Example 1

The glutaraldehyde-treated biological tissue (porcine pericardium was used in this experiment) was cut into three groups of similar size, 6 pieces in each group. Measure the size and record it. The pig pericardium was completely submerged in solution A for 12 hours, completely submerged in solution B for 12 hours after taking it out, and completely submerged in solution C for 12 hours after taking it out. Wherein solution A, B, the volume ratio of C and pig pericardium are all 80:1, and its distribution ratio of each group is as shown in Table 1.

Table 1

Solution combination Solution A Solution B Solution C glycerin 75% 80% 85% ethanol 25% 20% 15%

Test items:

1. Measure and record the diameter and thickness of the pericardial slices before treatment, after drying and after hydration, and read after the data is stable for 5 seconds. The same pericardial slice was tested 6 times at different positions, and the average value was taken. The specific test results are shown below.

Wherein table 2-3 has shown adopting solution A, B and C in the embodiment 1 to process successively the diameter and the thickness and the rate of change of the pericardial slices before processing, after drying and after hydration; The three solutions of solution A, B or C in 1 were treated separately, and used as a comparative example to measure the diameter and thickness of these three solutions before treatment, after drying and after hydration, and the specific results are shown in Table 4- 5.

Table 2 adopts the concrete result that solution A, B and C are successively processed successively

Table 3 adopts solution A, B and C to process successively the change rate of the result

Table 4 adopts the concrete result of solution A, B or C processing alone

Table 5 adopts the rate of change of the result of solution A, B or C processing alone

By comparing the data in Table 2-3 with the data in Table 4-5, it can be seen that when the solutions A, B and C in Example 1 are used to process sequentially, the diameter of the pericardial slice after drying and after hydration The rate of change of thickness and thickness are all less than the rate of change when using separate solution A, B or C to process, thus also explaining that the present invention adopts when three kinds of solutions are processed sequentially, compared with adopting single solution, it can make The size of biological tissue after treatment is closer to the size before dehydration.

2. Use the leather shrinkage temperature measuring instrument to measure and record the thermal shrinkage temperature of the pericardial slices before and after hydration using solutions A, B and C in Example 1 in sequence. The specific results are shown in Table 6.

Table 6

3. Measure respectively and record adopting solution A, B and C in embodiment 1 to process the pericardium sheet successively before processing, drying and the maximum tensile force after hydration so as to calculate the yield strength of the pig pericardium, wherein, table 7 is the pericardium sheet Longitudinal: the data obtained by cutting parallel fiber orientation, Table 8 is the pericardial slice transverse: the data obtained by perpendicular fiber orientation cutting.

Table 7

Table 8

It can be seen that, by using the above-mentioned solutions A, B and C to contact biological tissues in the order of solution A → B → C, it can be concluded that there is no significant difference in the data obtained before and after hydration, so that after treatment The size of the biological tissue is very close to the size before dehydration.

Example 2-7

The components shown in Table 9 are used to prepare solutions A, B and C, and the order of solutions A→B→C is used to contact biological tissues. After testing, the diameter and thickness changes before treatment, after drying and after hydration The rates are shown in Table 10.

Table 9

Table 10

It can be seen from the above results that in Examples 1-7, the solutions A, B and C are used to contact the biological tissue in the order of solution A→B→C, which can make the size of the treated biological tissue closer to the size before dehydration. , so that the dry tissue is less susceptible to microorganisms; in the embodiments 1-7, compared with the other embodiments 2-7, the embodiment 1 can make the size of the biological tissue after treatment the closest to the size before dehydration.

The present invention has also verified other animal pericardium (such as cattle, sheep, kangaroos, etc.), aorta, mitral valve, tricuspid valve, pulmonary artery, ligament, skin, ligament, peritoneum, pleura, Achilles tendon, venous valved conduit, etc. obtained similar results.

The applicant declares that the present invention illustrates the detailed process equipment and process flow of the present invention through the above-mentioned examples, but the present invention is not limited to the above-mentioned detailed process equipment and process flow, that is, it does not mean that the present invention must rely on the above-mentioned detailed process equipment and process flow process can be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims (8)

1. a kind of method of kept dry biological tissue, it is characterised in that methods described includes：

(1) solution A, B and C comprising organic solvent and glycerol are prepared；

5%～50% organic solvent and by volume 50%～95% glycerol by volume are included in the solution A；

5%～30% organic solvent and by volume 70%～95% glycerol by volume are included in the solution B；

1%～28% organic solvent and by volume 72%～99% glycerol by volume are included in the solution C；

(2) biological tissue and the solution A, B and C are made by the progressive contact of solution A → B → C.

2. the method for claim 1, it is characterised in that in step (1), it is preferable that comprising pressing volume in the solution A The organic solvent of meter 5%～25% and by volume 75%～95% glycerol, the preferably solution A is by by volume 25% Organic solvent and by volume 75% glycerol composition；

Preferably, comprising 16%～20% organic solvent by volume and by volume 80%～84% in the solution B Glycerol, the preferably solution B by by volume 20% organic solvent and by volume 80% glycerol is constituted；

Preferably, in the solution C comprising 5%～15% organic solvent by volume and by volume 85%～95% it is sweet Oil, the preferably solution C by by volume 15% organic solvent and by volume 85% glycerol is constituted.

3. method as claimed in claim 1 or 2, it is characterised in that in step (1), the organic solvent be ethanol, positive third In alcohol, 2- propanol, isopropanol or methanol any one or at least two mixture, preferred alcohol, normal propyl alcohol or 2- propanol In any one or at least two mixture, further preferred ethanol.

4. the method as described in one of claims 1 to 3, it is characterised in that in step (2), the biological tissue is dynamic for suckling Thing is organized；

Preferably, the mammalian tissues be animal pericardium, aorta, Bicuspid valve, Tricuspid valve, pulmonary artery, ligament, skin, In peritoneum, pleura, heel string or vein valved conduit any one or at least two mixture, further preferred bovine pericardium.

5. the method as described in one of Claims 1 to 4, it is characterised in that in step (2), the biological tissue is molten with described The time of liquid A, B and C contact is all higher than 40min, preferably greater than 1h；

Preferably, the temperature that the biological tissue contacts with the solution A, B and C be 0 DEG C～80 DEG C, preferably 10 DEG C～30 DEG C, Further preferred 30 DEG C.

6. the method as described in one of claim 1-5, it is characterised in that methods described also includes the biology processed from solution The part solution A, B and C are removed in tissue.

7. the biological tissue that the solution of the sterilizing being used in Srgery grafting to people is processed, it is characterised in that the solution of the sterilizing The biological tissue of process is prepared by the method for comprising the following steps：

(1) solution A, B and C comprising organic solvent and glycerol are prepared；

5%～50% organic solvent and by volume 50%～95% glycerol by volume are included in the solution A；

5%～30% organic solvent and by volume 70%～95% glycerol by volume are included in the solution B；

1%～28% organic solvent and by volume 72%～99% glycerol by volume are included in the solution C；

(2) biological tissue and the solution A, B and C are made by the progressive contact of solution A → B → C.

8. biological tissue as claimed in claim 7, it is characterised in that the biological tissue is mammalian tissues；

Preferably, the mammalian tissues be animal pericardium, aorta, Bicuspid valve, Tricuspid valve, pulmonary artery, ligament, skin, In peritoneum, pleura, heel string or vein valved conduit any one or at least two mixture, further preferred bovine pericardium.