WO2024098892A1

WO2024098892A1 - Anti-calcification animal-derived biomedical material, preparation method therefor, and use thereof in artificial heart valves and biological patches

Info

Publication number: WO2024098892A1
Application number: PCT/CN2023/115278
Authority: WO
Inventors: 李希恂; 杨威; 魏勇强
Original assignee: 江苏臻亿医疗科技有限公司
Priority date: 2022-11-08
Filing date: 2023-08-28
Publication date: 2024-05-16
Also published as: CN115779150B; CN115779150A

Abstract

The present invention provides an anti-calcification animal-derived biomedical material, a preparation method therefor, and use thereof in artificial heart valves and biological patches. For the anti-calcification animal-derived biomaterial, an appropriate permeabilization liquid and treatment parameters are selected to moderately increase the permeability of the cell membrane and to reduce the damage to tissue proteins while it is guaranteed that the permeability of the cell membrane is increased, that is, removing glycoproteins in cells more completely becomes easier, and then enzymes capable of specifically degrading Neu5Gc and αGal and appropriate enzymolysis parameters are selected to reduce the influence of the process on collagen fibers to a greater extent, so that the glycoproteins in the cells are completely removed while not influencing the mechanical properties of tissues. On the basis of original anti-calcification treatment, the present invention removes tissue immunogenic glycan, so that the calcification of biological tissues is reduced, and the service life of the biological tissues is prolonged.

Description

Anti-calcification animal-derived biomaterial, preparation method and application thereof in artificial heart valve and biological patch

Technical Field

The present invention relates to the technical field of medical biomaterials, and in particular to an anti-calcification animal-derived biomaterial, a preparation method and application thereof in artificial heart valves and biological patches.

Background technique

Biomedical materials are materials used to diagnose, treat, repair or replace damaged tissues and organs or enhance their functions. They are the basis for studying artificial organs and medical devices, and have become an important branch of contemporary materials science. Especially with the vigorous development and major breakthroughs in biotechnology, biomedical materials have become a hot spot for scientists from all over the world to compete in research and development.

Biomedical materials are widely used and can be used for skeletal-muscle system repair materials such as bones, teeth, joints, tendons, soft tissue materials such as skin, esophagus, respiratory tract, bladder, cardiovascular system materials such as artificial heart valves, blood vessels, cardiovascular catheters, blood purification membranes and separation membranes, gas selective permeable membranes, corneal contact lenses and other medical membrane materials, etc.

Biomedical materials can be divided into biomedical metal materials, biomedical inorganic non-metallic materials, biomedical polymer materials, biomedical composite materials and bio-derived materials according to the composition and properties of the materials. Among them, bio-derived materials are biomedical materials formed by specially treated natural biological tissues, also known as bioregenerative materials. Biological tissues can be taken from tissues of the same or different species of animals. Special treatments include fixation, sterilization and mild treatments to eliminate antigenicity to maintain the original configuration of the tissue, as well as strong treatments to dismantle the original configuration and rebuild a new physical form. Since the treated biological tissue has lost its vitality, bio-derived materials are lifeless materials. However, since bio-derived materials either have a configuration and function similar to natural tissues, or their composition is similar to natural tissues, they play an important role in maintaining the repair and replacement of the dynamic processes of the human body.

At present, bio-derived materials commonly used in clinical applications such as artificial heart valves and biological patches are derived from animal-derived collagen fiber tissue. After implantation, such products are prone to calcification, which can cause hardening and tearing of biological tissue materials, leading to failure of the tissue materials.

There are many reasons for calcification of biological tissues. For example, many biological tissue materials use The valve tissue is stored in glutaraldehyde and/or formaldehyde, or is cross-linked and fixed with these chemical reagents. After the valve tissue is cross-linked with glutaraldehyde, the residual glutaraldehyde after implantation will promote the calcification of the valve. If the glutaraldehyde cross-linking is insufficient, the residual amino groups, aldehyde groups, carboxyl groups, etc. will form calcification binding points, which are prone to calcification. In addition, glutaraldehyde solution preserves biological tissues and easily oxidizes biological tissues to form carboxylic acids, produce calcification binding points, and lead to calcification. For example, certain substances in biological tissues may cause calcification.

The current anti-calcification solutions in the prior art focus more on reducing the impact of chemical reagents such as glutaraldehyde, or reducing or even eliminating the factors of calcification by removing substances that may cause calcification in tissues, such as phospholipid residues.

However, animal tissues also contain sugars that the human body cannot produce, such as Neu5Gc. If these tissues are not treated, the non-human sugars in them will induce harmful immune responses in the recipients after they are implanted in the human body, and the antibodies produced will mediate the calcification of the leaflets of the biological valve. The antibody response to these foreign sugars appears as early as one month after implantation and may last for a certain period of time, ultimately affecting the service life of the valve.

Summary of the invention

The purpose of the present invention is to provide a method for preparing an anti-calcification animal-derived biomaterial to address the problem that immunogenic glycans in biological tissues induce receptors to produce harmful immune responses, and the antibodies produced will mediate the calcification of the leaflets of biological valves. On the basis of the original anti-calcification treatment, the tissue immunogenic glycans are removed to extend the service life of the valve.

The first aspect of the present invention relates to a method for preparing an anti-calcification animal-derived biomaterial, which reduces the antibody response produced by the receptor after the biological tissue is implanted by removing immunogenic polysaccharides in the biological tissue, thereby reducing immunosaccharide antibody-mediated calcification and improving the anti-calcification ability of the biological tissue.

As an optional embodiment, the preparation method comprises the following steps:

Process the fresh biological tissues, remove residual fat, blood stains, and blood vessels on the surface, and clean them;

Performing cross-linking and fixation treatment on the treated biological tissue to obtain a first biological tissue;

The first biological tissue is permeabilized to increase the permeability of the cell membrane without changing the cell morphology, thereby promoting the penetration of foreign solutions and obtaining a second biological tissue;

The second biological tissue is subjected to dissociation treatment, and the biological tissue skeleton structure is maintained. Degrading immunogenic glycans in the extracellular matrix, on the cell membrane and in the cell to obtain a third biological tissue;

Physically treating the third biological tissue to further decompose immunogenic glycans on the cell surface and in the cells to obtain a fourth biological tissue;

The fourth biological tissue is washed and the dissociation solution is removed to obtain the anti-calcification animal-derived biological material.

As an optional embodiment, the immunogenic glycans include Neu5Gc and αGal.

As an optional embodiment, the biological tissue includes one of bovine/porcine/horse pericardium/valve/intestinal membrane/blood vessel/ligament.

As an optional embodiment, the permeabilization solution used in the permeabilization treatment is a buffer solution prepared by using at least one of a surfactant, a chelating agent or a membrane permeation enhancer.

As an optional embodiment, when the permeabilization solution contains a surfactant, the mass percentage of the surfactant is 0.1% to 2%. Wherein, the surfactant includes at least one of Tween80, Triton X-100, dodecyl maltoside, digitonin, SDS, sodium deoxycholate, sodium cholate or sarcosine.

As an optional embodiment, when the permeabilization solution contains a chelating agent, the mass percentage of the chelating agent is 0.01% to 4%. Wherein, the chelating agent includes at least one of EDTA, STPP, NTA, DTPA, CA, TA, GA or DEG.

As an optional embodiment, when the permeabilization solution contains a membrane permeation enhancer, the mass percentage of the membrane permeation enhancer is 6% to 10%, wherein the membrane permeation enhancer includes dimethyl sulfoxide.

As an optional embodiment, the permeabilization solution further contains an organic solvent, and the mass percentage of the organic solvent is 0% to 40%, that is, at least one of a surfactant, a chelating agent or a membrane permeation enhancer is mixed with the organic solvent to prepare the permeabilization solution. The organic solvent is at least one of methanol, ethanol, hexane, toluene or chloroform.

As an optional embodiment, permeabilizing the first biological tissue comprises:

The treatment temperature is 4 to 14°C and the treatment time is 6 to 24 hours.

As an optional embodiment, the dissociation solution used in the dissociation treatment is a buffer solution containing enzymes with dissociation effect.

As an optional embodiment, the mass percentage of the enzyme in the dissociation solution is 0.6% to 10%, wherein the enzyme is at least one of melibiase, inulinase, maltase or β-galactosidase.

As an optional embodiment, the dissociation solution further contains an organic acid, and the mass percentage of the organic acid is 0% to 3%. That is, the dissociation solution is prepared by mixing the enzymes with dissociation effect and the organic acid. The organic acid is at least one of citric acid, acetic acid, lactic acid, gluconic acid, tartaric acid, glycyrrhetinic acid, oxalic acid, sorbic acid or benzoic acid.

As an optional embodiment, the second biological tissue is subjected to a dissociation process, comprising:

The pH of the dissociation solution was adjusted to 4.5-6.3;

The treatment temperature is 20-42°C and the treatment time is 3-17h.

As an optional embodiment, the method of physically treating the third biological tissue includes one or more of oscillation treatment, microwave treatment, and ultrasonic treatment.

As an optional embodiment, the cross-linking fixation treatment uses a buffer solution containing glutaraldehyde at a concentration of 0.6-0.7 (w/v)%.

The second aspect of the present invention relates to an anti-calcification animal-derived biomaterial prepared according to the aforementioned preparation method.

The technical scheme of the present invention provides a method for preparing anti-calcification animal-derived biomaterials. For the cross-linked and fixed tissue, permeabilization treatment is first performed to increase the permeability of the cell membrane and promote liquid penetration while reducing the damage to tissue proteins without changing the cell morphology, thereby providing a basis for the subsequent dissociation process and improving the treatment effect of the dissociation solution.

On the basis of the above, the dissociation solution is used to catalyze the decomposition of immunogenic glycans, and minimize the impact of the dissociation process on the collagen fibers in the extracellular matrix, maintain the skeletal structure of the biological tissue, and thus ensure the mechanical properties of the biological tissue. Under this premise, the immunogenic glycans in the extracellular matrix, on the cell membrane, and especially in the cells are completely catalytically hydrolyzed, thereby reducing the calcification mediated by immune carbohydrate antibodies and improving the anti-calcification ability of biological tissues.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a process flow chart of the method for preparing the anti-calcification animal-derived biomaterial of the present invention.

FIG. 2 is a microscopic image of fresh biological tissue according to Example 1 of the present invention.

FIG. 3 is a microscopic image of the tissue of fresh biological tissue after cross-linking and fixation treatment according to Example 1 of the present invention.

FIG. 4 is a microscopic image of biological tissue after permeabilization treatment according to Example 2 of the present invention.

FIG. 5 is a microscopic image of biological tissue after dissociation and physical treatment according to Example 2 of the present invention.

FIG. 6 is a tissue picture of the samples of Examples 1-3 of the present invention and the comparative example after calcification test and drying.

Detailed ways

In order to better understand the technical content of the present invention, specific embodiments are given and described as follows in conjunction with the accompanying drawings.

Various aspects of the present invention are described in this disclosure with reference to the accompanying drawings, in which many illustrative embodiments are shown. The embodiments of the present disclosure are not necessarily intended to include all aspects of the present invention. It should be understood that the various concepts and embodiments introduced above, as well as those described in more detail below, can be implemented in any of many ways.

Glycoproteins are complex sugars composed of branched oligosaccharide chains covalently linked to polypeptide chains. Neu5Gc and αGal are glycoproteins synthesized by most carnivorous animals other than humans. Therefore, when animal tissues containing glycoproteins are implanted into the human body, the body will produce corresponding antibodies. The antibodies produced will mediate the calcification of the leaflets of biological valves. Therefore, they can also be called immunogenic glycans or non-human glycans.

In animal tissues, glycoproteins are found in the extracellular matrix, on cell membranes, and within cells.

Glycoproteins on the cell membrane are located on the outer surface of the cell membrane, that is, the side facing the extracellular matrix. Therefore, the glycoproteins on the cell membrane and the glycoproteins in the extracellular matrix are both exposed outside the cell and are easier to handle when using solvents. However, due to the influence of the cell membrane, the glycoproteins inside the cell are more difficult to remove than those on the cell membrane and in the extracellular matrix.

When considering the removal of glycoproteins inside cells, on the one hand, it is necessary to consider whether it will change the cell morphology. Changes in cell morphology will cause cell lysis and necrosis, and exist in the extracellular matrix in the form of fragments. The fragments will cause an immune response, thereby forming calcification points; at the same time, lysed and necrotic cells will form vacancies in the matrix, affecting mechanical properties and forming calcification points. On the other hand, it is necessary to consider whether it will affect the tissue skeleton formed by collagen fibers in the extracellular matrix. The destruction of collagen fibers will affect the structure of the skeleton, thereby affecting the mechanical properties of the entire biological tissue.

Glycoproteins on the cell membrane and inside the cells can be removed by decellularization, but the currently commonly used decellularization methods will also damage the fiber structure while decellularizing, and the degree of damage will increase with the cleanliness of the decellularization. It is also easy to cause biological tissues to absorb water and swell, reduce flexibility, and reduce the mechanical properties of biological tissues, which is not conducive to use.

Therefore, the present invention proposes a method for preparing an anti-calcification animal-derived biomaterial by selecting Appropriate permeabilization solution and processing parameters are used to moderately increase cell membrane permeability and reduce damage to tissue proteins while ensuring that the increased cell membrane permeability makes it easier and more complete to process the glycoproteins in the cells.

By using permeabilization solution to moderately increase the permeability of the cell membrane so that the subsequent dissociation solution can better penetrate into the cells, an enzyme that can specifically degrade Neu5Gc and αGal is selected, and appropriate enzymatic hydrolysis parameters are selected to minimize the impact of this process on collagen fibers, i.e., the tissue skeleton, thereby achieving the complete removal of glycoproteins in the cells without affecting the mechanical properties of the tissue.

As shown in Figure 1, in an exemplary embodiment of the present invention, a method for preparing an anti-calcification animal-derived biomaterial is proposed, which reduces the antibody response produced by the receptor after the biological tissue is implanted by removing the immunogenic glycans in the biological tissue, thereby reducing the calcification mediated by the immune sugar antibody and improving the anti-calcification ability of the biological tissue.

The obtained fresh biological tissue is processed to remove residual fat, blood stains, and blood vessels on the surface, and then cleaned; wherein the biological tissue can be selected from one of the pericardium, valve, intestinal membrane, blood vessel, and ligament of cattle, pigs, or horses;

Performing permeabilization treatment on the first biological tissue to increase the permeability of the cell membrane without changing the cell morphology, thereby obtaining a second biological tissue;

The second biological tissue is subjected to a dissociation treatment to decompose immunogenic glycans in the extracellular matrix, on the cell membrane and in the cell under the premise of maintaining the skeleton structure of the biological tissue, thereby obtaining a third biological tissue;

Physically treating the third biological tissue to further decompose immunogenic glycans on the cell surface and in the cell to obtain a fourth biological tissue;

Below, we describe in more detail the implementation process and/or results obtained of various aspects of the aforementioned method of the present invention with reference to the accompanying drawings.

The obtained fresh biological tissue is processed, and the method and process of removing residual fat, blood stains, and blood vessels can be processed according to conventional means in the art, and the detailed process is not repeated here. After cleaning, it is set aside.

In the aforementioned method, the immunogenic glycans decomposed and removed by the dissociation treatment/physical treatment are Neu5Gc and αGal.

As an optional embodiment, the permeabilization solution used in the permeabilization treatment is a buffer solution prepared by using at least one of a surfactant, a chelating agent or a membrane permeation enhancer. It should be understood that the buffer solvent used in the buffer solution can be an existing commercial buffer solvent, including but not limited to PBS, HEPES, etc.

In another embodiment, the aforementioned permeabilization solution may also be prepared by mixing at least one of a surfactant, a chelating agent or a membrane permeation enhancer with an organic solvent, wherein the mass percentage of the organic solvent is 0% to 40%.

As an optional example, the organic solvent is at least one of methanol, ethanol, hexane, toluene or chloroform.

The permeabilization solution contains 0% to 40% by weight of an organic solvent, which means that the permeabilization solution may contain no organic solvent or contain at most 40 wt.% of an organic solvent.

For example, in one embodiment, the permeabilization solution is a buffer solution containing a surfactant; in another embodiment, the permeabilization solution is a buffer solution containing a surfactant and an organic solvent.

As an optional example, when the permeabilization solution contains an organic solution and at least one of a surfactant/chelating agent/membrane permeabilizer, the permeation-promoting effect can be enhanced through synergistic effects while maintaining the cell morphology.

As an optional example, since phospholipids have the function of transporting and enriching calcium ions and provide a microenvironment for the formation and protection of calcium phosphate microcrystals, when the permeabilization solution contains a surfactant, it can also act as a barrier to prevent phospholipids from penetrating into biological tissues, thereby helping to better reduce calcification.

As an optional embodiment, when the permeabilization solution contains a surfactant, the mass percentage of the surfactant is 0.1% to 2%, wherein the surfactant is at least one of Tween 80, Triton X-100, dodecyl maltoside, digitonin, SDS, sodium deoxycholate, sodium cholate or sarcosine.

As an optional embodiment, when the permeabilization solution contains a chelating agent, the mass percentage of the chelating agent is 0.01% to 4%, wherein the chelating agent is at least one of EDTA, STPP, NTA, DTPA, CA, TA, GA or DEG.

As an optional embodiment, when the permeabilization solution contains a membrane permeation enhancer, the mass percentage of the membrane permeation enhancer is 6% to 10%, wherein the membrane permeation enhancer is dimethyl sulfoxide.

It should be understood that the surfactant may also be a mixture of two or more of the substances listed above, and those skilled in the art may adjust the ratio between the mixed substances according to actual conditions.

Of course, the types of chelating agents and organic solvents may also be a mixture of two or more of the above-listed substances, and the ratio may be adjusted according to actual conditions.

As an optional embodiment, the dissociation solution contains an organic acid, the mass percentage of the organic acid is 0% to 3%, and the organic acid is citric acid, acetic acid, lactic acid, gluconic acid, tartaric acid, glycyrrhetinic acid, oxalic acid, sorbic acid or benzoic acid.

As an optional embodiment, the mass percentage of enzymes in the dissociation solution is 0.6% to 10%, and the enzymes are melibiase, inulinase, maltase or β-galactosidase.

The dissociation solution contains 0% to 3% by weight of organic acid, which means that the dissociation solution may not contain organic acid, or may contain at most 3 wt.% of organic acid.

For example, in one embodiment, the dissociation solution is a buffer solution containing melibiase; in another embodiment, the dissociation solution is a buffer solution containing melibiase and an organic acid.

When the dissociation solution contains both enzymes and organic acids, adding organic acids on the basis of enzymes can enhance the decomposition effect of glycoproteins and improve the decomposition efficiency while maintaining the structure of biological tissues.

It should be understood that the enzymes with dissociation effect can also be a combination of two or more of the above listed, and those skilled in the art can adjust the ratio between the mixed substances according to actual conditions.

By analogy, the type of organic acid can also be a mixture of two or more of the above-listed substances, and the proportion can be adjusted according to actual conditions.

The pH of the dissociation solution is adjusted to 4.5-6.3. In the low pH range, the dissociation solution can exert a better dissociation effect;

The treatment temperature is 20-42°C and the treatment time is 3-17h.

Physical treatment itself has a certain effect in decomposing sugars. Adding physical treatment on the basis of dissociation treatment can further effectively improve the decomposition effect.

As an optional embodiment, the cross-linking fixation treatment uses a buffer solution containing glutaraldehyde at a concentration of 0.6-0.7 (w/v)%, and particularly preferably 0.625 (w/v)%.

In the buffer solution used above, 20mmol/L HEPES is selected as the buffer solvent, and the pH is adjusted using conventional hydrochloric acid aqueous solution and sodium hydroxide aqueous solution. The concentrations of hydrochloric acid and sodium hydroxide can be selected according to actual conditions and are not limited here.

It should be understood that the role of the buffer solution is to maintain the stability of the system. As a balanced salt solution, those skilled in the art can select it according to actual conditions. For example, in some embodiments, phosphate buffered saline (PBS) can also be used.

Therefore, according to the aforementioned method of the present invention, fresh biological tissues of animals are processed to achieve anti-calcification treatment, obtain anti-calcification animal-derived biomaterials, reduce the antibody response produced by the receptor after the implantation of the biological valve, improve the anti-calcification ability of the biological valve, and have excellent durability.

According to an embodiment of the present invention, there is also proposed an anti-calcification animal-derived biomaterial prepared according to the above, which is used to prepare an artificial heart valve or a biological patch, for example, to improve the anti-calcification performance of the artificial heart valve or the biological patch, and to reduce defects such as hardening and tearing of biological tissue materials when injected into the human body or used on the human body.

For better understanding, the present invention is further described below in conjunction with specific examples, but the preparation method is not limited thereto, and the content of the present invention is not limited thereto.

The manufacturers of the reagents and equipment used in the following examples are as follows:

Glutaraldehyde: Sigma-Aldrich

Physiological saline: Huaren Pharmaceutical

Isopropyl alcohol: Shanghai test

Triton X-100: Shanghai trial

Melibiase: Sigma-Aldrich

Ethanol: Shanghai test

Twain 80: Shanghai Test

Inulinase: Sigma-Aldrich

EDTA: Sigma-Aldrich

SDS: Sigma-Aldrich

Citric acid: ALFA AESAR

Biochemical incubator: Shanghai Yiheng LRH-150

Ultrasonic cleaning machine: Shanghai Kedao SK3310HP

Constant temperature oscillator: Shanghai Jingqi CHA-2

Example 1

[Sample pretreatment]

Fresh tissue processing: obtain fresh biological tissue, remove residual fat, blood stains, blood vessels, etc. on the surface, and clean it.

Tissue fixation: The processed fresh tissue was placed in a buffer solution of 0.625 (w/v)% glutaraldehyde (20 mmol/L HEPES) for fixation.

The cross-linked bovine pericardium was selected with uniform thickness and cut into small pieces of 50 mm x 50 mm, and then thoroughly washed with physiological saline at room temperature.

Example 2

Prepare permeabilization solution: 0.45 (w/v)% Triton X-100 buffer solution (20 mmol/L HEPES).

The dissociation solution was prepared as follows: a 7.0 (w/v)% melibiase buffer solution (20 mmol/L HEPES), and the pH was adjusted to 6.3.

The pericardium cross-linked according to the method of Example 1 was immersed in 200 mL of permeabilization solution, placed in a biochemical incubator, set at 4° C., and treated for 9 hours.

The permeabilized pericardium was added into 200 mL of dissociation solution, and placed into an ultrasonic cleaning machine together with the solution, with the power set at 40% for 3 hours.

The tissue piece was taken out from the solution, and washed in an isopropanol aqueous solution and distilled water for 30 minutes respectively to fully wash and remove the residual dissociation solution. The treatment was completed and sample 1 was obtained.

Example 3

Prepare permeabilization solution: 35.0 (w/v)% ethanol + 2.0 (w/v)% Tween 80 buffer solution (20 mmol/L HEPES).

Prepare the dissociation solution: a 5.3 (w/v)% inulinase buffer solution (20 mmol/L HEPES), and adjust the solution pH to 5.5.

The cross-linked sample according to the method of Example 1 was immersed in 200 mL of permeabilization solution, placed in a biochemical incubator, set at 10° C., and treated for 6 hours.

The permeabilized sample was added to 200 mL of dissociation solution and placed in a thermostatic oscillator along with the solution, set at 37° C. and 500 rpm for 9 h.

The tissue piece was taken out from the solution, and washed in an isopropanol aqueous solution and distilled water for 30 minutes respectively to thoroughly remove the residual dissociation solution. The treatment was completed and sample 2 was obtained.

Example 4

Prepare permeabilization solution: 2.9 (w/v)% EDTA + 0.8 (w/v)% SDS buffer solution (20 mmol/L HEPES).

Prepare the dissociation solution: a buffer solution of 3.7 (w/v)% melibiase + 0.6 (w/v)% citric acid (20 mmol/L HEPES), and adjust the pH of the solution to 4.5.

The cross-linked sample according to the method of Example 1 was immersed in 200 mL of permeabilization solution, placed in a biochemical incubator, set at 14° C., and treated for 20 hours.

The permeabilized sample was added to 200 mL of dissociation solution and placed in a thermostatic oscillator along with the solution at 20° C. and 300 rpm for 17 h.

The tissue piece was taken out from the solution, and washed in an isopropanol aqueous solution and distilled water for 30 minutes respectively to fully wash and remove the residual dissociation solution. The treatment was completed and sample 3 was obtained.

Comparative Example 1

90 small samples treated according to the method of Example 1 were taken, immersed in 200 mL of physiological saline, placed in a biochemical incubator, set at 14° C., and treated for 24 hours.

The pericardium treated for 24 h was transferred into a new 200 mL saline solution and placed at room temperature for 17 h.

After the treatment, the pericardium was taken out from the solution, and washed in an isopropyl alcohol aqueous solution and distilled water for 30 minutes respectively to fully wash and remove the residual physiological saline to obtain a control sample.

Micrograph of tissue

Microscopic observation was performed on the fresh biological tissue (fresh biological tissue), the tissue after cross-linking and fixation treatment (first biological tissue) in Example 1, the biological tissue after permeabilization treatment (second biological tissue), and the biological tissue after dissociation and physical treatment (fourth biological tissue) in Example 2, and the results are shown in Figures 2, 3, 4 and 5.

As can be seen from the figure, compared with the fresh biological tissue (Figure 2), the fibers of the first biological tissue (Figure 3) are arranged regularly after cross-linking and fixation, while the fibers of the second biological tissue (Figure 3) and the fourth biological tissue (Figure 4) The pericardial tissue fibers were still arranged in parallel in a wavy shape, with good continuity and no obvious breaks. Compared with the first biological tissue, the structure was not obviously loose, and there was no obvious cell reduction or cell structure damage. It can be seen that the permeabilization solution, dissociation solution and physical treatment had no significant effect on the tissue strength of the pericardium.

Performance measurement

Calcium content determination

The control samples were implanted subcutaneously in the left side of 90 rats as the control group. The rats were killed after 90 days and the pericardial calcium content was determined by atomic flame absorption spectrometry, and the average value was calculated.

Sample 1 was implanted subcutaneously into the right side of 30 rats that had been implanted with the control group sample. The rats were killed 90 days later and the pericardial calcium content was measured by atomic flame absorption spectrometry, and the average value was calculated.

Sample 2 was implanted subcutaneously into the right side of 30 rats that had been implanted with the control group sample. The rats were killed 90 days later and the pericardial calcium content was measured by atomic flame absorption spectrometry, and the average value was calculated.

Sample 3 was implanted subcutaneously into the right side of 30 rats that had been implanted with the control group sample. The rats were killed 90 days later and the pericardial calcium content was measured by atomic flame absorption spectrometry, and the average value was calculated.

At the same time, the mechanical properties of the control sample and samples 1-3 were tested.

The results are shown in the following table

From the test results, we can know that:

The results of subcutaneous implantation in rats of samples 1 to 3 were significantly better than those of the control sample, and the calcium content thereof could be reduced by more than 99%.

5 , the pericardium slices taken from the subcutaneous tissue of rats showed obvious calcification in the control sample, while no visible calcium deposition was found in samples 1, 2, and 3. Therefore, it can be shown that the removal of sugars from biological tissues by the method of the present invention can play an anti-calcification role.

The tensile strength and elongation of samples 1 to 3 are not significantly different from those of the control sample. Combined with the tissue micrographs, it can be shown that the desugaring method of the present invention has no significant effect on the mechanical properties of biological tissues.

Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. A person with ordinary knowledge in the technical field to which the present invention belongs may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the definition of the claims.

Claims

A method for preparing anticalcification animal-derived biomaterial, characterized in that the preparation method comprises the following steps:

Process the fresh biological tissues, remove residual fat, blood stains, and blood vessels on the surface, and wash them;

Performing cross-linking and fixation treatment on the treated biological tissue to obtain a first biological tissue;

Performing permeabilization treatment on the first biological tissue to increase the permeability of the cell membrane without changing the cell morphology, thereby obtaining a second biological tissue;

performing a dissociation treatment on the second biological tissue to decompose immunogenic glycans in the extracellular matrix, on the cell membrane and in the cell while maintaining the skeleton structure of the biological tissue, thereby obtaining a third biological tissue;

Physically treating the third biological tissue to further decompose immunogenic glycans on the cell surface and in the cells to obtain a fourth biological tissue;

The fourth biological tissue is washed and the dissociation solution is removed to obtain an anti-calcification animal-derived biological material.
The method for preparing an anti-calcification animal-derived biomaterial according to claim 1 or 2, characterized in that the immunogenic polysaccharides include Neu5Gc and αGal.
The method for preparing an anti-calcification animal-derived biomaterial according to claim 1 or 2 is characterized in that the biological tissue includes at least one of bovine/pig/horse pericardium/valve/intestinal membrane/blood vessel/ligament.
The method for preparing an anti-calcification animal-derived biomaterial according to claim 1 is characterized in that the permeabilization solution used in the permeabilization treatment is a buffer solution prepared containing any one of a surfactant, a chelating agent or a membrane permeation enhancer.
The method for preparing an anti-calcification animal-derived biomaterial according to claim 4, characterized in that the permeabilization solution contains a surfactant, and the mass percentage of the surfactant is 0.1% to 2%.
The method for preparing an anti-calcification animal-derived biomaterial according to claim 5 is characterized in that the surfactant includes at least one of Tween 80, Triton X-100, dodecyl maltoside, digitonin, SDS, sodium deoxycholate, sodium cholate or sarcosine.
The method for preparing the anti-calcification animal-derived biomaterial according to claim 4 is characterized in that The permeabilization solution contains a chelating agent, and the mass percentage of the chelating agent is 0.01% to 4%.
The method for preparing anticalcification animal-derived biomaterials according to claim 7, characterized in that the chelating agent comprises at least one of EDTA, STPP, NTA, DTPA, CA, TA, GA or DEG.
The method for preparing anticalcification animal-derived biomaterials according to claim 4, characterized in that the permeabilization solution contains a membrane permeation enhancer, and the mass percentage of the membrane permeation enhancer is 6% to 10%.
The method for preparing anticalcification animal-derived biomaterial according to claim 9, characterized in that the membrane permeability enhancer comprises dimethyl sulfoxide.
The method for preparing an anti-calcification animal-derived biomaterial according to any one of claims 4 to 10, characterized in that the permeabilization solution further comprises an organic solvent, and the mass percentage of the organic solvent is 0% to 40%.
The method for preparing anticalcification animal-derived biomaterial according to claim 11, characterized in that the organic solvent comprises at least one of methanol, ethanol, hexane, toluene or chloroform.
The method for preparing an anti-calcification animal-derived biomaterial according to claim 1, characterized in that the first biological tissue is subjected to permeabilization treatment, comprising:

The permeabilization temperature is 4 to 14°C, and the permeabilization time is 6 to 24 hours.
The method for preparing an anti-calcification animal-derived biomaterial according to claim 1 is characterized in that the dissociation solution used in the dissociation treatment is a buffer solution containing enzymes with a dissociation effect.
The method for preparing anticalcification animal-derived biomaterials according to claim 10, characterized in that the mass percentage of the enzymes with dissociation effect contained in the dissociation solution is 0.6% to 10%.
The method for preparing an anti-calcification animal-derived biomaterial according to claim 15, characterized in that the enzyme is at least one of melibiase, inulinase, maltase or β-galactosidase.
The method for preparing anticalcification animal-derived biomaterial according to any one of claims 14 to 16, characterized in that the dissociation solution further comprises an organic acid, and the mass percentage of the organic acid is 0% to 3%.
The method for preparing anticalcification animal-derived biomaterial according to claim 17, wherein The characteristic is that the organic acid is at least one of citric acid, acetic acid, lactic acid, gluconic acid, tartaric acid, glycyrrhetinic acid, oxalic acid, sorbic acid or benzoic acid.
The method for preparing anticalcification animal-derived biomaterial according to claim 1, characterized in that the second biological tissue is subjected to a dissociation treatment, comprising:

The pH of the dissociation solution was adjusted to 4.5-6.3;

The dissociation treatment temperature is 20 to 42° C., and the dissociation treatment time is 3 to 17 hours.
The method for preparing anticalcification animal-derived biomaterial according to claim 1, characterized in that the method of physically treating the third biological tissue comprises:

One or more of oscillation treatment, microwave treatment, and ultrasonic treatment.
The method for preparing an anti-calcification animal-derived biomaterial according to claim 1 is characterized in that the cross-linking fixation treatment uses a buffer solution containing glutaraldehyde at a concentration of 0.6 to 0.7 (w/v)%.
An anti-calcification animal-derived biomaterial prepared according to the preparation method according to any one of claims 1 to 21.
A use of the anti-calcification animal-derived biomaterial as claimed in claim 22 in artificial heart valves and biological patches.