阅读说明：本技术 一种心脏脱细胞基质修饰仿生膜及其制备和应用 (Heart acellular matrix modified bionic membrane and preparation and application thereof ) 是由 孙广炜 刘洋 张英 赵姗 于 2019-11-22 设计创作，主要内容包括：本发明涉及一种心脏脱细胞基质修饰仿生膜及其制备方法,其特征在于制备含有猪心脏脱细胞基质颗粒(粒径为10-100μm)、壳聚糖、透明质酸、明胶、聚乙烯醇及甘油的成胶水溶液,其中猪心脏脱细胞基质颗粒浓度为0.2-5％(w/v,g/ml),之后加入交联剂进行交联反应,获得凝胶,再经过中和、清洗及干燥后即得到心脏脱细胞基质修饰仿生膜。该心脏脱细胞基质修饰仿生膜具有良好的生物活性,能促进心肌细胞的粘附、生长。因此,该心脏脱细胞基质修饰仿生膜可用于心肌组织损伤的治疗,提高组织修复的效果。(The invention relates to a heart acellular matrix modified bionic membrane and a preparation method thereof, which are characterized in that a gelling aqueous solution containing pig heart acellular matrix particles (the particle size is 10-100 mu m), chitosan, hyaluronic acid, gelatin, polyvinyl alcohol and glycerol is prepared, wherein the concentration of the pig heart acellular matrix particles is 0.2-5% (w/v, g/ml), then a cross-linking agent is added for cross-linking reaction to obtain gel, and the gel is neutralized, washed and dried to obtain the heart acellular matrix modified bionic membrane. The bionic membrane modified by the heart acellular matrix has good biological activity and can promote adhesion and growth of myocardial cells. Therefore, the cardiac acellular matrix modified bionic membrane can be used for treating myocardial tissue injury and improving the effect of tissue repair.)

1. A preparation method of a cardiac acellular matrix modified bionic membrane is characterized by comprising the following steps: preparing a gelling aqueous solution containing pig heart acellular matrix particles (the particle size is 10-100 mu m), chitosan, hyaluronic acid, gelatin, polyvinyl alcohol and glycerol, wherein the concentration of the pig heart acellular matrix particles is 0.2-5% (w/v, g/ml), adding a cross-linking agent for cross-linking reaction to obtain gel, and neutralizing, cleaning and drying to obtain the heart acellular matrix modified bionic membrane.

2. The method of claim 1, wherein: the preparation of the pig heart acellular matrix particles comprises the following two processes:

the preparation process of the pig heart tissue section comprises the following steps: perfusing the heparin-treated in-vivo pig heart with 1000-3000ml of phosphate buffer solution containing 0.25-0.5% (w/v, g/ml) trypsin and 0.02-0.1% (w/v, g/ml) ethylenediaminetetraacetic acid (EDTA) at 37 ℃, freezing at-80 ℃ for more than or equal to 24h, and slicing into tissue slices with the thickness of 1-4 mm;

the acellular treatment process of the heart tissue slice comprises the following steps: the heart tissue slices and the liquids used in the decellularization process were measured at 1: 15-1: mixing at a ratio of 25(w/v, g/ml), sequentially adopting liquid and time for a cell removing process of double distilled water for 24 hours, 0.1-0.3% (v/v) ammonia water solution containing 2-4% (v/v) Triton X-100 for 96-120 hours, 0.1-0.3% (w/v, g/ml) Sodium Dodecyl Sulfate (SDS) solution for 48-72 hours, and double distilled water for 72-96 hours, and carrying out vacuum freeze drying; placing the freeze-dried acellular tissue slices into a ball mill, crushing into particles with the particle size range of 10-100 mu m, sterilizing by gamma ray irradiation, and freezing and storing at-80 ℃ for later use.

3. The method of claim 1, wherein:

the molecular weight of the chitosan is 5000-300000kDa, and the concentration of the chitosan in the gel-forming aqueous solution is 1-10% (w/v, g/ml);

the molecular weight of the hyaluronic acid is 1000000-4000000kDa, and the concentration of the hyaluronic acid in the gelling aqueous solution is 0.1-1% (w/v, g/ml).

4. The method of claim 1, wherein:

the gelatin comprises one or two of alkaline gelatin and acidic gelatin;

the gelatin has a jelly strength of greater than 100Bloom g and a concentration in aqueous gelling solution of 3-15% (w/v, g/ml).

5. The method of claim 1, wherein:

the polymerization degree of the polyvinyl alcohol is 2-5 ten thousand, and the concentration of the polyvinyl alcohol in the gelling aqueous solution is 1-5% (w/v, g/ml);

the volume ratio of the glycerol in the gelling aqueous solution is 1-10% (v/v);

the cross-linking agent is one or more than two of formaldehyde, glutaraldehyde, genipin and carbodiimide;

the concentration of the cross-linking agent after the cross-linking agent is mixed with the gel-forming aqueous solution is 0.01-3% (w/v, g/ml).

6. The method of claim 1, wherein:

the crosslinking conditions are 18-25 ℃ of temperature and 50-80% of humidity.

7. The method of claim 1, wherein:

the neutralization process is to soak the gel formed by crosslinking with 0.1-1.0M glycine solution.

8. The method of claim 1, wherein:

the drying condition is that the temperature is 18-25 ℃ and the humidity is 5-30%.

9. A decellularized matrix modified biomimetic membrane or a decellularized matrix modified biomimetic membrane prepared by any of the methods of claims 1-7, wherein:

comprises pig heart acellular matrix particles, chitosan, hyaluronic acid, gelatin, polyvinyl alcohol, glycerol and a cross-linking agent, wherein the mass percentages of the pig heart acellular matrix particles, the chitosan, the hyaluronic acid, the gelatin, the polyvinyl alcohol, the glycerol and the cross-linking agent are 2.2-62.1% (w/w), 0.2-6.2% (w/w), 6.6-93% (w/w), 2.2-31% (w/w), 2.2-62.1% (w/w) and 0.02-18.6% (w/w), and the balance is water.

10. An application of a cardiac acellular matrix modified bionic membrane in preparing a medicament for treating myocardial tissue injury.

Technical Field

The invention relates to the field of regenerative medicine, in particular to a cardiac acellular matrix modified bionic membrane and preparation and application thereof.

Background

The heart is the most important organ of the human body, and the scaffold material with high affinity with the heart can better promote the repair of damaged myocardial tissues. However, the conventional scaffold material is far from the extracellular matrix of heart tissue in terms of composition and structure, so how to obtain the bionic scaffold similar to the heart muscle tissue in vivo is one of the core problems facing the field of myocardial tissue engineering.

Research shows that the heart acellular matrix retains important matrix components and structures of heart muscle tissues in vivo and has higher biological activity. The acellular matrix is not easy to process and form, so that the acellular matrix has wider application prospect when being combined with other materials. However, how to prepare the bionic scaffold modified by the cardiac acellular matrix and having more uniform components and structure is a bottleneck problem to be solved.

In order to overcome the bottleneck of the prior art, the invention discloses a cardiac acellular matrix modified bionic membrane. The heart acellular matrix is prepared into particles, and the particles are integrated into the membrane scaffold, so that the heart acellular matrix modified bionic membrane is prepared, the problem of uneven components and structures in the acellular matrix modification technology is solved, and the biological activity of the membrane is further improved. The present invention thus overcomes the important drawbacks of the prior art and will play an important role in the field of myocardial tissue repair.

Disclosure of Invention

The invention discloses a cardiac acellular matrix modified bionic membrane.

The heart acellular matrix modified bionic membrane is prepared by the following specific technical scheme: preparing a gelling aqueous solution containing pig heart acellular matrix particles (the particle size is 10-100 mu m), chitosan, hyaluronic acid, gelatin, polyvinyl alcohol and glycerol, wherein the concentration of the pig heart acellular matrix particles is 0.2-5% (w/v, g/ml), adding a cross-linking agent for cross-linking reaction to obtain gel, and neutralizing, cleaning and drying to obtain the heart acellular matrix modified bionic membrane;

the preparation of the pig heart acellular matrix particles comprises the following two processes:

the preparation process of the pig heart tissue section comprises the following steps: perfusing the heparin-treated in-vivo pig heart with 1000-3000ml of phosphate buffer solution containing 0.25-0.5% (w/v, g/ml) trypsin and 0.02-0.1% (w/v, g/ml) ethylenediaminetetraacetic acid (EDTA) at 37 ℃, freezing at-80 ℃ for more than or equal to 24h, and slicing into tissue slices with the thickness of 1-4 mm;

the acellular treatment process of the heart tissue slice comprises the following steps: the heart tissue slices and the liquids used in the decellularization process were measured at 1: 15-1: mixing at a ratio of 25(w/v, g/ml), sequentially adopting liquid and time for a cell removing process of double distilled water for 24 hours, 0.1-0.3% (v/v) ammonia water solution containing 2-4% (v/v) Triton X-100 for 96-120 hours, 0.1-0.3% (w/v, g/ml) Sodium Dodecyl Sulfate (SDS) solution for 48-72 hours, and double distilled water for 72-96 hours, and carrying out vacuum freeze drying; placing the freeze-dried acellular tissue slices into a ball mill, crushing into particles with the particle size range of 10-100 mu m, sterilizing by gamma ray irradiation, and freezing and storing at-80 ℃ for later use;

the molecular weight of the chitosan is 5000-300000kDa, and the concentration of the chitosan in the gel-forming aqueous solution is 1-10% (w/v, g/ml);

the molecular weight of the hyaluronic acid is 1000000-4000000kDa, and the concentration of the hyaluronic acid in the gelling aqueous solution is 0.1-1% (w/v, g/ml);

the gelatin comprises one or two of alkaline gelatin and acidic gelatin;

the gelatin has a jelly strength of greater than 100Bloom g and a concentration in aqueous gelling solution of 3-15% (w/v, g/ml);

the polymerization degree of the polyvinyl alcohol is 2-5 ten thousand, and the concentration of the polyvinyl alcohol in the gelling aqueous solution is 1-5% (w/v, g/ml);

the volume ratio of the glycerol in the gelling aqueous solution is 1-10% (v/v);

the cross-linking agent is one or more than two of formaldehyde, glutaraldehyde, genipin and carbodiimide;

the concentration of the cross-linking agent after the cross-linking agent is mixed with the gelling aqueous solution is 0.01-3% (w/v, g/ml);

the crosslinking condition is that the temperature is 18-25 ℃ and the humidity is 50-80%;

the neutralization process is to use 0.1-1.0M glycine solution to soak the gel formed by crosslinking;

the drying condition is that the temperature is 18-25 ℃ and the humidity is 5-30%;

the acellular matrix modified bionic membrane is characterized in that:

comprises pig heart acellular matrix particles, chitosan, hyaluronic acid, gelatin, polyvinyl alcohol, glycerol and a cross-linking agent, wherein the mass percentages of the pig heart acellular matrix particles, the chitosan, the hyaluronic acid, the gelatin, the polyvinyl alcohol, the glycerol and the cross-linking agent are 2.2-62.1% (w/w), 0.2-6.2% (w/w), 6.6-93% (w/w), 2.2-31% (w/w), 2.2-62.1% (w/w) and 0.02-18.6% (w/w), and the balance is water;

the application of the cardiac acellular matrix modified bionic membrane in preparing a medicine for treating myocardial tissue injury.

THE ADVANTAGES OF THE PRESENT INVENTION

1. The acellular matrix particles and the membrane are used in a composite way, so that the acellular matrix modified bionic membrane is prepared, and the biological activity of the membrane is further improved;

2. in addition to the acellular matrix particles, the biomimetic membranes of the present invention also contain hyaluronic acid and gelatin. Hyaluronic acid is one of the important components of extracellular matrix, gelatin is a collagen hydrolysis product and has a function similar to collagen, so that the addition of hyaluronic acid and gelatin can enable the acellular matrix modified bionic membrane to be closer to the extracellular matrix of myocardial tissue in terms of components, has better affinity with myocardial cells and myocardial tissue, and is beneficial to adhesion, growth and migration of the myocardial cells.

Example 1:

anaesthetizing the small pig, unhairing, sterilizing, performing a cross incision in the center of the abdomen, performing anticoagulation on the heart artery by perfusion of a heparin solution, perfusing 2L of a PBS solution containing 0.25 percent (w/v, g/mL) trypsin and 0.02 percent (w/v, g/mL) EDTA at 37 ℃ by a peristaltic pump at the speed of 5mL/min after intravenous intubation and fixation, freezing and storing for 24h at the temperature of minus 80 ℃, cutting into small tissue slices with the thickness of 3mm and the thickness of 2cm multiplied by 2cm by a slicer, mixing the myocardial tissue slices and the acellular according to the ratio of 1:15(w/v, g/mL) in a bottle, placing in a constant temperature shaking table at the temperature of 4 ℃ at 200 r/min. Performing cell removal on a cell-free tissue piece by double distilled water for 24h, 0.1% (v/v) ammonia water containing 2% (v/v) Triton X-100 for 96h, 0.1% (w/v) SDS for 48h and double distilled water for 72h in sequence, performing freeze drying on the cell-free tissue piece, performing ball milling on the cell-free tissue piece to obtain particles with the size of 10-20 mu m, and performing gamma ray irradiation sterilization.

An aqueous gelling solution containing 0.2% (w/v, g/ml)10-20 μm cardiac acellular matrix particles, 1% (w/v, g/ml) chitosan (molecular weight 300000kDa), 1% (w/v, g/ml) hyaluronic acid (1000000kDa), 15% (w/v, g/ml) acidic gelatin (jelly strength 150Bloom g), 1% (w/v, g/ml) polyvinyl alcohol (degree of polymerization 2 ten thousand), 10% (v/v) glycerol was prepared. Then, a crosslinking agent glutaraldehyde is added into the gelling aqueous solution, the final concentration of the glutaraldehyde is 3% (w/v, g/ml), and crosslinking reaction is carried out to obtain gel. The crosslinking conditions were a temperature of 23 ℃ and a humidity of 80%. Thereafter, the gel formed by crosslinking was soaked with a 0.1M glycine solution, neutralized, and washed with water. Then dried at a temperature of 20 ℃ and a humidity of 20% to obtain the acellular matrix modified biomimetic membrane containing 0.2% (w/v, g/ml) of particles. Two control groups were set: control 1 was a membrane without added particles, and the other conditions were identical to the above conditions; control 2 was a film without hyaluronic acid and gelatin, and the other conditions were identical to those described above.

The prepared acellular matrix modified bionic membrane and the two control group membranes are cut into a round piece with the diameter of 3cm, and the growth difference of the myocardial cells on the surface of the membrane is utilized to compare the affinity of the myocardial cells. The three membranes were placed in a dish bottom of 3.5cm diameter, soaked overnight with PBS, and then seeded 3X10 onto the membranes⁵For each myocardial cell line cell, the cell adherence condition is observed at 24h, and the cell proliferation condition is observed at 5 days.

Experiments show that cells adhere to three membranes at 24h of cell inoculation, however, the sequence of the adherent cells is from more to less: the acellular matrix modified bionic membrane, the control group 2 and the control group 1 show that the membrane simultaneously containing the particles, hyaluronic acid and gelatin remarkably promotes the adhesion of myocardial cells, and in addition, the effect of adding the particles is larger than that of adding the hyaluronic acid and the gelatin. In addition, when the cells are cultured for 5 days, the number of the cells on the acellular matrix modified bionic membrane reaches 3.1x10⁶Control 2 was 2.2 × 10⁶Control 1 was 1.5 × 10⁶This shows that the acellular matrix modified biomimetic membrane significantly supports the proliferation of cardiac muscle cells, and in addition, the effect of adding the particles is larger than the effect of adding hyaluronic acid and gelatin.

Example 2:

anaesthetizing the small pig, unhairing, sterilizing, performing a cross incision in the middle of the abdomen, performing anticoagulation by a heparin solution for cardiac artery perfusion, performing venous intubation and fixation, perfusing a PBS solution containing 0.5 percent of trypsin and 0.1 percent of EDTA at 37 ℃ by a peristaltic pump at the speed of 5mL/min for perfusing the heart by about 3L, freezing and storing at the temperature of minus 80 ℃ for 24h, cutting the small tissue slices into 2cm × 2cm large tissue slices with the thickness of 4mm by a slicer, mixing the myocardial tissue slices and the cell removal solution according to the ratio of 1:25(w/v, g/mL) in a bottle, placing in a constant temperature shaking table at the temperature of 4 ℃ and 200 rpm. Sequentially decellularizing 24h by double distilled water, 120h by 0.3% (v/v) ammonia water containing 4% (v/v) Triton X-100, 72h by 0.3% (w/v) SDS and 96h by double distilled water, freeze-drying the decellularized tissue piece, ball-milling and crushing the decellularized tissue piece into particles with the size of 80-100 mu m, and sterilizing by gamma ray irradiation.

An aqueous gelling solution containing 5% (w/v, g/ml)80-100 μm cardiac acellular matrix particles, 10% (w/v, g/ml) chitosan (molecular weight 5000kDa), 0.1% (w/v, g/ml) hyaluronic acid (4000000kDa), 3% (w/v, g/ml) basic gelatin (jelly strength 220Bloom g), 5% (w/v, g/ml) polyvinyl alcohol (degree of polymerization 5 ten thousand), 1% (v/v) glycerol was prepared. Then, a crosslinking agent of glutaraldehyde is added into the gelling aqueous solution, the final concentration of the glutaraldehyde is 0.01% (w/v, g/ml), and crosslinking reaction is carried out to obtain gel. The crosslinking conditions were a temperature of 25 ℃ and a humidity of 50%. Thereafter, the gel formed by crosslinking was soaked with a 1.0M glycine solution, neutralized, and washed with water. Then dried at a temperature of 22 ℃ and a humidity of 5% to obtain the acellular matrix modified biomimetic membrane containing 5% (w/v, g/ml) of particles. 4 control groups were set:

control 1 had no added particles and the other conditions were identical to those described above;

control 2 added 1% (w/v, g/ml) of particles, other conditions were identical to those described above;

control 3 was added with 3% (w/v, g/ml) of particles, and the other conditions were identical to the above conditions;

control 4 was supplemented with 6% (w/v, g/ml) of particles, and the other conditions were identical to those described above.

The control group 4 was found to have irregular film morphology and non-uniform structure, which indicates that the content of 6% (w/v, g/ml) particles was too high and the effect was too highSince the film is stably produced, the content of particles of 6% (w/v, g/ml) or more is not suitable for use. The remaining 4 membranes were cut into 3cm diameter discs, and the difference in cardiomyocyte growth on the membrane surface was used to compare the degree of affinity of the cardiomyocytes. 4 membranes were placed in a dish bottom of 3.5cm diameter, soaked overnight with PBS, and then seeded 3X10 on the membrane⁵For each myocardial cell line cell, the cell adherence condition is observed at 24h, and the cell proliferation condition is observed at 5 days.

Experiments show that cells are attached to 4 membranes at 24h of cell inoculation, however, the attached cells are in the order of more cells to less cells: the acellular matrix modified bionic membrane containing 5% (w/v, g/ml) of particles, the control group 3, the control group 2 and the control group 1 show that the affinity of the myocardial cells of the bionic membrane is enhanced along with the increase of the concentration of the particles, and the effect of 5% (w/v, g/ml) is the best. In addition, the number of cells on the acellular matrix modified biomimetic membrane containing 5% (w/v, g/ml) particles was found to reach 4.2X10 after 5 days of cell culture⁶Control 3 was 3.9 × 10⁶Control 2 was 3.5 × 10⁶Control 1 was 1.6 × 10⁶This indicates that the faster cardiomyocytes proliferate with increasing particle concentration, the best 5% (w/v, g/ml) effect.