阅读说明：本技术 一种心脏脱细胞基质复合温敏凝胶及其应用 (Heart acellular matrix composite temperature-sensitive gel 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复合温敏凝胶,其特征在于此复合温敏凝胶由壳聚糖温敏凝胶和猪心脏脱细胞基质颗粒(粒径为10-100μm)所构成。本发明还提供上述复合温敏凝胶的制备方法,步骤为：(1)制备壳聚糖溶液,在冰浴及搅拌条件下将β-甘油磷酸钠溶液逐滴加入到壳聚糖溶液中,制备得到低温的壳聚糖温敏凝胶成胶溶液；(2)制备猪心脏脱细胞基质颗粒,粒径为10-100μm；(3)将猪心脏脱细胞基质颗粒加入上述低温的壳聚糖温敏凝胶成胶溶液,颗粒浓度为0.2-6％(w/v,g/ml),从而得到心脏脱细胞基质复合温敏凝胶成胶溶液；(4)低温的心脏脱细胞基质复合温敏凝胶成胶溶液在处于33-37℃条件时可快速转变为凝胶态。该心脏脱细胞基质复合温敏凝胶成胶溶液可通过注射到达心脏受损部位,原位快速形成复合凝胶,起到修复受损心肌组织的效果。(The invention relates to a heart acellular matrix composite temperature-sensitive gel, which is characterized by consisting of chitosan temperature-sensitive gel and pig heart acellular matrix particles (the particle size is 10-100 mu m). The invention also provides a preparation method of the composite temperature-sensitive gel, which comprises the following steps: (1) preparing a chitosan solution, dropwise adding a beta-sodium glycerophosphate solution into the chitosan solution under the conditions of ice bath and stirring, and preparing a low-temperature chitosan temperature-sensitive gel forming solution; (2) preparing pig heart acellular matrix particles with the particle size of 10-100 mu m; (3) adding the pig heart acellular matrix particles into the low-temperature chitosan temperature-sensitive gel forming solution, wherein the particle concentration is 0.2-6% (w/v, g/ml), so as to obtain the heart acellular matrix composite temperature-sensitive gel forming solution; (4) the low-temperature heart acellular matrix composite temperature-sensitive gel forming solution can be quickly converted into a gel state at the temperature of 33-37 ℃. The gel forming solution of the heart acellular matrix composite temperature-sensitive gel can reach the damaged part of the heart through injection, and the composite gel is rapidly formed in situ, so that the effect of repairing the damaged myocardial tissue is achieved.)

1. A heart acellular matrix composite temperature-sensitive gel is characterized in that: the composite temperature-sensitive gel is composed of chitosan temperature-sensitive gel and porcine heart acellular matrix particles (the particle size is 10-100 mu m), and the preparation method comprises the following steps: (1) preparing a chitosan solution, dropwise adding a beta-sodium glycerophosphate solution into the chitosan solution under the conditions of ice bath and stirring, and preparing a low-temperature chitosan temperature-sensitive gel forming solution; (2) preparing pig heart acellular matrix particles with the particle size of 10-100 mu m; (3) adding the pig heart acellular matrix particles into the low-temperature chitosan temperature-sensitive gel forming solution, wherein the particle concentration is 0.2-6% (w/v, g/ml), so as to obtain the heart acellular matrix composite temperature-sensitive gel forming solution; (4) the low-temperature heart acellular matrix composite temperature-sensitive gel forming solution can be quickly converted into a gel state at the temperature of 33-37 ℃.

2. The composite temperature-sensitive gel according to claim 1, wherein:

the deacetylation degree of the chitosan is 95% or more;

the molecular weight of the chitosan is 10000-300000Da, preferably 200000 Da.

3. The composite temperature-sensitive gel according to claim 1, wherein:

the concentration of the chitosan solution is 1-3% (w/v, g/ml), preferably 2% (w/v, g/ml).

4. The composite temperature-sensitive gel according to claim 1, wherein:

the concentration of the beta-sodium glycerophosphate solution is 56% (w/v, g/ml);

the volume ratio of the chitosan solution to the sodium beta-glycerophosphate solution is 35:1-8:1, preferably 14: 1.

5. The composite temperature-sensitive gel according to 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: 20(w/v, g/ml) dosage ratio, adopting liquid and time in the cell removing process to be double distilled water for 24 hours, 0.1-0.2% (v/v) ammonia water solution containing 1-3% (v/v) Triton X-100 for 96-120 hours, 0.1-0.2% (w/v, g/ml) Sodium Dodecyl Sulfate (SDS) solution for 24-48 hours, 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.

6. Use of a cardiac acellular matrix composite temperature sensitive gel according to any one of claims 1 to 5 for the treatment of damaged myocardial tissue.

Technical Field

The invention relates to the field of regenerative medicine, in particular to a cardiac acellular matrix composite temperature-sensitive gel and application thereof.

Background

The injection type temperature-sensitive gel is liquid at low temperature, is converted into hydrogel at 37 ℃, is suitable for being used as a transfer carrier for in vivo injection treatment, effectively avoids secondary damage to organisms, and has wide clinical application prospect. The chitosan is from shells of crabs and shrimps, has wide sources, no toxicity, excellent biocompatibility and degradability, and is applied to clinic as a biological material. Chitosan is insoluble in neutral and alkaline environments, and can be dissolved in acidic solutions such as hydrochloric acid and acetic acid, but precipitates appear when the pH value exceeds about 6.2. However, when the beta-sodium glycerophosphate solution is used for neutralizing chitosan, a mixed solution with the pH value of 6.8-7.2 can be prepared, no precipitation occurs, and the mixed solution is suitable for cell culture, particularly, the mixed solution has temperature-sensitive characteristics, is gelatinized at 37 ℃, and is suitable for in vivo injection. However, the chitosan temperature-sensitive and myocardial tissue extracellular matrix components and structures are greatly different, and the affinity of myocardial tissue is not enough, so that the application of the chitosan temperature-sensitive and myocardial tissue extracellular matrix components in the treatment of damaged myocardial tissue is greatly limited. Research shows that the heart acellular matrix retains important matrix components and structures of heart muscle tissues in vivo and has higher biological activity. In order to overcome the defect of low affinity of myocardial tissues of the conventional chitosan temperature-sensitive gel, the invention discloses a preparation method of a heart acellular matrix composite temperature-sensitive gel, namely pig heart acellular matrix particles are added into a gelling solution of the chitosan temperature-sensitive gel, so that the affinity of the myocardial tissues can be obviously improved, the repair of damaged myocardial tissues is accelerated, and the preparation method has an important application prospect.

Disclosure of Invention

The invention discloses a heart acellular matrix composite temperature-sensitive gel, which is composed of chitosan temperature-sensitive gel and pig heart acellular matrix particles (the particle size is 10-100 mu m).

The preparation method of the composite temperature-sensitive gel is realized by the following specific technical scheme: (1) preparing a chitosan solution, dropwise adding a beta-sodium glycerophosphate solution into the chitosan solution under the conditions of ice bath and stirring, and preparing a low-temperature chitosan temperature-sensitive gel forming solution; (2) preparing pig heart acellular matrix particles with the particle size of 10-100 mu m; (3) adding the pig heart acellular matrix particles into the low-temperature chitosan temperature-sensitive gel forming solution, wherein the particle concentration is 0.2-6% (w/v, g/ml), so as to obtain the heart acellular matrix composite temperature-sensitive gel forming solution; (4) the low-temperature heart acellular matrix composite temperature-sensitive gel forming solution can be quickly converted into a gel state at the temperature of 33-37 ℃.

The deacetylation degree of the chitosan is 95% or more;

the molecular weight of the chitosan is 10000-300000Da, preferably 200000 Da;

the concentration of the chitosan solution is 1-3% (w/v, g/ml), preferably 2% (w/v, g/ml);

the concentration of the beta-sodium glycerophosphate solution is 56% (w/v, g/ml);

the volume ratio of the chitosan solution to the sodium beta-glycerophosphate solution is 35:1-8:1, preferably 14: 1;

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: 20(w/v, g/ml) dosage ratio, adopting liquid and time in the cell removing process to be double distilled water for 24 hours, 0.1-0.2% (v/v) ammonia water solution containing 1-3% (v/v) Triton X-100 for 96-120 hours, 0.1-0.2% (w/v, g/ml) Sodium Dodecyl Sulfate (SDS) solution for 24-48 hours, 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 cardiac acellular matrix composite temperature-sensitive gel is applied to the treatment of damaged myocardial tissues.

THE ADVANTAGES OF THE PRESENT INVENTION

1. According to the invention, the pig heart acellular matrix particles are added into the chitosan temperature-sensitive gel system, so that the affinity of the myocardial tissue is improved, and the repair of the damaged myocardial tissue is accelerated;

2. the invention has simple process, low cost and wide clinical application prospect.

Example 1:

200ml of 0.1mol/L hydrochloric acid solution is prepared, 2g of chitosan (degree of deacetylation 95%, molecular weight 10000Da) is slowly added under the stirring condition (400rpm), and the mixture is stirred at room temperature to obtain 1% (w/v, g/ml) chitosan solution. Preparing a 56% (w/v, g/ml) beta-sodium glycerophosphate solution, dropwise adding the beta-sodium glycerophosphate solution into the chitosan solution under the conditions of ice bath and stirring (the volume ratio of the chitosan solution to the beta-sodium glycerophosphate solution is 35:1), and preparing to obtain the chitosan temperature-sensitive gel forming solution.

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 0.25 percent of trypsin and 0.02 percent of EDTA at 37 ℃ by a peristaltic pump at the speed of 5mL/min for perfusing the heart by about 2L, freezing and storing at the temperature of minus 80 ℃ for 24h, cutting the small tissue slices into 2cm multiplied by 2cm with the thickness of 3mm by a slicer, mixing the myocardial tissue slices and the cell removal solution in a bottle according to the ratio of 1:15(w/v, g/mL) and placing in a constant temperature shaking table at the temperature of 4 ℃ at 200 rpm. Performing cell removal in sequence 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, g/ml) SDS for 24h and double distilled water for 72h, freeze-drying the cell-removed tissue slices, performing ball milling on the cell-removed tissue slices to obtain particles with the size of 10-20 mu m, and performing gamma ray irradiation sterilization.

And then adding the 10-20 mu m pig heart acellular matrix particles into the low-temperature-sensitive gel forming solution, wherein the particle concentration is 0.2% (w/v, g/ml), so as to obtain the heart acellular matrix composite temperature-sensitive gel forming solution. 20 mul of the low-temperature compound temperature-sensitive gel forming solution is injected into a rat myocardial ischemia model (ligation of left coronary artery anterior descending branch modeling, ST-segment elevation in electrocardiogram proves successful modeling), and myocardial infarction area is evaluated after four weeks. PBS group and injection notThe control group contained porcine heart acellular matrix particles (chitosan temperature-sensitive gel forming solution) and other conditions were the same. After four weeks of injection, the infarct size of the group injected with the composite temperature-sensitive gel containing the granules was found to be minimal (3.3 cm)²) Followed by injection of the non-granule group (chitosan temperature sensitive gel forming solution) (4.2 cm)²) While the group injected with PBS had the largest infarct size (4.5 cm)²) The results show that the compound temperature-sensitive gel injection can accelerate the repair of damaged cardiac muscle tissues in vivo and the porcine heart acellular matrix particles play a main role.

Example 2:

200ml of 0.1mol/L hydrochloric acid solution is prepared, 6g of chitosan (degree of deacetylation 97%, molecular weight 300000Da) is slowly added under stirring (400rpm), and stirring is carried out at room temperature to obtain 3% (w/v, g/ml) chitosan solution. Preparing a 56% (w/v, g/ml) beta-sodium glycerophosphate solution, dropwise adding the beta-sodium glycerophosphate solution into the chitosan solution under the conditions of ice bath and stirring (the volume ratio of the chitosan solution to the beta-sodium glycerophosphate solution is 8:1), and preparing to obtain the chitosan temperature-sensitive gel forming solution.

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 the heart at the temperature of minus 80 ℃ for 24 hours, cutting the heart into small tissue slices with the thickness of 4mm and the thickness of 2cm multiplied by 2cm by a slicer, and mixing the myocardial tissue slices with the acellular solution according to the ratio of 1: 20(w/v, g/ml) in a flask and placed in a shaker at 200 rpm and a constant temperature of 4 ℃. Performing double-distilled water for 24h, sequentially performing cell removal on 0.2% (v/v) ammonia water 96h containing 3% (v/v) Triton X-100 and 0.1% (w/v) SDS 48h, performing freeze-drying on the cell-removed tissue slices, performing ball-milling on the cell-removed tissue slices, crushing the cell-removed tissue slices into particles with the size of 90-100 mu m, and performing gamma-ray irradiation sterilization.

And then adding the pig heart acellular matrix particles of 90-100 mu m into the low-temperature-sensitive gel forming solution, wherein the particle concentration is 6% (w/v, g/ml), so as to obtain the heart acellular matrix composite temperature-sensitive gel forming solution. Injecting 20 μ l of the low-temperature composite temperature-sensitive gel-forming solution into rat myocardial ischemia model (before ligation of left coronary artery)Lowering the branch and making the model, and raising ST segment in electrocardiogram to confirm the success of the model), and evaluating the myocardial infarction area after four weeks. The control group containing 0.2% (w/v, g/ml), 3% (w/v, g/ml), 7% (w/v, g/ml) of porcine heart acellular matrix particles was injected, and the other conditions were the same. Experiments show that 7% (w/v, g/ml) of the pig heart acellular matrix particles cannot be uniformly suspended after being added into the temperature-sensitive gel-forming solution, and a serious particle concentration gradient exists, so that the 7% (w/v, g/ml) or more pig heart acellular matrix particles are not suitable for use. The experimental result after four weeks of injection shows that the infarct size of the group injected with the composite temperature-sensitive gel containing 6% (w/v, g/ml) of particles is the smallest (1.9 cm)²) The 3% (w/v, g/ml) particle group was 2.5cm²0.2% (w/v, g/ml) of the particle group was 3.4cm²These results show that the repair effect of the damaged myocardial tissue in vivo is improved along with the increase of the content of the particles, and the composite temperature-sensitive gel containing 6% (w/v, g/ml) of the particles has the best effect.