Manufacturing process of a large-sized myocardial patch

Abstract

The present invention discloses a manufacturing process for a large-sized myocardial patch, comprising the following steps: (1) screening water-soluble porogens within a certain particle size range, and after drying, making the screened porogens into a porogen scaffold by means of alcohol immersion pressing; (2) filling a polymer solution into the porogen scaffold, and fully drying to remove the solvent to obtain a porogen-polymer composite scaffold; (3) continuously slicing the porogen-polymer composite scaffold using a slicing machine to obtain thin slices of the porogen-polymer composite scaffold; (4) placing the thin slices of the porogen-polymer composite scaffold in deionized water to remove the porogens, and then performing freeze-drying to obtain a large-sized myocardial patch with a connected three-dimensional structure. The preparation process of the present invention has high stability, a small product shrinkage rate, and better connectivity of the internal pores of the prepared myocardial patch.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a manufacturing process of a large-sized myocardial patch. Background Art

[0002] In recent years, myocardial patches have made considerable progress in the treatment of myocardial infarction, heart failure, etc., and are considered an effective treatment tool for improving ventricular remodeling. Compared with traditional methods such as drugs and cell therapy, myocardial patches can provide mechanical support for damaged myocardium, so as to achieve the purpose of inhibiting left ventricular remodeling, slowing down heart failure, and improving or maintaining heart function. On the other hand, myocardial patches can serve as carriers for transporting drugs, proteins, and cells, so they are a very promising treatment plan.

[0003] Common preparation methods for polymer / composite myocardial patches include electrospinning, 3D printing, thermally induced phase separation, porogen method, etc.

[0004] Among them, the porogen method can stably prepare a three-dimensional structure with high porosity and high pore connectivity, and can well simulate the three-dimensional structure of natural extracellular matrix. For example, the Chinese patent document with the publication number CN113425897A written by this research group discloses a preparation method of a reactive oxygen species-responsive degradable polyurethane cardiac patch for myocardial infarction repair, and discloses the operation process of preparing a porous membrane: dissolving polyurethane in tetrahydrofuran, dioxane or hexafluoroisopropanol, adding sodium chloride or gelatin particles to the polyurethane solution, and then pouring the solution into a polytetrafluoroethylene mold and removing the solvent; then washing away the sodium chloride or gelatin particles with water to obtain a polyurethane porous cardiac patch.

[0005] Although the above materials have prepared small-sized patches for small animal experiments, the preparation of large-sized and quality-stable polymer-based myocardial patches with practical application value has always been a current research and development difficulty.

[0006] Currently, only the acellular matrix-based myocardial patch prepared by CorMatrix Cardiovascular Company in the United States has been commercialized on the market (Reference: An off-the-shelf artificial cardiac patch improves cardiac repair after myocardial infarction in rats and pigs, 2020), but the raw material cost is high and the source is single and limited. Although synthetic myocardial patches have strong function regulation ability, wide raw material sources, and low cost, they have not been industrialized. Summary of the Invention

[0007] The present invention provides a manufacturing process for large-sized myocardial patches that can be industrially produced. The product quality of the preparation process is highly controllable, and the connectivity of the internal pores of the prepared myocardial patches is better.

[0008] The technical solution of the present invention is as follows:

[0009] A manufacturing process for large-sized myocardial patches, comprising the following steps:

[0010] (1) Screen water-soluble porogens, and after drying, press the screened porogens into a porogen scaffold by the alcohol immersion pressing method;

[0011] (2) Fill the polymer solution into the porogen scaffold, and freeze-dry it thoroughly to remove the solvent to obtain a porogen-polymer composite scaffold;

[0012] (3) Continuously slice the porogen-polymer composite scaffold by a machine to obtain thin slices of the porogen-polymer composite;

[0013] (4) Immerse the thin slices of the porogen-polymer composite in deionized water at 37-50 °C to remove the porogen, and then perform freeze-drying to obtain large-sized myocardial patches with a connected three-dimensional structure. Preferably, the porogen is water-soluble protein particles; the size range of the screened porogen is 70-300 μm.

[0014] Furthermore, the porogen is gelatin particles and / or collagen particles.

[0015] In step (1), pressing the screened porogens into a porogen scaffold by the alcohol immersion pressing method includes: drying the porogens at a constant temperature, weighing a certain amount of porogen particles according to the diameter of the required scaffold and pouring them into a mold to level, pouring an alcohol aqueous solution to cover the porogens, combining the molds overnight, and putting them into an oven for drying to obtain a porogen scaffold with a certain thickness.

[0016] Furthermore, the temperature of the constant-temperature drying is 25-40 °C; the concentration of the alcohol aqueous solution is 70-90%; the drying temperature of the oven is 40-70 °C, and the drying time is 5-24 hours.

[0017] Under this condition, a scaffold with moderate porogen bonding strength and that will not fall apart when ultrasonically infiltrated with the polymer solution can be prepared, and finally, myocardial patches with high pore connectivity can be obtained.

[0018] Furthermore, the size of the porogen scaffold is a cylindrical porogen scaffold with a diameter of 40-100 mm and a thickness of 10-20 mm.

[0019] The polymer described above is a biocompatible polymer material. Preferably, the polymer is selected from polycaprolactone (PCL), polyglycerol sebacate (PGS), poly(lactic-co-glycolic acid) (PLGA), poly(lactic-co-caprolactone) (PLCL), PC-3572D, PC-3555D, PC-3595A, PC-3585A, PC-3575A of Lubrizol Advanced Materials (Shanghai) Co., Ltd., and reactive oxygen-responsive degradable polyurethane.

[0020] The reactive oxygen-responsive degradable polyurethane described above can be prepared by the preparation method disclosed in the Chinese patent document with the publication number CN113425897A.

[0021] The solvent of the polymer solution is at least one of 1,4-dioxane, tetrahydrofuran, and hexafluoroisopropanol; in the polymer solution, the concentration of the polymer is 4-20 wt%.

[0022] In step (2), the method of filling the polymer solution into the porogen scaffold is: immersing the scaffold in the polymer solution and performing infiltration by the negative pressure method.

[0023] The infiltration conditions are: the infiltration temperature is 30-50 °C, the infiltration time by the negative pressure method is 0.5-1 h, and the vacuum degree is 0.5-0.1 MPa.

[0024] In step (2), the method of removing the solvent is: vacuum freeze-drying at -40 °C for 12-24 hours.

[0025] In step (3), the slice thickness can be determined according to specific usage conditions. Preferably, the thickness of the prepared porogen-polymer composite scaffold thin slice is 0.5-1.2 mm.

[0026] A numerical control cryostat can be used for slicing. The slicing temperature is 0-37 °C, the slicing speed is 20-200 mm / min, and the slicing angle is 1-6 degrees.

[0027] In step (4), the freeze-drying time is 12-36 h, and the freeze-drying temperature is -40--20 °C.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] (1) In the method disclosed in the Chinese patent document with the publication number CN113425897A, the porogen scaffold is not completely bonded, resulting in a low connectivity of some pores inside the prepared myocardial patch. However, in the present invention, the surface of the porogen is slightly dissolved by the alcohol immersion and pressing method, and the contact bonding can improve the connectivity of the pores formed by the porogen. When the connectivity of the pores inside the myocardial patch is high during actual use, it can improve the transmission of nutrients and wastes inside and outside the myocardial patch and can better promote the migration and growth of cells.

[0030] (2) The present invention uses the alcohol immersion and pressing method to replace the casting method for preparing the porogen scaffold, which can improve the dimensional stability of the relatively thick gelatin scaffold, thereby reducing the dimensional error of the final patch product, improving the stability of the preparation process, and significantly shortening the production cycle.

[0031] (3) The present invention uses the method of vacuum pumping to introduce the polymer solution, which improves the wetting ability of the polymer solution to the porogen scaffold, removes the bubbles in the middle part, enables the relatively thick porogen scaffold to be fully wetted, and thus improves the quality of the product.

[0032] (4) The present invention uses the method of machine slicing. By controlling the slicing parameters, large-size myocardial patches can be mass-produced, significantly improving the production efficiency and the stability of the preparation process. Description of the Drawings

[0033] Figure 1 is the myocardial patch prepared by the alcohol immersion-slicing method in Example 1;

[0034] Figure 2 is the SEM image of the cross-section of the myocardial patches prepared in Example 1 and Comparative Example 1, where (a) is Example 1 and (b) is Comparative Example 1.

[0035] Figure 3 is the schematic diagram of the used mold, where (a) is the upper cover of the mold and (b) is the lower groove of the mold. Detailed Embodiments

[0036] Example 1

[0037] A manufacturing process for large-size myocardial patches, as Figure 1 shown, includes the following steps:

[0038] (1) Crush and screen gelatin particles with a particle size of 100 - 200 μm. After drying at 37°C for 6 hours, pour them into a cylindrical mold with a diameter of 4 cm, cover the porogen with a 75% alcohol solution, combine the molds overnight, and place them in an oven at 50°C for drying for 5 hours. A cylindrical gelatin scaffold with a diameter of 4 cm and a thickness of 15 mm is obtained.

[0039] (2) Fill the 10% tetrahydrofuran solution of PC-3572D (Lubrizol) into the scaffold by the negative pressure method at a temperature of 50°C for 60 minutes with a vacuum degree of 0.5 MPa; after discharging the air, freeze-dry to remove the solvent to obtain the porogen-polymer composite, with a freeze-drying temperature of -40°C and a freeze-drying time of 24 hours;

[0040] (3) Using a cryostat, cut the porogen-polymer composite scaffold into thin slices with a thickness of 0.6 mm. The sectioning temperature is 37 °C, the sectioning angle used is 1 degree, and the sectioning speed is 100 rpm / min (discard the parts about 1 mm at both ends of the composite scaffold).

[0041] (4) Place the porogen-polymer composite thin slice in deionized water for constant-temperature ultrasonic treatment. The ultrasonic temperature is 50 °C, and the ultrasonic time is 20 minutes to remove the porogen. Then freeze and dry the polymer thin slice to obtain a myocardial patch with a connected three-dimensional structure. The freeze-drying temperature is -40 °C, and the time is 24 hours. The prepared myocardial patch is as Figure 1 shown.

[0042] Example 2

[0043] A manufacturing process for a large-sized myocardial patch, comprising the following steps:

[0044] (1) Screen gelatin particles with a particle size of 70 - 150 μm. After drying at 30 °C for 12 hours, pour them into a cylindrical mold with a diameter of 4 cm, immerse the porogen with a 90% alcohol solution, combine the molds overnight, and air-dry at room temperature for 10 hours. Obtain a cylindrical gelatin scaffold with a diameter of 4 cm and a thickness of 15 mm;

[0045] (2) Fill a 1,4-dioxane solution of 10% PLCL (poly(L-lactide-co-caprolactone), random copolymer, ratio 1:1) into the scaffold by the negative pressure method at a temperature of 37 °C for 30 minutes, with a vacuum degree of 0.3 MPa; after discharging the air, freeze-dry to remove the solvent to obtain a porogen-polymer composite. The freeze-drying temperature is -40 °C, and the freeze-drying time is 16 hours;

[0046] (3) Use a slicing machine to cut the porogen-polymer composite into thin slices with a thickness of 1 mm. The sectioning temperature is 20 °C, the sectioning speed is 60 rpm / min, and the sectioning angle is 2 degrees;

[0047] (4) Place the porogen-polymer composite thin slice in deionized water for constant-temperature ultrasonic treatment. The ultrasonic temperature is 40 °C, and the ultrasonic time is 30 minutes to remove the porogen. Then freeze and dry the polymer thin slice to obtain a myocardial patch with a connected three-dimensional structure. The freeze-drying temperature is -40 °C, and the time is 36 hours.

[0048] Example 3

[0049] A manufacturing process for a large-sized myocardial patch, comprising the following steps:

[0050] (1) Crush and screen gelatin particles with a particle size of 150 - 200 μm. After drying at 40 °C for 6 hours, pour them into a cylindrical mold with a diameter of 6 cm. Use an 80% alcohol solution to submerge the porogen, combine the molds overnight, and place them in an oven to dry at 70 °C for 2 hours. A cylindrical gelatin scaffold with a diameter of 6 cm and a thickness of 15 mm is obtained;

[0051] (2) Fill the scaffold with a 12% hexafluoroisopropanol solution of 3595A (Lubrizol) by ultrasonic method at room temperature for 120 minutes with a vacuum degree of 0.1 MPa. After discharging the air, freeze-dry to remove the solvent to obtain a porogen-polymer composite. The freeze-drying temperature is -80 °C and the freeze-drying time is 24 hours;

[0052] (3) Use a slicing machine to cut the porogen-polymer composite into thin slices with a thickness of 1 mm at a slicing temperature of 0 °C, a slicing speed of 120 rpm / min, and a slicing angle of 4 degrees;

[0053] (4) Place the thin slices of the porogen-polymer composite in deionized water for constant-temperature ultrasonic treatment at a ultrasonic temperature of 60 °C and a ultrasonic time of 25 minutes to remove the porogen. Then freeze and dry the polymer thin slices to obtain a myocardial patch with a connected three-dimensional structure. The freeze-drying temperature is -40 °C and the time is 48 hours.

[0054] Comparative Example 1

[0055] The preparation method disclosed in the patent document with the publication number CN113425897A is used for preparation. The particle leaching method is as follows: Dissolve 3572D in dioxane to obtain a 10% solution, add gelatin particles with a size of 180 - 250 μm accounting for 50% by volume, then pour the solution into a polytetrafluoroethylene mold, remove the solvent, and wash away the gelatin particles with water to obtain a porous myocardial patch.

[0056] In the preparation method of Comparative Example 1, the porogen scaffold was not completely bonded, resulting in a lower connectivity of some pores inside the prepared myocardial patch (as shown in (b) of Figure 2 ), while in Example 1, the connectivity of the pores formed by the porogen can be improved by alcohol immersion pressing (as shown in (a) of Figure 2 ). In actual use, a high connectivity of the pores inside the myocardial patch can improve the transport of nutrients and waste inside and outside the myocardial patch, and can better promote the migration and growth of myocardial cells.

[0057] The performance of the myocardial patches prepared in Comparative Example 1 and Example 1 is shown in Table 1:

[0058] Table 1

[0059]

[0060] In Example 1, the alcohol immersion and pressing method can be used to prepare a product with a patch shrinkage rate almost equal to 0. At the same time, the modulus measurement error of the obtained patch is smaller, ensuring the quality and dimensional stability of the product. Meanwhile, the process cycle is significantly shortened and the production efficiency is improved.

[0061] The above-described embodiments have detailed the technical solutions and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, supplements, equivalent replacements, etc. made within the principle scope of the present invention should be included within the protection scope of the present invention.

Claims

1. A manufacturing process for a large-sized myocardial patch, characterized in that, It includes the following steps: (1) Screen water-soluble porogens. After drying, press the screened porogens into a porogen scaffold by the alcohol immersion pressing method. The alcohol immersion pressing method includes pouring the porogens into a mold and leveling them, pouring an aqueous alcohol solution into the mold to submerge the porogens, then combining the mold overnight and putting it into an oven to dry to obtain a porogen scaffold. The particle size of the screened porogens is 70-150μm, 100-200μm or 150-250μm, and the diameter of the porogen scaffold is 30-100mm. The porogen is a water-soluble protein particle; (2) Fill the polymer solution into the porogen scaffold, and freeze-dry it thoroughly to remove the solvent to obtain a porogen-polymer composite scaffold; (3) Cut the porogen-polymer composite scaffold to obtain a thin slice of the porogen-polymer composite; (4) Immerse the thin slice of the porogen-polymer composite in deionized water at 37-50°C to remove the porogen, and then perform freeze-drying to obtain a large-sized myocardial patch with a connected three-dimensional structure.

2. The manufacturing process of the large-sized myocardial patch according to claim 1, characterized in that, The concentration of the aqueous alcohol solution is 70-90%; the drying temperature of the oven is 40-70°C, and the drying time is 5-24 hours.

3. The manufacturing process of the large-sized myocardial patch according to claim 1, characterized in that, The polymer is selected from polycaprolactone, polyglycerol sebacate, poly(lactic-co-glycolic acid), poly(lactic-co-caprolactone), and reactive oxygen-responsive degradable polyurethane.

4. The manufacturing process of the large-sized myocardial patch according to claim 1, characterized in that, In the polymer solution, the concentration of the polymer is 4-20wt%.

5. The manufacturing process of the large-sized myocardial patch according to claim 1, characterized in that, In step (2), the method of filling the polymer solution into the porogen scaffold is: submerge the scaffold in the polymer solution and perform infiltration by the negative pressure method.

6. The manufacturing process of the large-sized myocardial patch according to claim 5, characterized in that, The infiltration conditions are: the infiltration temperature is 30-50°C, the infiltration time by the negative pressure method is 0.5-1h, and the vacuum degree is 0.5-0.1MPa.

7. The manufacturing process of the large-sized myocardial patch according to claim 1, characterized in that, In step (3), the sectioning temperature is 0-37°C, the sectioning speed is 20-200rpm / min, and the sectioning angle is 1-6 degrees; the thickness of the prepared thin slice of the porogen-polymer composite scaffold is 0.5-1.2mm.

8. The manufacturing process of the large-sized myocardial patch according to claim 1, characterized in that, In step (4), the freeze-drying time is 12-36h, and the freeze-drying temperature is -40--20°C.

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