CN118806793A - Use of composition and preparation method thereof in the treatment of myocardial infarction - Google Patents

Abstract

The application relates to a composition and application of a preparation method thereof in myocardial infarction treatment, belonging to the technical field of biology. The aforementioned composition comprises: mesenchymal stem cells and decellularized amniotic membrane matrix particles, wherein the ratio of the number of the mesenchymal stem cells to the mass of the decellularized amniotic membrane matrix particles is 10≡4: 0.25mg-1mg. The aforementioned composition is effective in improving MSC survival, enhancing cardiac function, and reducing cardiac fibrosis.

Description

Use of composition and preparation method thereof in the treatment of myocardial infarction

Technical Field

The present application belongs to the field of biotechnology, and specifically, the present application relates to the use of a composition and a preparation method thereof in the treatment of myocardial infarction.

Background Art

Myocardial infarction (MI) remains the most common cause of cardiovascular death worldwide. MI causes ischemia, hypoxia and functional loss of myocardial tissue, leading to ventricular dysfunction and eventually heart failure. Traditional drug therapy and surgical treatment can only slow the progression of the disease but cannot prevent the death of myocardial tissue. It is generally believed that the regenerative capacity of the heart is very limited, which limits the effectiveness of various treatments.

Stem cell transplantation is a breakthrough treatment for myocardial infarction. Mesenchymal stem cells (MSCs) are the most commonly used cell type, with self-renewal ability, directed differentiation, anti-inflammatory, anti-fibrotic and pro-angiogenic effects, and are beneficial for cardiac repair after MI. However, due to the dense arrangement of cardiac tissue and the ischemic and hypoxic environment after infarction, the transplanted MSCs are poorly retained in the infarcted tissue, resulting in poor therapeutic effect.

Recently, the use of biomatrices as MSC carriers has emerged as a new strategy to enhance the efficacy of MSCs after MI. For example, conductive nanocomposite hydrogels improved the electromechanical coupling between MSCs and host myocardial tissue after transplantation. Graphene oxide/alginate composite microgels enhanced the antioxidant activity and cell survival of MSCs after transplantation. Myocardial patches formed by loading MSCs into collagen or fibrin enhanced their therapeutic effects by promoting the paracrine secretion of MSCs. An increasing number of studies have shown that synthetic or natural biomaterials not only provide scaffolds for the growth and attachment of MSCs, but also mimic the internal microenvironment to provide growth factors, further improving the survival of MSCs after in vivo transplantation.

Human amniotic membrane (HAM) has attracted widespread attention as an excellent natural extracellular matrix (ECM) material. HAM is considered to be an easily available postpartum medical waste without ethical issues. Human acellular amniotic membrane matrix (HAAM) not only maintains the structural integrity of the amniotic matrix, but also retains ECM components and a variety of active factors, such as transforming growth factor β1 (TGFβ1), vascular endothelial growth factor (VEGF), and fibroblast growth factor 2 (FGF-2). In addition, decellularized HAAM minimizes immune response after MSC transplantation and has antibacterial, anti-inflammatory, and anti-fibrotic properties.

So far, HAAM has been mainly used to deliver MSCs to myocardial infarction treatment and tissue repair through cardiac dressings or myocardial patches to enhance the efficacy of MSCs. However, myocardial patches mainly deliver active factors released by HAAM through the outer cardiac membrane, rather than directly acting on specific damaged sites, which lacks accuracy. Secondly, most MSCs loaded into cardiac dressings stay on the outer cardiac membrane and can hardly reach the infarct area deep in the heart, greatly reducing the therapeutic effect.

Therefore, there is a need in the art for drugs that can treat myocardial infarction.

Summary of the invention

The present application aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, one object of the present application is to provide a drug that effectively treats or prevents myocardial infarction.

Specifically, this application provides the following technical solutions:

In the first aspect of the present application, the present application proposes a composition. According to an embodiment of the present application, the composition comprises: mesenchymal stem cells and decellularized amniotic matrix microparticles, and the ratio of the number of the mesenchymal stem cells to the mass of the decellularized amniotic matrix microparticles is 10^4: 0.25mg-1mg. In some examples of the present application, the aforementioned composition can effectively improve MSC survival, enhance cardiac function and reduce cardiac fibrosis.

In the second aspect of the present application, the present application proposes a preparation. According to an embodiment of the present application, the preparation comprises: the composition described in the first aspect. In some examples of the present application, the aforementioned preparation can effectively improve MSC survival, enhance cardiac function and reduce cardiac fibrosis.

In the third aspect of the present application, the present application proposes a method for preparing a composition. According to an embodiment of the present application, the method comprises: incubating mesenchymal stem cells (MSC) with decellularized amniotic matrix microparticles (HAAM); separating the incubation product to obtain the composition; wherein, the ratio of the number of mesenchymal stem cells to the mass of decellularized amniotic matrix microparticles is 10^4: 0.25mg-1mg. In some examples of the present application, the composition prepared based on the aforementioned method can effectively improve MSC survival, enhance cardiac function and reduce cardiac fibrosis.

In the fourth aspect of the present application, the present application proposes a composition. According to an embodiment of the present application, the composition is prepared by the method described in the third aspect. In some examples of the present application, the aforementioned composition can effectively improve MSC survival, enhance cardiac function and reduce cardiac fibrosis.

In the fifth aspect of the present application, the present application proposes a drug. According to an embodiment of the present application, the drug includes: the composition described in the first aspect and the fourth aspect, and the drug is used to treat or prevent myocardial infarction-related diseases. In some examples of the present application, the aforementioned drug can effectively treat or prevent myocardial infarction.

In the sixth aspect of the present application, the present application proposes the use of the composition described in the first aspect and the fourth aspect in the preparation of a drug, wherein the drug is used to treat or prevent myocardial infarction-related diseases. In some examples of the present application, the drug prepared based on the aforementioned composition can effectively treat or prevent myocardial infarction.

In the seventh aspect of the present application, the present application proposes a kit. According to an embodiment of the present application, the kit is used to implement the composition preparation method described in the third aspect. In some examples of the present application, based on the aforementioned kit, a composition for treating or preventing myocardial infarction can be obtained simply and quickly.

Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and/or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

FIG1 is a schematic diagram of the SEM image results of HAAM provided in an embodiment of the present application;

FIG2 is a schematic diagram of the particle size distribution results of HAAM provided in an embodiment of the present application;

FIG3 is a schematic diagram of the thermal stability results of HAAM provided in the examples of the present application;

FIG4 is a schematic diagram of TEM image results of MSCs provided in an embodiment of the present application;

FIG5 is a schematic diagram of flow cytometry results of MSCs provided in an embodiment of the present application;

FIG6 is a schematic diagram of the differentiation results (adipogenesis, osteogenic and chondrogenic) of MSCs provided in the examples of the present application;

FIG. 7 is a schematic diagram showing the effect of HAAM on the proliferation and viability of MSCs provided in the examples of the present application;

FIG8 is a schematic diagram of the differentiation results (adipogenesis, osteogenic and chondrogenic) of HAAM-MSCs provided in the examples of the present application;

FIG9 is a schematic diagram of in vivo tracking results provided in an embodiment of the present application;

FIG. 10 is a schematic diagram of cardiac ultrasound imaging results provided in an embodiment of the present application.

DETAILED DESCRIPTION

The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

In this document, unless otherwise specified, the terms "first" and "second" are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

In this document, unless otherwise specified, the terms “comprise” or “include” are open-ended expressions, that is, including the contents specified in the present invention, but not excluding other contents.

As used herein, unless otherwise specified, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes cases where the event or circumstance occurs and cases where it does not occur.

In the present application, the term "pharmaceutically acceptable" ingredients are substances that are suitable for use in humans and/or mammals without excessive adverse side effects (such as toxicity, irritation and allergic response), ie, substances with a reasonable benefit/risk ratio.

In the present application, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. In addition to any conventional excipients incompatible with the compounds of the present application, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of consideration of the present application.

In the present application, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by a suitable method. The composition, medicine or preparation of the present application can be administered by any common route as long as it can reach the desired target tissue. Various modes of administration are expected, including cardiac injection, etc., but the present application is not limited to the exemplified modes of administration.

In the present application, the term "treatment" is used to refer to obtaining a desired pharmacological and/or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and/or may be therapeutic in terms of partially or completely curing a disease and/or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, especially humans, including: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit an individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

In the present application, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and/or animals and can be accepted by humans and/or animals.

In view of the shortcomings of existing myocardial infarction treatment methods in terms of treatment accuracy and treatment effect, the present application proposes a composition, a preparation, a method for preparing the composition, a drug and its use and a kit. The following describes them respectively:

Composition

In one aspect of the present application, the present application proposes a composition, which includes: mesenchymal stem cells and decellularized amniotic matrix particles, wherein the ratio of the number of the mesenchymal stem cells to the mass of the decellularized amniotic matrix particles is 10^4:0.25mg-1mg.

In some examples of the present application, the ratio of the number of mesenchymal stem cells to the mass of the decellularized amniotic matrix microparticles is optionally 10^4: 0.25 mg, 10^4: 0.3 mg, 10^4: 0.35 mg, 10^4: 0.4 mg, 10^4: 0.45 mg, 10^4: 0.5 mg, 10^4: 0.55 mg, 10^4: 0.6 mg, 10^4: 0.65 mg, 10^4: 0.7 mg, 10^4: 0.75 mg, 10^4: 0.8 mg, 10^4: 0.85 mg, 10^4: 0.9 mg, 10^4: 0.95 mg or 10^4: 1 mg. In some preferred examples of the present application, the ratio of the number of mesenchymal stem cells to the mass of the decellularized amniotic matrix microparticles is 10^4: 0.5 mg.

The composition prepared by using the aforementioned ratio of mesenchymal stem cells and decellularized amniotic matrix microparticles can effectively improve MSC survival, enhance cardiac function and reduce cardiac fibrosis.

preparation

In another aspect of the present application, the present application provides a preparation comprising any one of the above-mentioned exemplified compositions.

In some examples of the present application, the aforementioned preparation further includes: PBS buffer.

In some examples of the present application, the aforementioned preparation type is an injection.

The injection prepared based on the above composition can accurately deliver MSCs to the infarct area, help repair myocardial tissue in situ after myocardial infarction, and significantly improve the accuracy and therapeutic effect of treatment.

Composition preparation method

In another aspect of the present application, the present application proposes a method for preparing a composition, the method comprising:

First, mesenchymal stem cells are incubated with decellularized amniotic matrix microparticles; wherein the ratio of the number of the mesenchymal stem cells to the mass of the decellularized amniotic matrix microparticles is 10^4: 0.25 mg-1 mg.

In some examples of the present application, the aforementioned mesenchymal stem cells can be obtained through laboratory culture or through commercial channels.

In some examples of the present application, the aforementioned mesenchymal stem cells are selected from mesenchymal stem cells (MSCs) of passages 3 to 5 that have good growth.

In one example of the present application, the aforementioned 3rd-5th generation mesenchymal stem cells are obtained by the following steps: Step 1: Use PBS buffer to clean the blood on the surface of the umbilical cord, and immerse the cleaned umbilical cord in 75% ethanol for 1-2 minutes for disinfection; Step 2: Remove excess blood vessels and the outer amniotic membrane of the umbilical cord, and divide it into ^{2-3 mm3} tissue blocks; Step 3: Incubate the tissue blocks in 5% _CO2 and 37°C for 10-30 minutes, add culture medium and incubate for one week, and replace the culture medium every three days; Step 4: Digest the product of step 3 with 0.25% trypsin, and perform cell passaging when the culture reaches 80% confluence.

In some examples of the present application, the aforementioned decellularized amniotic matrix microparticles are obtained by the following steps: 1) using physiological saline to clean the blood on the surface of the placental tissue under sterile conditions; 2) peeling off the amniotic membrane in the placental tissue and repeatedly rinsing it with pure water; 3) repeatedly freezing and thawing and digesting the amniotic membrane; 4) using a non-ionic surfactant to wash the digested amniotic membrane; 5) freeze-drying the washed product.

In some examples of the present application, the aforementioned placental tissue is selected from hospital waste or voluntary donation.

In step 3), the aforementioned repeated freezing and thawing is performed alternately at a freezing temperature of -80°C (±2°C) and a temperature of 37°C (±2°C).

In step 3), the aforementioned digestion treatment is performed in a 10 U/mL DNase solution.

In step 4), the nonionic surfactant is selected from at least one of Triton X-100, Brij-35, Polysorbate 20, Laureth-23, sodium fatty alcohol ether sulfate and sodium fatty alcohol polyether sulfate. In some preferred examples of the present application, the nonionic surfactant is Triton X-100. In some more preferred examples of the present application, the nonionic surfactant is 0.3% Triton X-100.

In step 4), the washing treatment time is 8-16 hours.

After step 5), the method further comprises: performing a first resuspension treatment on the freeze-dried product. In some examples of the present application, the first resuspension treatment is performed in a 0.9% sodium chloride solution.

In some examples of the present application, the aforementioned incubation treatment includes: 5% CO ₂ , 37° C., incubation for 5-7 hours. In some preferred examples of the present application, the aforementioned incubation treatment includes: 5% CO ₂ , 37° C., incubation for 6 hours. Incubation treatment under such conditions can ensure effective binding of MSCs and HAAM, maintain cell activity, and promote cell-to-cell interaction.

In some examples of the present application, the ratio of the number of mesenchymal stem cells to the mass of the decellularized amniotic matrix microparticles is optionally 10^4: 0.25 mg, 10^4: 0.3 mg, 10^4: 0.35 mg, 10^4: 0.4 mg, 10^4: 0.45 mg, 10^4: 0.5 mg, 10^4: 0.55 mg, 10^4: 0.6 mg, 10^4: 0.65 mg, 10^4: 0.7 mg, 10^4: 0.75 mg, 10^4: 0.8 mg, 10^4: 0.85 mg, 10^4: 0.9 mg, 10^4: 0.95 mg or 10^4: 1 mg. In some preferred examples of the present application, the ratio of the number of mesenchymal stem cells to the mass of the decellularized amniotic matrix microparticles is 10^4: 0.5 mg. Based on the composition obtained with this quantity-volume ratio, the cell morphology remains unchanged, and the cell proliferation, ATP content and mitochondrial activity are all increased.

Secondly, the incubation product is subjected to separation treatment to obtain the aforementioned composition.

In some examples of the present application, the aforementioned separation process includes a centrifugation process and a second resuspension process, wherein the aforementioned centrifugation process includes: centrifugation at 3000 rpm for 5 minutes; and the aforementioned second resuspension process is performed in a phosphate buffered saline (PBS buffer).

The composition prepared based on the above steps can accurately deliver MSCs to the infarct area by injection, which helps to repair the in situ myocardial tissue after myocardial infarction and significantly improves the accuracy and therapeutic effect of the treatment.

Composition

In another aspect of the present application, the present application proposes a composition, which is prepared by any of the above-mentioned composition preparation methods. In some examples of the present application, the above-mentioned composition needs to be administered to a subject as a whole to achieve in situ myocardial tissue repair after myocardial infarction.

drug

In another aspect of the present application, the present application proposes a drug, which includes any of the above-mentioned examples of the composition. In some examples of the present application, the above-mentioned drug treats or prevents myocardial infarction-related diseases by improving MSC survival, enhancing cardiac function and reducing cardiac fibrosis.

In some examples of the present application, the aforementioned drug further includes: a pharmaceutically acceptable excipient.

In some examples of the present application, the auxiliary material includes: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

In some examples of the present application, the pharmaceutical composition is an injection.

The drug of the present application contains a safe and effective amount of the active ingredient of the present application (i.e., HAAM-MSC) and pharmaceutically acceptable excipients. Such excipients include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the drug preparation should match the mode of administration, and the dosage form of the drug of the present application is an injection. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. The drug is preferably manufactured under sterile conditions.

The effective amount of the active ingredient described in the present application may vary with the mode of administration and the severity of myocardial infarction. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The aforementioned factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment situation, several divided doses may be administered daily, or the dose may be reduced proportionally.

The pharmaceutically acceptable excipients described in the present application include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptide substances, cellulose, nanogels, or combinations thereof. The choice of carrier should match the mode of administration, which are well known to those of ordinary skill in the art.

Use of the composition in preparing medicines

In another aspect of the present application, the present application proposes the use of the aforementioned composition or preparation in the preparation of a drug for treating or preventing myocardial infarction-related diseases.

Reagent test kit

In another aspect of the present application, the present application proposes a kit for implementing the aforementioned composition preparation method. Based on the kit, the aforementioned composition can be prepared quickly and portablely.

In some examples of the present application, the aforementioned kit includes: at least one of mesenchymal stem cells and decellularized amniotic matrix microparticles.

In some examples of the present application, the aforementioned kit further includes: at least one of a non-ionic surfactant, a DNA enzyme, a phosphate buffer, and an instruction manual.

In some examples of the present application, the aforementioned nonionic surfactant is selected from at least one of Triton X-100, Brij-35, Polysorbate 20, Laureth-23, sodium fatty alcohol ether sulfate and sodium fatty alcohol polyether sulfate.

Disease treatment methods

In yet another aspect of the present application, the present application provides a method for treating or preventing myocardial infarction, the method comprising: administering a pharmaceutically acceptable amount of the aforementioned composition or drug to a test animal.

It should be noted that the term "subject animal" refers to a mammal that is evaluated for treatment and/or treated. In one embodiment, the mammal is a rat. In other examples of the present application, the subject may also be a human, but also includes other mammals, such as mice, rabbits, etc.

The effective amount of the composition or drug described in the present application may vary depending on the mode of administration and the severity of myocardial infarction. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The aforementioned factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment situation, several divided doses may be administered daily, or the dose may be reduced proportionally.

Disease treatment uses

In another aspect of the present application, the present application proposes the use of the aforementioned composition or drug in treating or preventing myocardial infarction. In some examples of the present application, administering an effective dose of the composition or drug to a test animal can effectively treat myocardial infarction.

Beneficial Effects

The injectable preparation HAAM-MSC of the present application can be used to significantly improve the cardiac function of rats with myocardial infarction. Compared with the traditional myocardial patch method (https://www.sciencedirect.com/science/article/pii/S1525001621002057?via%3Dihub#fig1), the HAAM-MSC injectable preparation can significantly improve the retention rate of MSC in the heart in the later stage of transplantation. 28 days after myocardial patch transplantation, most of the transplanted hAM-MSCs still remained on the surface of the heart, failed to effectively migrate into the myocardial tissue, and the proportion retained in the heart was extremely low (<1%). The HAAM-MSC injectable preparation prepared in the present application can significantly improve the retention rate of MSC in the heart through minimally invasive surgical injection, thereby enhancing its therapeutic effect.

In addition, the traditional myocardial patch treatment method requires open-chest surgery, while the HAAM-MSC injection preparation of the present application only requires minimally invasive surgery, reducing surgical risks and postoperative recovery time. Myocardial patches are suitable for the treatment of large areas of infarction, while the injection preparation of the present application can be used for more precise local treatment, suitable for the specific conditions of various infarction areas, thereby providing more flexible and effective treatment options. In summary, this patent provides a more efficient, safer and more flexible method for treating myocardial infarction, which has broad clinical application prospects.

The scheme of the present application will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If no specific technology or conditions are indicated in the embodiments, the technology or conditions described in the literature in this area or the product specification are carried out. The reagents used or the instruments that do not indicate the manufacturer are all conventional products that can be obtained commercially.

Example 1: Preparation of HAAM-MSCs

1. Preparation of acellular amniotic membrane matrix (HAAM)

Amniotic membrane tissue donated by healthy pregnant women was collected, and the residual blood on the surface of fresh placental tissue was cleaned with physiological saline under sterile conditions. The amniotic membrane was mechanically peeled off from the placenta and then repeatedly rinsed in pure water; the amniotic membrane was placed at a freezing temperature of -80°C (±2°C) and a temperature of 37°C (±2°C) and alternately frozen and thawed, and then placed in a 10U/mL DNA enzyme (DNase) solution and shaken at 37°C for 15 hours; the amniotic membrane treated with the DNA enzyme solution was repeatedly rinsed with injection water, and then placed in 0.3% Triton X-100 for washing overnight; then it was repeatedly washed with injection water, and after removing the residual detergent, it was placed in a freeze dryer for freeze drying; it was ground and sieved under liquid nitrogen to prepare decellularized amniotic membrane matrix particles, which were resuspended with water in a 0.9% sodium chloride aqueous solution to prepare 10% decellularized amniotic membrane matrix material for injection, and sterilized by electron beam for use.

The DNA of HAAM was quantified, and the specific steps included: using the PicoGreeen DNA quantification kit (ThermoFisher Scientific, Waltham, MA) to measure the residual DNA content in the HAAM matrix suspension and untreated HAM tissue. The samples were digested with proteinase K solution at 56°C for 4 hours until completely digested, and the DNA content was detected according to the manufacturer's instructions. The results are shown in Table 1. The residual DNA content in HAAM was 8.5±3.0ng/mg, indicating that the decellularization process completely removed the cell components and DNA residues, leaving only the extracellular matrix scaffold of the amniotic tissue, which is more suitable as a stem cell delivery carrier and reduces the occurrence of adverse immune reactions.

Table 1

The morphology of HAAM microparticles was identified, and the specific steps included: HAAM microparticles were fixed with 4% paraformaldehyde for at least 24 hours. After dehydration with increasing concentrations of ethanol, the samples were critical point dried and gold-sprayed before observation, and then observed using a scanning electron microscope (SEM). The results are shown in Figure 1. The SEM image shows that HAAM is in the form of irregular porous particles with uniform size, indicating that the main components and structure maintain the original integrity of the amniotic membrane. In addition, the extracellular matrix fibers are continuously entangled into a three-dimensional network structure, the gaps are interconnected, and the pore size is about 20-50μm. This pore size is conducive to the combination of materials and mesenchymal stem cells, providing a supportive microenvironment for mesenchymal stem cells.

The particle size distribution of HAAM was identified, and the specific steps included: the HAAM matrix suspension was diluted 30 to 50 times by volume with 0.9% NaCl. After 3 minutes of ultrasonic dispersion, the volume-based particle size distribution was detected using a laser scattering method (particle size analyzer, Malvern, UK). The results are shown in Figure 2. The HAAM matrix material Dx (90) ≤ 300 μm, indicating that the particle size distribution is uniform and the material homogeneity is good.

The thermal stability of HAAM was identified, specifically including: placing the HAAM matrix suspension into a pair of sealed crucibles and sealing them. The measurement was performed in the range of 10 to 100°C at a heating rate of 2°C/min, using dry nitrogen as the purge gas (DSC-1, Mettler Toledo, Switzerland). The development temperature and enthalpy were detected. The results are shown in Figure 3. The thermal changes of HAAM decellularized amniotic membrane matrix and non-decellularized amniotic membrane tissue are basically the same, indicating that HAAM maintains the original main components and structure of the amniotic membrane.

2. Preparation of Mesenchymal Stem Cells (MSC)

Discarded human umbilical cords were used to isolate and culture mesenchymal stem cells (MSCs). The umbilical cord was soaked in 75% ethanol for 1-2 minutes. It was washed with sterile saline, 1 cm was removed from both ends of the umbilical cord, and the remaining umbilical cord was cut into 2-4 cm long segments. Each segment was dissected longitudinally to remove excess blood vessels and the outer amniotic membrane of the umbilical cord. Then, the fragments were cut into ^2-3 mm3 tissue blocks, and each block was attached to a culture dish with a 0.5 cm interval around it, covering 70-80%. The tissue blocks were incubated in 5% _CO2 and 37°C for 10-30 minutes. After incubation, culture medium (CytoNiche, China) was added until the tissue blocks were completely covered, and the incubation was continued for 7 days. After incubation, long spindle-shaped cells crawled out around the tissue blocks, and then the culture medium was shaken and replaced with fresh culture medium, and culture was continued. The culture medium was renewed every 3 days. When the cell clusters around the tissue blocks were dense, the tissue blocks were discarded, digested with 0.25% trypsin, and passaged, recorded as P1 generation. The cells were passaged when the culture reached 80% confluence, and experiments were performed using passage 3-5 MSCs (HU-MSCs).

Transmission electron microscopy (TEM) analysis was performed on the subcultured MSCs. The specific steps were as follows: the MSC cell mass was fixed with electron microscopy fixative (Servicebio, China) at 4°C for 24 hours, and then embedded and sliced after gradient ethanol dehydration. The ultrastructure of MSCs was observed using a transmission electron microscope (HITACHI, Japan). The results are shown in Figure 4, indicating that MSCs have large oval nuclei, a large nuclear-cytoplasmic ratio, and are adherent and grow in colonies.

The MSCs after passage were identified by flow cytometry, and the specific steps were as follows: The third generation MSCs (1x10^7 cells/mL) were collected in PBS and labeled with CD90, CD105, CD73, CD44, CD4, CD34, CD11b, and HLA-DR+ (BD StemflowTM, USA) according to the concentration recommended in the instructions. Data were collected using a BD flow cytometer (Beckman CytoFLEX, USA) and analyzed using FlowJo software. The results are shown in Figure 5, indicating that the flow cytometry results showed that CD90, CD44, CD105, and CD73 were positively expressed, while CD34, CD11b, CD19, CD45, and HLA-DR were negatively expressed.

The MSC differentiation ability was determined in the following steps: Based on the instructions (Fuyuanbio, FY200007 (adipogenic), FY2000006 (osteogenic), FY200008 (chondrogenic)), the third generation MSC cells were seeded into 6-well plates at a density of 2×10 ⁴ cells/cm ² and 3×10 ⁴ cells/cm ² , respectively, and osteogenic and adipogenic differentiation was performed in sequence (Fuyuanbio, China). In addition, the cell pellet of 2×10 ⁶ cells/ml was resuspended in a centrifuge tube and incubated at 5% CO ₂ and 37°C for chondrogenic differentiation (Fuyuanbio, China). The results are shown in Figure 6. Oil red O staining showed that a large number of lipid droplets appeared in the cells after 10 days of adipogenic culture. Alizarin red staining showed that obvious granular calcium precipitation appeared in the cells after 3 weeks of osteogenic culture. Alcian blue staining showed that the cells turned blue after 2 weeks of chondrogenic culture.

3. Preparation of HAAM-MSCs

Take well-grown 3rd-5th generation mesenchymal stem cells (MSC, the subculturing method is the common laboratory method) and mix them evenly with the decellularized amniotic membrane matrix (HAAM) prepared in step 1, and inoculate them in a 3D Mesenchymal stem cells were placed in a 6-well plate with serum-free complete medium (purchased from Huakan Bio) and incubated in a 5% CO ₂ , 37°C incubator for 6 hours. The cell seeding density was 1x10^5 cells/well, and the HAAM addition volume was 25μl, 50μl and 100μl, of which the optimal volume was 50μl. The cell viability after co-culture of HAAM and MSC was detected by mitochondrial staining (Thermo, USA), CCK8 (Dojindo, Japan) and ATP kit (Biyuntian, China). The results are shown in Figure 7. After 1x10^5 MSCs were co-cultured with 50μl of HAAM for 6 hours, the cell morphology remained unchanged, and cell proliferation, ATP content and mitochondrial activity were all increased.

Then, 1x10^5 MSCs at passage 35 were mixed with 50 μl of HAAM in 3D Mesenchymal stem cells were placed in serum-free complete medium (purchased from Huakan Bio) and incubated in a 5% CO ₂ , 37° C. incubator for 6 hours. After incubation, the mixture (MSC and HAAM co-culture) was centrifuged at 3000 rpm for 5 minutes, the supernatant was discarded, the pellet was resuspended in phosphate buffer and stored at 4° C. for subsequent in vivo experiments, i.e., HAAM-MSC.

The HAAM-MSC differentiation ability was determined, and the specific steps were as follows: the HAAM-MSC prepared above was seeded into a 6-well plate, and osteogenic and adipogenic differentiation was performed in sequence (Fuyuanbio, China). In addition, the cell pellet of 2×10 ⁶ cells/ml was resuspended in a centrifuge tube and incubated at 5% CO ₂ and 37°C for chondrogenic differentiation (Fuyuanbio, China). The results are shown in Figure 8. After HAAM incubation, obvious oil droplets, calcium precipitation and Alcian blue-stained chondrocyte spheres appeared in MSCs. This shows that MSCs still have the potential for adipogenic, osteogenic, and chondrogenic differentiation after incubation with HAAM.

Example 2: Study on the therapeutic effect of HAAM-MSC on myocardial infarction rat model

The HAAM-MSC and MSC prepared in Example 1 were labeled with DiR (Thermo, USA) for in vivo tracking. According to the instructions, MSC or HAAM-MSC were suspended in a phosphate buffer containing 10 μg/mL DiR. Incubate at 37°C in the dark for 30 minutes. Subsequently, complete medium was added to terminate the reaction, the cells were centrifuged at 1200 rpm for 3 minutes, and washed twice with phosphate buffer, and the labeled MSC or HAAM-MSC were resuspended with 50 μl of phosphate buffer, respectively, and stored on ice.

The rat model of myocardial infarction induced by permanent ligation of the left anterior descending coronary artery (LAD) was specifically operated as follows: male Sprague Dawley rats (180-220g) were anesthetized by inhalation of 2% isoflurane, then intubated with a 16G polyethylene endotracheal tube and connected to a small animal ventilator (KW-10, China). Subsequently, the chest cavity was opened between the third and fourth ribs, and the pericardium was incised to expose the heart. A 6-0 polypropylene suture was used to ligate approximately 3 mm from the lower edge of the left atrial appendage. When the anterior wall of the left ventricle turned white, the ligation was considered successful. After successful ligation, the labeled MSCs or HAAM-MSCs prepared above were immediately injected into the myocardium around the infarct. The chest cavity was sutured after injection. Sham group: no ligation treatment was performed. Surgery group: no treatment was performed after ligation. During the entire operation, the animals were placed on a 37°C heating pad to maintain body temperature.

Cardiac function was assessed by in vivo tracking using small animal imaging and by cardiac ultrasound imaging.

The results are shown in Figures 9 and 10. Compared with MSCs, HAAM enhanced the engraftment of MSCs in the infarcted hearts and improved the cardiac function of rats after infarction.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (14)

1. A composition, characterized in that it comprises: mesenchymal stem cells and decellularized amniotic matrix particles, wherein the ratio of the number of the mesenchymal stem cells to the mass of the decellularized amniotic matrix particles is 10^4:0.25mg-1mg. 2. The composition according to claim 1, characterized in that the ratio of the number of the mesenchymal stem cells to the mass of the decellularized amniotic matrix particles is 10^4:0.5 mg. 3. A preparation, characterized in that it comprises: the composition according to any one of claims 1 to 2. 4. The preparation according to claim 3, further comprising: PBS buffer; Optionally, the preparation is an injection. 5. A method for preparing a composition, comprising: The mesenchymal stem cells are incubated with decellularized amniotic matrix microparticles; The incubation product is subjected to separation treatment to obtain the composition; The ratio of the number of the mesenchymal stem cells to the mass of the decellularized amniotic matrix particles is 10^4: 0.25 mg-1 mg. 6 . The method according to claim 5 , wherein the ratio of the number of the mesenchymal stem cells to the mass of the decellularized amniotic matrix particles is 10^4:0.5 mg. 7. The method according to claim 5, characterized in that the decellularized amniotic matrix microparticles are obtained by the following steps: 1) Under sterile conditions, use physiological saline to clean the blood on the surface of the placenta tissue; 2) stripping the amniotic membrane from the placental tissue and repeatedly washing the amniotic membrane with pure water; 3) subjecting the amniotic membrane to repeated freezing and thawing treatments and digestion treatments; 4) washing the digested amniotic membrane with a non-ionic surfactant; 5) freeze-drying the washed product; Optionally, the repeated freezing and thawing is performed alternately at a freezing temperature of -80°C (±2°C) and a temperature of 37°C (±2°C); Optionally, the digestion treatment is performed in a 10U/mL DNase solution; Optionally, the nonionic surfactant is selected from at least one of Triton X-100, Brij-35, Polysorbate 20, Laureth-23, sodium fatty alcohol ether sulfate and sodium fatty alcohol polyether sulfate, preferably Triton X-100, more preferably 0.3% Triton X-100; Optionally, the washing treatment time is 8-16 hours; Optionally, further comprising a first resuspension process after the freeze-drying process; Preferably, the first resuspension treatment is performed in a 0.9% sodium chloride solution. 8. The method according to claim 5, characterized in that the incubation treatment is carried out at 5% CO ₂ and 37°C for 5-7 hours; Preferably, the incubation treatment is performed at 5% CO ₂ and 37°C for 6 hours. 9. The method according to claim 5, characterized in that the separation process comprises a centrifugation process and a second resuspension process; Optionally, the centrifugation is performed at a rotation speed of 3000 rpm for 5 minutes; Optionally, the second resuspension treatment is performed in phosphate buffer. 10. A composition, characterized in that the composition is prepared by the method according to any one of claims 5 to 9. 11. A medicine, characterized in that it comprises the composition according to any one of claims 1 to 2 and 10. 12. The drug according to claim 11, characterized in that the drug further comprises a pharmaceutically acceptable excipient; Optionally, the excipients include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers; Optionally, the drug is an injection. 13. Use of the composition according to any one of claims 1 to 2 and 11 or the preparation according to any one of claims 3 to 4 in the preparation of a medicament for treating or preventing a disease related to myocardial infarction. 14. A kit, characterized in that the kit is used to implement the method according to any one of claims 5 to 9.