A bacterial cellulose in-situ synthesis of cerium dioxide plga composite myocardial patch, its preparation method and purpose

Abstract

A PLGA composite myocardial patch for in-situ synthesis of cerium dioxide by bacterial cellulose, a preparation method and application thereof, wherein the preparation method of the PLGA composite myocardial patch for in-situ synthesis of cerium dioxide by bacterial cellulose comprises the following steps: S1, inoculating Acetobacter xylinum seed liquid into a container containing liquid HS culture medium for first culture; S2, adding trivalent cerium salt solution into the container for in-situ synthesis of cerium dioxide nanoparticles and second culture, finally obtaining a bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate; S3, constructing a polylactic acid-glycolic acid copolymer nanofiber layer on the surface of the bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate obtained in S2 through electrospinning, and obtaining the PLGA composite myocardial patch. The PLGA composite myocardial patch has good mechanical matching, cell compatibility, antioxidant capacity and interface bonding force.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a PLGA composite myocardial patch made by in-situ synthesis of cerium dioxide from bacterial cellulose, its preparation method and uses. Background Technology

[0002] Following a myocardial infarction, a large number of myocardial cells undergo irreversible necrosis, and the damaged areas are gradually replaced by fibrous scar tissue. The ventricular walls thin and dilate, ultimately leading to a sustained decline in cardiac function and even heart failure. Current clinical treatment strategies mainly include drug therapy, interventional therapy, and coronary artery bypass grafting. While these methods can improve myocardial blood supply to some extent, they lack effective structural repair methods for already formed necrotic areas and scar tissue. To improve the local mechanical environment of the myocardium and provide scaffolds for cell attachment, various myocardial patch or epicardial patch solutions have been proposed in the prior art. These include: non-degradable patches based on inert polymer films such as polytetrafluoroethylene (ePTFE) and polyethylene terephthalate (PET); bio-patches based on natural biomaterials such as collagen, gelatin, decellularized myocardium, or small intestinal submucosa; and porous scaffolds and electrospun nanofiber membranes prepared from biodegradable synthetic polymers such as PLGA, PCL, and PLA, as shown in Chinese Patent Publication Nos. CN112999418A and CN113521389A.

[0003] The aforementioned patch materials still have the following problems and defects in practical applications: 1. Poor mechanical compatibility: Some synthetic polymer films are too rigid and do not match the compliance of myocardial tissue, easily causing mechanical stimulation and inflammatory reactions under long-term pulsation conditions; while some natural materials lack mechanical strength and are prone to deformation, tearing, or failure during repeated contraction and relaxation of the heart. 2. Insufficient microenvironment and cell compatibility: Some inert materials lack hydrophilicity and cell recognition sites on their surface, which is not conducive to the adhesion, extension, and orientation of cardiomyocytes, mesenchymal stem cells, or cardiac progenitor cells, making it difficult to form an ordered structure similar to the natural muscle bundles of the myocardium. 3. Simple structure and limited function: Most existing myocardial patches are single-layer structures, making it difficult to simultaneously provide long-term mechanical support, controllable degradation, and bioactive delivery; although some electrospun PLGA scaffolds have good three-dimensional porous structures, their own mechanical strength is insufficient, resulting in limited stability when used as standalone myocardial patches. 4. Limited Improvement in Electro-Mechanical Coupling: Myocardial tissue possesses anisotropic electrical and mechanical properties. Most existing patch designs fail to achieve precise control over fiber orientation, pore size, and thickness, making it difficult to effectively guide cardiomyocytes to align along the direction of myocardial fibers. Research and applications that could improve electrical signal conduction and synchronized contraction remain limited. 5. Weak Bonding at Composite Material Interfaces: Some reports use simple lamination or adhesive bonding to combine multiple materials into composite patches. The interlayer bonding strength is limited, and delamination and lifting may occur under long-term mechanical load, affecting implantation stability and safety. 6. Insufficient Antioxidant Stress Capacity: Local ROS (reactive oxygen species) levels increase after myocardial infarction, leading to increased oxidative stress, apoptosis, and inflammation. Existing patch materials often lack intrinsic antioxidant function and cannot effectively scavenge ROS, limiting repair efficacy.

[0004] Therefore, in view of the shortcomings of the existing technology, it is necessary to provide a PLGA composite myocardial patch with in situ bacterial cellulose synthesis of cerium dioxide, its preparation method and uses to solve the shortcomings of the existing technology. Summary of the Invention

[0005] The first objective of this invention is to overcome the shortcomings of existing technologies by providing a method for preparing a PLGA composite myocardial patch using bacterial cellulose in situ synthesis of cerium dioxide. The PLGA composite myocardial patch prepared by this method exhibits good mechanical compatibility, cell compatibility, antioxidant capacity, and interfacial bonding.

[0006] The above-mentioned objectives of the present invention are achieved through the following technical measures: A method for preparing PLGA composite myocardial patches based on in-situ bacterial cellulose synthesis of cerium dioxide is provided, comprising the following steps: S1. Inoculate the Acetobacter xylinum seed culture into a container containing liquid HS medium for the first culture. S2. Add trivalent cerium salt solution to a container to prepare cerium dioxide nanoparticles in situ and then culture them for a second time to finally obtain bacterial cellulose-cerium dioxide composite-bacterial cellulose substrate. S3. A polylactic acid-glycolic acid copolymer nanofiber layer is constructed on the surface of the bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate obtained in S2 by electrospinning, thereby obtaining the PLGA composite myocardial patch.

[0007] Preferably, the above S2 is performed by the following steps: S2.1 Add trivalent cerium salt solution to the container in S1, and then incubate statically in a constant temperature incubator at 36℃~37℃ for 6d~8d, then proceed to S2.2; S2.2 Remove the solid from the container, add a NaOH aqueous solution with a concentration of 0.4mol / L to 0.6mol / L to the solid, control the temperature to 140℃ to 150℃ and react for 1h to 4h, then proceed to S2.3; S2.3 The solid is repeatedly rinsed with deionized water until neutral, and then dried to obtain the bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate.

[0008] Preferably, the above S3 is performed by the following steps: S3.1 Dissolve polylactic acid-glycolic acid copolymer (PLGA) in an organic solvent to obtain a spinning solution with a concentration of 8 wt% to 15 wt%. S3.2. Electrospinning the spinning solution directly onto the surface of the bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate to obtain the PLGA composite myocardial patch with a deposition layer thickness of 1 mm to 2 mm.

[0009] Preferably, the above S2 is performed by the following steps: S2.1 Add trivalent cerium salt solution to the container in S1, and then incubate it statically in a constant temperature incubator at 37℃ for 7 days. During the incubation period, keep the ambient humidity >80% before proceeding to S2.2. S2.2 Remove the solid from the container, add a 0.5 mol / L NaOH aqueous solution to the solid, control the temperature to 148℃ and react for 3 hours, then proceed to S2.3; S2.3 The solid is repeatedly rinsed with deionized water until neutral, and then dried to obtain the bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate.

[0010] Preferably, the above-mentioned trivalent cerium salt solution is an aqueous solution of cerium chloride heptahydrate or an aqueous solution of cerium nitrate hexahydrate; and the concentration of the trivalent cerium salt solution is 80 mg / ml to 120 mg / ml.

[0011] Preferably, the ratio of the above-mentioned trivalent cerium salt solution, the liquid HS culture medium of S1 and the NaOH aqueous solution of S2.2 is (1 μl ~ 5 μl): (1 ml ~ 2 ml): (2 ml ~ 3 ml).

[0012] Preferably, the above-mentioned liquid HS culture medium contains 5g-10g mannitol, 2.5g-5g peptone, and 2.5g-5g yeast extract per 1L of deionized water. In step S3.2, the electrospinning parameters are: voltage of 10 kV to 20 kV, receiving distance of 10 cm to 20 cm, and flow rate of 0.035 mL / h to 0.05 mL / h. Preferably, the organic solvent is hexafluoroisopropanol.

[0013] Preferably, S1 specifically involves inoculating the Acetobacter xylinum seed culture into a container containing liquid HS culture medium at an inoculation amount of 3% (v / v) to 7% (v / v), and then incubating it statically in a constant temperature incubator at 36℃ to 37℃ for 8h to 16h.

[0014] Preferably, the above-mentioned Acetobacter xylophilus seed solution is obtained by the following steps: streaking Acetobacter xylophilus into 50 mL of liquid HS medium, and shaking culture at 28°C and 1000 rpm for 24 h to obtain the Acetobacter xylophilus seed solution.

[0015] The second objective of this invention is to overcome the shortcomings of existing technologies and provide a PLGA composite myocardial patch based on in-situ bacterial cellulose synthesis of cerium dioxide. This PLGA composite myocardial patch based on in-situ bacterial cellulose synthesis of cerium dioxide exhibits good mechanical compatibility, cell compatibility, antioxidant capacity, and interfacial bonding strength.

[0016] The above-mentioned objectives of the present invention are achieved through the following technical measures: A PLGA composite myocardial patch based on in-situ bacterial cellulose synthesis of cerium dioxide is provided, which is prepared by the aforementioned method for preparing the PLGA composite myocardial patch based on in-situ bacterial cellulose synthesis of cerium dioxide.

[0017] The third objective of this invention is to overcome the shortcomings of the prior art by providing a PLGA composite myocardial patch made from bacterial cellulose in situ synthesized with cerium dioxide for use in the preparation of products supporting damaged myocardium. This PLGA composite myocardial patch, made from bacterial cellulose in situ synthesized with cerium dioxide, exhibits good mechanical compatibility, cell compatibility, antioxidant capacity, and interfacial bonding strength, and therefore can be used for supporting damaged myocardium.

[0018] The above-mentioned objectives of the present invention are achieved through the following technical measures: This invention provides the use of a PLGA composite myocardial patch, which is synthesized in situ from bacterial cellulose to cerium dioxide, in the preparation of a product for supporting damaged myocardium. The PLGA composite myocardial patch is prepared by the aforementioned method for preparing the PLGA composite myocardial patch.

[0019] This invention discloses a PLGA composite myocardial patch based on in-situ synthesis of cerium dioxide from bacterial cellulose, its preparation method, and its uses. The preparation method comprises the following steps: S1, inoculating Acetobacter xylinum seed culture into a container containing liquid HS culture medium for a first culture; S2, adding trivalent cerium salt solution to the container to prepare cerium dioxide nanoparticles via in-situ synthesis and conducting a second culture, ultimately obtaining a bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate; S3, constructing a polylactic acid-glycolic acid copolymer nanofiber layer on the surface of the bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate obtained in S2 via electrospinning, thus obtaining the PLGA composite myocardial patch. The PLGA composite myocardial patch of this invention exhibits good mechanical compatibility, cell compatibility, antioxidant capacity, and interfacial bonding strength. Furthermore, this PLGA composite myocardial patch uses a high-strength, high-water-content bacterial cellulose membrane as a base, on which a PLGA nanofiber layer with controllable orientation, pore size, and thickness is constructed in situ. It can also be loaded with bioactive factors as needed, thereby achieving: 1. Providing long-term, flexible, and tissue-matched mechanical support to damaged myocardial areas; 2. Providing a biomimetic three-dimensional microenvironment for cell adhesion, migration, and orderly arrangement; 3. Achieving local sustained release of growth factors or drugs through the degradability of PLGA, promoting angiogenesis and myocardial regeneration; 4. Enhancing the interfacial bonding strength between bacterial cellulose and PLGA, ensuring stability and safety during implantation and long-term use. Attached Figure Description

[0020] The invention will be further described with reference to the accompanying drawings, but the contents of the drawings do not constitute any limitation on the invention.

[0021] Figure 1 This is a flowchart illustrating the preparation method of the PLGA composite myocardial patch using bacterial cellulose in situ synthesis of cerium dioxide, as described in this invention.

[0022] Figure 2 This is a magnified microscopic image of the PLGA composite myocardial patch synthesized in situ from bacterial cellulose according to the present invention.

[0023] Figure 3 This is an electron micrograph of a PLGA composite myocardial patch synthesized from cerium dioxide in situ using bacterial cellulose, as described in this invention.

[0024] Figure 4This is a biocompatibility diagram of the PLGA composite myocardial patch synthesized from cerium dioxide in situ using bacterial cellulose according to the present invention. Detailed Implementation

[0025] The technical solution of the present invention will be further described in conjunction with the following embodiments.

[0026] Example 1 A method for preparing a PLGA composite myocardial patch using bacterial cellulose in situ synthesis of cerium dioxide, such as... Figure 1 The process is as follows: S1. Inoculate the Acetobacter xylinum seed culture into a container containing liquid HS medium for the first culture. S2. Trivalent cerium salt solution was added to a container to prepare cerium dioxide nanoparticles by in-situ synthesis and then cultured for a second time to finally obtain bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate. S3. A polylactic acid-glycolic acid copolymer nanofiber layer is constructed on the surface of the bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate obtained in S2 by electrospinning, thus obtaining a PLGA composite myocardial patch.

[0027] It should be noted that the bacterial cellulose (BC) of this invention, as a pure cellulose nanofiber network produced by bacterial fermentation, possesses high water content, high strength, and excellent biocompatibility, and has already seen preliminary applications in skin wound dressings and soft tissue repair. Cerium nanoparticles, as biomimetic nanoenzymes, exhibit activities mimicking superoxide dismutase (SOD) and catalase (CAT), effectively scavenging reactive oxygen species (ROS). Therefore, the cerium nanoparticles in the PLGA composite myocardial patch of this invention also effectively scavenge ROS. The polylactic acid-glycolic acid copolymer nanofiber layer is a commonly used biodegradable medical synthetic polymer, and its degradation products can be metabolized and absorbed by the body.

[0028] Specifically, S1 involves inoculating a container filled with liquid HS culture medium with Acetobacter xylinum seed solution at an inoculation rate of 3% (v / v) to 7% (v / v), and then incubating it statically in a 37°C incubator for 8 to 16 hours.

[0029] The Acetobacter xylinum seed culture was obtained by the following steps: Acetobacter xylinum was streaked into 50 mL of liquid HS medium and cultured with shaking at 28℃ and 1000 rpm for 24 h to obtain the Acetobacter xylinum seed culture.

[0030] The liquid HS culture medium contains 5g-10g mannitol, 2.5g-5g peptone and 2.5g-5g yeast extract per 1L of deionized water.

[0031] S2 is performed through the following steps: S2.1 Add trivalent cerium salt solution to the container in S1, and then incubate statically in a constant temperature incubator at 36℃~37℃ for 6d~8d, then proceed to S2.2; S2.2 Remove the solid from the container, add a NaOH aqueous solution with a concentration of 0.4mol / L to 0.6mol / L to the solid, control the temperature to 140℃ to 150℃ and react for 1h to 4h, then proceed to S2.3; S2.3 The solid is repeatedly rinsed with deionized water until neutral, and then dried to obtain a bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate.

[0032] The trivalent cerium salt solution is either an aqueous solution of cerium chloride heptahydrate or an aqueous solution of cerium nitrate hexahydrate; and the concentration of the trivalent cerium salt solution is 80 mg / ml to 120 mg / ml. The ratio of the amount of trivalent cerium salt solution, liquid HS medium of S1 and NaOH aqueous solution of S2.2 added is (1 μl to 5 μl): (1 ml to 2 ml): (2 ml to 3 ml).

[0033] It should be noted that this invention oxidizes cerium ions to cerium dioxide by reacting a trivalent cerium salt solution at high temperature under alkaline conditions. The cerium dioxide nanoparticles obtained by this invention have a size of 5 nm to 50 nm. In S2.1, this invention achieves the deposition and crystal growth of cerium dioxide nanoparticles, which are uniformly distributed on the surface of bacterial cellulose fibers, avoiding aggregation and improving antioxidant activity.

[0034] Specifically, S3 is performed through the following steps: S3.1 Dissolve polylactic acid-glycolic acid copolymer (PLGA) in an organic solvent to obtain a spinning solution with a concentration of 8 wt% to 15 wt%; wherein the organic solvent is hexafluoroisopropanol. S3.2. Electrospinning the spinning solution directly onto the surface of the bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate to obtain a PLGA composite myocardial patch with a deposition layer thickness of 1 mm to 2 mm.

[0035] In S3.2, the electrospinning parameters are a voltage of 10 kV to 20 kV, a receiving distance of 10 cm to 20 cm, and a flow rate of 0.035 mL / h to 0.05 mL / h.

[0036] This embodiment is a general process description. Specific parameters can be adjusted by referring to the preferred range or optimal value of embodiments 2-4 below.

[0037] The method for preparing PLGA composite myocardial patches by in-situ synthesis of cerium dioxide from bacterial cellulose yields PLGA composite myocardial patches with good mechanical compatibility, cell compatibility, antioxidant capacity, and interfacial bonding.

[0038] Example 2 A method for preparing a PLGA composite myocardial patch by in situ synthesis of cerium dioxide from bacterial cellulose is the same as that in Example 1, except that: S1 specifically involves inoculating a container containing liquid HS culture medium with Acetobacter xylinum seed liquid at an inoculation amount of 3% (v / v), and then incubating it statically in a constant temperature incubator at 36°C for 16 hours.

[0039] The liquid HS culture medium contains 5g mannitol, 5g peptone and 2.5g yeast extract per 1L of deionized water.

[0040] S2 is performed through the following steps: S2.1 Add trivalent cerium salt solution to the container in S1, and then incubate it statically in a constant temperature incubator at 36℃ for 8 days. During the incubation period, maintain the ambient humidity >80% before proceeding to S2.2. The trivalent cerium salt solution is an aqueous solution of cerium chloride heptahydrate, and the concentration of the trivalent cerium salt solution is 80 mg / ml. S2.2 Remove the solid from the container, add a 0.4 mol / L NaOH aqueous solution to the solid, control the temperature to 150℃ and react for 1 hour, then proceed to S2.3; S2.3 The solid is repeatedly rinsed with deionized water until neutral, and then dried to obtain a bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate.

[0041] Specifically, S3 is performed through the following steps: S3.1 Dissolve polylactic acid-glycolic acid copolymer (PLGA) in an organic solvent to obtain a spinning solution with a concentration of 8 wt%; wherein the organic solvent is hexafluoroisopropanol. S3.2. Electrospinning the spinning solution directly onto the surface of the bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate to obtain a PLGA composite myocardial patch with a deposition layer thickness of 1 mm.

[0042] The ratio of trivalent cerium salt solution, liquid HS medium of S1, and NaOH aqueous solution of S2.2 is 1 μl: 1 ml: 2 mL.

[0043] In S3.2, the electrospinning parameters are a voltage of 10 kV, a receiving distance of 10 cm, and a flow rate of 0.035 mL / h.

[0044] Example 3 A method for preparing a PLGA composite myocardial patch based on in-situ bacterial cellulose synthesis of cerium dioxide, with other features the same as in Example 1, except that: Specifically, S1 involves inoculating a 7% (v / v) seed culture of Acetobacter xylinum into a container containing liquid HS medium, and then incubating it statically in a 37°C incubator for 8 hours.

[0045] The liquid HS culture medium contains 10g mannitol, 2.5g peptone and 5g yeast extract per 1L of deionized water.

[0046] S2 is performed through the following steps: S2.1 Add trivalent cerium salt solution to the container in S1, and then incubate it statically in a constant temperature incubator at 37℃ for 6 days. During the incubation period, maintain the ambient humidity >80%. The trivalent cerium salt solution is an aqueous solution of cerium nitrate hexahydrate, and the concentration of the trivalent cerium salt solution is 120 mg / ml. S2.2 Remove the solid from the container, add a 0.6 mol / L NaOH aqueous solution to the solid, control the temperature to 140℃ and react for 4 hours, then proceed to S2.3; S2.3 The solid is repeatedly rinsed with deionized water until neutral, and then dried to obtain a bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate.

[0047] Specifically, S3 is performed through the following steps: S3.1 Dissolve polylactic acid-glycolic acid copolymer (PLGA) in an organic solvent to obtain a spinning solution with a concentration of 15 wt%; wherein the organic solvent is hexafluoroisopropanol. S3.2. Electrospinning the spinning solution directly onto the surface of the bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate to obtain a PLGA composite myocardial patch with a deposition layer thickness of 2 mm.

[0048] The ratio of trivalent cerium salt solution to liquid HS medium of S1 was 5 μl: 1 ml.

[0049] The ratio of trivalent cerium salt solution, liquid HS medium of S1, and NaOH aqueous solution of S2.2 is 5 μl: 1 ml: 3 mL.

[0050] In S3.2, the electrospinning parameters are a voltage of 20kV, a receiving distance of 20cm, and a flow rate of 0.05mL / h.

[0051] Example 4 A method for preparing a PLGA composite myocardial patch based on in-situ bacterial cellulose synthesis of cerium dioxide, with other features the same as in Example 1, except that: Specifically, S1 involves inoculating a 5% (v / v) seed culture of Acetobacter xylinum into a container containing liquid HS medium, and then incubating it statically in a 37°C incubator for 12 hours.

[0052] The liquid HS culture medium contains 8g mannitol, 4g peptone and 3g yeast extract per 1L of deionized water.

[0053] S2 is performed through the following steps: S2.1 Add trivalent cerium salt solution to the container in S1, and then incubate it statically in a constant temperature incubator at 37℃ for 7 days. During the incubation period, maintain the ambient humidity >80% before proceeding to S2.2. The trivalent cerium salt solution is an aqueous solution of cerium chloride heptahydrate, and the concentration of the trivalent cerium salt solution is 100 mg / ml. S2.2, Remove the solid from the container, add a 0.5 mol / L NaOH aqueous solution to the solid, control the temperature to 148℃ and react for 3 hours, then proceed to S2.3; S2.3 The solid was repeatedly rinsed with deionized water until neutral, and then dried to obtain a bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate.

[0054] Specifically, S3 is performed through the following steps: S3.1 Dissolve polylactic acid-glycolic acid copolymer (PLGA) in an organic solvent to obtain a spinning solution with a concentration of 10 wt%; wherein the organic solvent is hexafluoroisopropanol. S3.2. Electrospinning the spinning solution directly onto the surface of the bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate to obtain a PLGA composite myocardial patch with a deposition layer thickness of 1 mm.

[0055] The ratio of trivalent cerium salt solution, liquid HS medium of S1, and NaOH aqueous solution of S2.2 added is 3 μl: 1.5 ml: 3 mL.

[0056] In S3.2, the electrospinning parameters are a voltage of 15kV, a receiving distance of 15cm, and a flow rate of 0.040mL / h.

[0057] Example of effect 1. Appearance The PLGA composite myocardial patch from Example 4 was magnified under a microscope to obtain... Figure 2 .

[0058] from Figure 2 As can be seen, the PLGA composite myocardial patch has a porous structure, thereby improving cell adhesion and guidance.

[0059] 2. Electron microscopy observation Electron microscopy was performed on the PLGA composite myocardial patch from Example 4 to obtain... Figure 3 ,in Figure 3 (a) shows the morphology at a 40 μm field of view. Figure 3 (b) shows the morphology under a 50 μm field of view.

[0060] from Figure 3 As can be seen, PLGA nanofibers are uniformly deposited on the surface of the composite substrate. The fiber diameter is 100-500 nm and the pore size is 5-20 μm, forming a continuous porous structure. This porous structure facilitates the passage of cerium ions, further repairing myocardial tissue. The PLGA nanofiber coating can form a hydrophobic side to prevent adhesion.

[0061] 3. Cell biocompatibility H9C2 cells were fluorescently stained using a Live / Dead Viability Assay Kit to observe cell viability; live cells stained green and dead cells stained red. The specific experimental procedures are as follows: 3.1. With pure bacterial cellulose as the control, the PLGA composite myocardial patch from Example 4 was used as the experimental group, and inoculated with 5 × 10 4 One H9C2 cell; 3.2. At 37℃, 5% CO 2 Incubate in a 95% humidity incubator for 24 hours; 3.3. Sterilize the biosafety cabinet with ultraviolet light for 30 minutes, and wipe the biosafety cabinet with 75% alcohol. Remove the 24-well plate from the incubator and place it in the biosafety cabinet. Remove the old culture medium, wash gently with PBS, and remove the PBS. 3.4. In a light-protected environment, prepare the Live / Dead staining working solution with a ratio of Calcein AM (solution A): Ethidium homodimer-1 (solution B): PBS = 1:1:1000, and mix well by pipetting. 3.5. Add 500 μl of staining working solution to a 24-well plate, incubate at room temperature for 30 min, discard the staining solution, and wash three times with PBS; 3.6. Observe and photograph on a live-cell workstation. Green fluorescence indicates live cells, and red fluorescence indicates dead cells. Below is a merge image, yielding the desired result. Figure 4 .

[0062] pass Figure 4 As can be seen, the PLGA composite myocardial patch (experimental group) and the pure bacterial cellulose myocardial patch (control group) of the present invention both achieved a survival rate of over 90% in HUVEC cells, proving that the PLGA composite myocardial patch of the present invention has good cell compatibility.

[0063] In summary, the PLGA composite myocardial patch synthesized from cerium dioxide in situ using bacterial cellulose of the present invention possesses a porous structure, thereby enhancing cell adhesion and guidance, and providing a biomimetic three-dimensional microenvironment for cell adhesion, migration, and orderly arrangement. The above data also demonstrate that this PLGA composite myocardial patch exhibits good cell biocompatibility.

[0064] Example 5 The use of a PLGA composite myocardial patch, which is synthesized in situ from bacterial cellulose to cerium dioxide, in the preparation of products supporting damaged myocardium, wherein the PLGA composite myocardial patch is prepared by the preparation method of the PLGA composite myocardial patch synthesized in situ from bacterial cellulose to cerium dioxide described in Examples 2 to 4.

[0065] The experimental results demonstrate that the PLGA composite myocardial patch possesses a porous structure, providing a biomimetic three-dimensional microenvironment for cell adhesion, migration, and orderly arrangement. Furthermore, the PLGA composite myocardial patch exhibits excellent biocompatibility. Because the PLGA composite myocardial patch synthesizes cerium dioxide in situ from bacterial cellulose, which itself has high water content, high strength, and excellent biocompatibility, and because cerium nanoparticles, as biomimetic nanozymes, possess activities mimicking superoxide dismutase (SOD) and catalase (CAT), it can effectively scavenge reactive oxygen species (ROS). Moreover, this PLGA composite myocardial patch utilizes the degradability of PLGA to achieve local sustained release of growth factors or drugs, promoting angiogenesis and myocardial regeneration; it also enhances the interfacial binding strength between bacterial cellulose and PLGA, ensuring stability and safety during implantation and long-term use.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a PLGA composite myocardial patch using bacterial cellulose in situ to synthesize cerium dioxide, characterized in that, The process is as follows: S1. Inoculate the Acetobacter xylinum seed culture into a container containing liquid HS medium for the first culture. S2. Add trivalent cerium salt solution to a container to prepare cerium dioxide nanoparticles in situ and then culture them for a second time to finally obtain bacterial cellulose-cerium dioxide composite-bacterial cellulose substrate. S3. A polylactic acid-glycolic acid copolymer nanofiber layer is constructed on the surface of the bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate obtained in S2 by electrospinning, thereby obtaining the PLGA composite myocardial patch.

2. The method for preparing PLGA composite myocardial patches based on in-situ bacterial cellulose synthesis of cerium dioxide according to claim 1, characterized in that, S2 is specifically performed by the following steps: S2.1 Add trivalent cerium salt solution to the container in S1, and then incubate statically in a constant temperature incubator at 36℃~37℃ for 6d~8d, then proceed to S2.2; S2.2 Remove the solid from the container, add a NaOH aqueous solution with a concentration of 0.4mol / L to 0.6mol / L to the solid, control the temperature to 140℃ to 150℃ and react for 1h to 4h, then proceed to S2.3; S2.3 The solid is repeatedly rinsed with deionized water until neutral, and then dried to obtain the bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate.

3. The method for preparing PLGA composite myocardial patches based on in-situ bacterial cellulose synthesis of cerium dioxide according to claim 1, characterized in that, S3 is specifically performed by the following steps: S3.1 Dissolve polylactic acid-glycolic acid copolymer (PLGA) in an organic solvent to obtain a spinning solution with a concentration of 8 wt% to 15 wt%. S3.

2. Electrospinning the spinning solution directly onto the surface of the bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate to obtain the PLGA composite myocardial patch with a deposition layer thickness of 1 mm to 2 mm.

4. The method for preparing PLGA composite myocardial patches based on in-situ bacterial cellulose synthesis of cerium dioxide according to claim 2, characterized in that, S2 is specifically performed by the following steps: S2.1 Add trivalent cerium salt solution to the container in S1, and then incubate it statically in a constant temperature incubator at 37℃ for 7 days. During the incubation period, keep the ambient humidity >80% before proceeding to S2.

2. S2.2 Remove the solid from the container, add a 0.5 mol / L NaOH aqueous solution to the solid, control the temperature to 148℃ and react for 3 hours, then proceed to S2.3; S2.3 The solid is repeatedly rinsed with deionized water until neutral, and then dried to obtain the bacterial cellulose-cerium dioxide-bacterial cellulose composite substrate.

5. The method for preparing PLGA composite myocardial patches based on in-situ bacterial cellulose synthesis of cerium dioxide according to any one of claims 1 to 4, characterized in that: The trivalent cerium salt solution is an aqueous solution of cerium chloride heptahydrate or cerium nitrate hexahydrate; and the concentration of the trivalent cerium salt solution is 80 mg / ml to 120 mg / ml; The ratio of the added trivalent cerium salt solution, the liquid HS culture medium of S1, and the NaOH aqueous solution of S2.2 is (1 μl ~ 5 μl): (1 ml ~ 2 ml): (2 ml ~ 3 ml).

6. The method for preparing PLGA composite myocardial patches based on in-situ bacterial cellulose synthesis of cerium dioxide according to any one of claims 1 to 4, characterized in that: The liquid HS culture medium contains 5g-10g mannitol, 2.5g-5g peptone and 2.5g-5g yeast extract per 1L of deionized water.

7. The method for preparing PLGA composite myocardial patches based on in-situ bacterial cellulose synthesis of cerium dioxide according to claim 3, characterized in that: In step S3.2, the electrospinning parameters are: voltage of 10kV to 20kV, receiving distance of 10cm to 20cm, and flow rate of 0.035mL / h to 0.05mL / h. The organic solvent is hexafluoroisopropanol.

8. The method for preparing PLGA composite myocardial patches based on in-situ bacterial cellulose synthesis of cerium dioxide according to claim 1, characterized in that: S1 specifically involves inoculating the Acetobacter xylinum seed solution into a container containing liquid HS culture medium at an inoculation amount of 3% (v / v) to 7% (v / v), and then incubating it statically in a constant temperature incubator at 36℃ to 37℃ for 8h to 16h. The Acetobacter xylophilus seed culture was obtained by the following steps: Acetobacter xylophilus was streaked into 50 mL of liquid HS medium and cultured with shaking at 28 °C and 1000 rpm for 24 h to obtain the Acetobacter xylophilus seed culture.

9. A PLGA composite myocardial patch using bacterial cellulose to synthesize cerium dioxide in situ, characterized in that: The PLGA composite myocardial patch, prepared by the method for in-situ synthesis of cerium dioxide from bacterial cellulose as described in any one of claims 1 to 8, was obtained.

10. The use of a PLGA composite myocardial patch, synthesized in situ from bacterial cellulose to cerium dioxide, in the preparation of products supporting damaged myocardium, characterized in that: The PLGA composite myocardial patch is prepared by the method for preparing PLGA composite myocardial patch by in-situ synthesis of cerium dioxide from bacterial cellulose as described in any one of claims 1 to 8.

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