3D bio-printing epicardial patch for treating myocardial infarction as well as preparation method and application of 3D bio-printing epicardial patch

Abstract

The invention discloses a 3D biological printing epicardial patch for treating myocardial infarction as well as a preparation method and application of the 3D biological printing epicardial patch. The 3D biological printing epicardial patch is an epicardial patch with a specific structure, and the epicardial patch is prepared from chitosan nanoparticles loaded with recombinant human vascular endothelial growth factors (rhVEGF165) and methylacryloyl gelatin (GelMA) biological ink loaded with human bone marrow mesenchymal stem cells (hMSCs) through 3D biological printing, wherein the methylacryloyl gelatin (GelMA) biological ink contains the chitosan nanoparticles loaded with the recombinant human vascular endothelial growth factors (rhVEGF165) and the human bone marrow mesenchymal stem cells (hMSCs). Through morphological observation, mechanical property detection, swelling property detection and degradation property detection on the prepared patch, good physical and chemical characteristics of the patch are verified. An in-vitro release test shows that the patch can continuously and stably release rhVEGF165 and promote cell proliferation and angiogenesis. In-vivo experiments prove that the traditional Chinese medicine composition can effectively promote cardiac function recovery, improve myocardial remodeling and remarkably increase angiogenesis of an ischemic area. The invention provides a new technical means for repairing after myocardial infarction. The method not only shows excellent clinical transformation potential, but also opens up a new path for treatment of cardiovascular diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to an epicardial patch for treating myocardial infarction and its preparation method and application, in particular to a 3D bioprinted epicardial patch containing recombinant human vascular endothelial growth factor (rhVEGF165) loaded chitosan nanoparticles, gelatin methacrylate (GelMA) and human mesenchymal stem cells (hMSCs) and its preparation method and application in treating myocardial infarction. The present application belongs to the technical field of medicine. BACKGROUND

[0002] In recent years, the treatment of myocardial infarction mainly relies on drugs, coronary intervention (PCI) and coronary artery bypass grafting (CABG). Although these methods can improve the early survival rate of patients, they have limited effect on the functional recovery of damaged myocardial tissue. In particular, stem cell transplantation therapy, especially the use of human mesenchymal stem cells (hMSCs), has become a research hotspot due to its low immunogenicity, easy accessibility and expansion characteristics. However, traditional stem cell transplantation methods such as intravenous injection, coronary injection and subepicardial injection generally have low cell delivery efficiency, low survival number after transplantation, large operation damage and poor targeting.

[0003] The existence of these problems is mainly due to the complexity of the cardiac microenvironment and the limitations of existing technology. The cardiac microenvironment after myocardial infarction involves multiple signal pathways and cell-cell interactions, and it is quite challenging to find a method that can both promote cell survival and targeted release of therapeutic factors. In addition, selecting the appropriate scaffold material not only needs to meet the requirements of biocompatibility and mechanical properties, but also needs to have good degradation characteristics and controllable drug release characteristics, which requires a large number of experimental verification and optimization. At the same time, combining 3D bioprinting technology and nanotechnology while ensuring cell activity and effective release of therapeutic factors puts high requirements on technology integration.

[0004] It is of great significance to solve the above problems. By developing a new type of 3D bioprinted epicardial patch, the survival rate and implantation efficiency of stem cells can be significantly improved, thereby better exerting their pro-angiogenic, anti-fibrotic and other effects, and ultimately improving the cardiac function after myocardial infarction. This new technology not only provides a new idea and technical means for the treatment of myocardial infarction, but also has important clinical translation value. Compared with traditional treatment methods, it is expected to reduce surgical trauma, reduce the risk of complications, alleviate the suffering of patients and improve the quality of life. SUMMARY

[0005] The present application aims to provide a novel 3D bioprinted epicardial patch, which is designed to significantly improve the therapeutic effect after myocardial infarction by combining recombinant human vascular endothelial growth factor (rhVEGF165) loaded chitosan nanoparticles, methacrylated gelatin (GelMA) and human mesenchymal stem cells (hMSCs).

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical means:

[0007] The 3D bioprinted epicardial patch for treating myocardial infarction of the present application is obtained by 3D bioprinting of methacrylated gelatin (GelMA) bio-ink containing recombinant human vascular endothelial growth factor (rhVEGF165) loaded chitosan nanoparticles and human mesenchymal stem cells (hMSCs).

[0008] Preferably, the recombinant human vascular endothelial growth factor (rhVEGF165) loaded chitosan nanoparticles are prepared by ion crosslinking method and uniformly dispersed in the methacrylated gelatin bio-ink.

[0009] Preferably, the bio-ink containing rhVEGF165 loaded chitosan nanoparticles and hMSCs is printed into an epicardial patch with a specific structure by 3D bioprinting; after printing, the patch is cured using a wavelength 405 nm blue light source to prepare a methacrylated gelatin (GelMA) epicardial patch containing recombinant human vascular endothelial growth factor (rhVEGF165) loaded chitosan nanoparticles and human mesenchymal stem cells (hMSCs), which has a good network structure and stable three-dimensional scaffold structure.

[0010] Further, the present application also proposes a method for preparing the epicardial patch, comprising the following steps:

[0011] (1) preparing GelMA bio-ink and mixing it with rhVEGF165 loaded chitosan nanoparticles and hMSCs;

[0012] (2) using a 3D bioprinter to print the above mixture into an epicardial patch with a specific structure;

[0013] (3) performing blue light curing treatment on the printed patch.

[0014] Preferably, the recombinant human vascular endothelial growth factor (rhVEGF165) loaded chitosan nanoparticles are prepared by the following method:

[0015] (1) dissolving low molecular weight chitosan in acetic acid solution to form a chitosan solution;

[0016] (2) Under stirring condition, rhVEGF165 solution was added, and the pH value was adjusted to 7.0-7.4, so that the chitosan nanoparticles were formed and loaded with rhVEGF165;

[0017] (3) The chitosan nanoparticles loaded with rhVEGF165 were purified by centrifugation and dialysis.

[0018] Preferably, the step of preparing the GelMA bio-ink comprises:

[0019] (1) Dissolving GelMA powder in PBS buffer, adding initiator LAP, and heating and dissolving under light-proof condition to obtain a GelMA solution;

[0020] (2) The obtained GelMA solution was sterilized and filtered, and then stored for standby use.

[0021] Preferably, the method comprises the following steps:

[0022] (1) Preparing chitosan nanoparticles loaded with rhVEGF165:

[0023] ① Weighing low molecular weight chitosan powder, dissolving it in acetic acid solution, and preparing chitosan acetic acid solution, and magnetically stirring overnight;

[0024] ② Adjusting the pH of the acetic acid solution to 5.0 with sodium hydroxide solution, and then heating the chitosan solution in a 60°C water bath for 10 minutes, and quickly filtering it with a 0.45μm filter to remove insoluble impurities, to obtain a chitosan stock solution;

[0025] ③ Weighing sodium tripolyphosphate (TPP) powder, preparing a 1.5mg / mL TPP aqueous solution, filtering impurities with a 0.22μm filter, and obtaining a TPP stock solution;

[0026] ④ Placing the chitosan stock solution and the TPP stock solution in an ultraclean bench in advance for ultraviolet sterilization, then taking 7.5ml of the chitosan stock solution and placing it in a 25ml round-bottom flask, placing the flask on a magnetic stirrer, and slowly adding 2ml of the TPP stock solution to the flask with a Pasteur pipette, continuing to stir for 15 minutes until a light blue opalescence appears in the solution, to obtain a blank chitosan nanoparticle suspension;

[0027] ⑤ Preparation of chitosan nanoparticles loaded with rhVEGF165

[0028] The rhVEGF165 powder was dissolved in sterile ultrapure water to prepare a rhVEGF protein solution with a concentration of 100 ng / μL. 5 μl of the rhVEGF165 protein solution was aliquoted into an EP tube to which 5 μl of PBS had been added in advance, diluted to 50 ng / μL, mixed thoroughly, and stored in a -80°C refrigerator. The chitosan stock solution and the TPP stock solution were placed in an ultraclean bench for ultraviolet sterilization for 30 minutes in advance. Then, 7.5 ml of the chitosan stock solution was placed in a round-bottomed beaker, 10 μl of the rhVEGF165 protein solution was added, and the beaker was placed on a magnetic stirrer with a stirring speed of 900 rpm for 5 minutes. Then, 2 ml of the TPP stock solution was slowly added to the beaker with a Pasteur pipette, and the solution was stirred for another 15 minutes until a light blue opalescence appeared. Thus, a chitosan nanoparticle suspension loaded with rhVEGF165 was prepared.

[0029] (2) Preparation of methacrylated gelatin (GelMA) bio-ink:

[0030] ① 20 ml of PBS was added to a brown bottle containing 0.05 g of initiator LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate);

[0031] ② The solution was dissolved in a 40-50°C water bath for 15 minutes, with shaking several times during the process. Note that the process should be carried out in the dark. After dissolution, the solution was wrapped in tin foil and stored at 4°C.

[0032] ③ The required amount of GelMA was placed in a 50 ml centrifuge tube, and the initiator standard solution was added to the centrifuge tube and shaken to ensure complete infiltration and swelling;

[0033] ④ The solution was heated in a 60-70°C water bath in the dark for 20-30 minutes, with shaking several times during the process. After complete dissolution, a GelMA solution was obtained.

[0034] ⑤ The GelMA solution was dissolved in PBS buffer, and then immediately sterilized with a 0.22 μm sterile filter and filtered into a 15 ml centrifuge tube. A GelMA solution with a concentration of 5% w / v was obtained, wrapped in tin foil, and stored at -20°C.

[0035] (3) Mixing materials

[0036] ① The chitosan nanoparticle suspension loaded with rhVEGF165 was mixed with 3 ml of the GelMA solution, mixed thoroughly to form a bio-ink containing chitosan nanoparticles loaded with rhVEGF165, and stored in a 37°C water bath for later use.

[0037] ②Take the third generation of hMSCs with 80%-90% cell fusion, and use 0.25% trypsin to digest, then use a cell counter to count the collected cells;

[0038] ③The hMSCs cell suspension is loaded into 3 15ml centrifuge tubes at a ratio of 3x10 6 cells per tube. After centrifugation at 1000rpm for 5 minutes, the supernatant is discarded;

[0039] ④Take 3ml of bio-ink containing rhVEGF165-loaded chitosan nanoparticles, and add it to the centrifuge tube containing the hMSCs cell pellet, resuspend the cells, mix thoroughly, avoid air bubbles, and finally prepare a bio-ink with a concentration of 1x10 6 cells / mL, and store at 37℃ for the next step of printing;

[0040] (4) 3D bioprinting

[0041] The bio-ink containing rhVEGF165-loaded chitosan nanoparticles and hMSCs is printed into an epicardial patch with a specific structure by 3D bioprinting. After printing, the patch is cured using a wavelength of 405nm blue light source to prepare a methacrylated gelatin (GelMA) epicardial patch containing rhVEGF165-loaded chitosan nanoparticles and human bone marrow mesenchymal stem cells (hMSCs), which has a good network structure and stable three-dimensional scaffold structure.

[0042] Further, the application also provides the use of the epicardial patch in the preparation of medical devices for treating myocardial infarction.

[0043] Preferably, the epicardial patch can effectively promote the recovery of heart function, improve myocardial remodeling, and significantly increase the angiogenesis in the ischemic area.

[0044] Compared with the prior art, the application has the following advantages:

[0045] (1) The application provides a new treatment method for myocardial infarction, which solves the problems of low cell survival rate, insufficient implantation, large operation damage and poor targeting in traditional stem cell transplantation methods.

[0046] (2) The application innovatively combines rhVEGF165-loaded chitosan nanoparticles, GelMA and hMSCs, and uses advanced 3D bioprinting technology to develop an efficient epicardial patch, which significantly improves the treatment effect and quality of life of patients with myocardial infarction.

[0047] (3) The prepared epicardial patch of the application is subjected to in vitro release efficiency test, which proves that it can release rhVEGF165 stably and continuously. Through rat myocardial infarction model experiment, it is verified that the patch not only can promote long-term survival of stem cells and paracrine effect, but also can significantly increase angiogenesis in ischemic myocardial area, improve ventricular remodeling, and thus improve the effect of heart function recovery. BRIEF DESCRIPTION OF DRAWINGS

[0048] In order to more clearly show the technical solutions of the application and its implementation effects, the following is a detailed description of the related drawings:

[0049] Figure 1 Surface marker analysis of hMSCs;

[0050] Figure 2 Preparation of schematic diagram and electron microscope morphology of chitosan nanoparticles;

[0051] (A) Nanoparticle preparation schematic diagram;

[0052] (B) Morphology of blank nanoparticles under microscope (scale bar = 1 um);

[0053] (C) Morphology of rhVEGF165-loaded nanoparticles under microscope (scale bar = 1 um);

[0054] Figure 3 Particle size distribution and Zeta potential of blank and rhVEGF165-loaded chitosan nanoparticles;

[0055] (A) Particle size distribution diagram of blank nanoparticles;

[0056] (B) Zeta potential diagram of blank nanoparticles;

[0057] (c) Particle size distribution diagram of rhVEGF165-loaded nanoparticles;

[0058] (D) Zeta potential diagram of rhVEGF165-loaded nanoparticles;

[0059] Figure 4 Release efficiency of rhVEGF165-loaded chitosan nanoparticles at different time points;

[0060] Figure 5 GelMA / GelMA+blank chitosan nanoparticle epicardial patch appearance and electron microscope morphology.

[0061] (A) Appearance and morphology of two kinds of patches after freeze-drying;

[0062] (B) Structure and morphology characteristics of two kinds of patches under electron microscope (scale bar is shown in the figure);

[0063] Figure 6Compressive modulus of GelMA / GelMA+ blank chitosan nanoparticle epicardial patch;

[0064] (A) Schematic diagram of compressive modulus detection;

[0065] (B) Stress-strain curves of two groups of patches;

[0066] (C) Comparison of compressive modulus of two groups of patches (*, P <0.05);

[0067] Figure 7 Swelling and degradation of GelMA / GelMA+ blank chitosan nanoparticle epicardial patch;

[0068] (A) Comparison of swelling rates of two groups of patches;

[0069] (B) Degradation curves of two groups of patches (*, P <0.05);

[0070] Figure 8 In vitro release of GelMA+ rhVEGF165-loaded chitosan nanoparticle epicardial patch;

[0071] Figure 9 Cell survival and proliferation of epicardial patch;

[0072] (A) Live and dead cell staining of 3 groups of patches (live cells: green; dead cells: red; scale bar = 100 um);

[0073] (B) Comparison of the proportion of live cells at different times of 3 groups of patches;

[0074] (C) Comparison of cell proliferation at different times of 3 groups of patches (*, P <0.05; ** P <0.01);

[0075] Figure 10 In vitro pro-angiogenic effect of epicardial patch;

[0076] (A) Angiogenesis images of 3 groups of patches (scale bar = 50 um);

[0077] (B) Comparison of the number of angiogenesis of 3 groups of patches (*, P <0.05);

[0078] Figure 11 Postoperative echocardiography of myocardial infarction rats;

[0079] (A) Representative M-mode echocardiogram of 4 groups of rats after myocardial infarction surgery (left ventricular short axis, long line indicates left ventricular end-diastolic diameter, and short line indicates left ventricular end-systolic diameter);

[0080] (B) Comparison of left ventricular ejection fraction of 4 groups of rats after myocardial infarction surgery;

[0081] (C) Comparison of left ventricular fractional shortening in rats after myocardial infarction in the four groups;

[0082] Figure 12 Myocardial fibrosis in rats after myocardial infarction;

[0083] (A) Myocardial fibrosis images in rats after myocardial infarction in the four groups (low magnification scale = 5 mm, high magnification scale = 500 μm);

[0084] (B) Comparison of infarct size (fibrosis area) in rats after myocardial infarction in the four groups. (*, P < 0.05; **, P < 0.01; ***, P < 0.001);

[0085] Figure 13 Inflammatory cell infiltration in rats after myocardial infarction;

[0086] (A) CD11b staining in rats after myocardial infarction in the four groups;

[0087] (B) Comparison of the number of inflammatory cells in rats after myocardial infarction in the four groups. (*, P < 0.05; ***, P < 0.001);

[0088] Figure 14 Myocardial cell apoptosis in rats after myocardial infarction;

[0089] (A) Myocardial cell Tunel staining in rats after myocardial infarction in the four groups;

[0090] (B) Comparison of myocardial cell apoptosis in rats after myocardial infarction in the four groups; (*, P < 0.05; ***, P < 0.001)

[0091] Figure 15 Neovascularization in rats after myocardial infarction;

[0092] (A) CD31 staining in rats after myocardial infarction in the four groups;

[0093] (B) Comparison of neovascularization in rats after myocardial infarction in the four groups; (*, P < 0.05; ***, P < 0.001)

[0094] Figure 16 Neovascularization in rats after myocardial infarction;

[0095] (A) α-SMA staining in rats after myocardial infarction in the four groups;

[0096] (B) Comparison of neovascularization in rats after myocardial infarction in the four groups; (*, P < 0.05; ***, P < 0.001)

[0097] Figure 17 Survival of transplanted patch hMSCs;

[0098] (A) Animals live imaging images at different times after patch transplantation;

[0099] (B) Comparison of bioluminescence intensity at different times. (*, P<0.05)

[0100] These figures not only demonstrate the specific embodiments and technical details of the present application, but also provide detailed data support to ensure the effectiveness and reliability of the technology. DETAILED DESCRIPTION

[0101] The preferred examples of the application will be described in detail below. The examples are given to better illustrate the application, and the application is not limited to the examples. Non-essential improvements and adjustments to the embodiments according to the application still fall within the scope of the application.

[0102] The sources of purchase of reagents and instruments involved in the examples, and the acquisition and separation and culture methods of hMSCs:

[0103] 1. Experimental reagents:

[0104] ① Recombinant human vascular endothelial growth factor (rhVEGF165) (Peprotech, USA)

[0105] ② Human VEGF Elisa kit detection kit (Hangzhou Lianke Biotechnology Co., Ltd.)

[0106] ③ Calcein / PI cell live and dead staining kit (Shanghai Biyun Tian Biotechnology Co., Ltd.)

[0107] ④ CCK-8 reagent (Beijing Lanjiekeli Technology Co., Ltd.)

[0108] ⑤ ECM special medium (Sciencell, USA)

[0109] ⑥ Matrigel matrix glue (Corning, USA)

[0110] ⑦ DAPI (Shanghai Biyun Tian Biotechnology Co., Ltd.)

[0111] 2. Experimental instruments:

[0112] ① Super clean bench (Henan Xingling Instrument and Equipment Co., Ltd.)

[0113] 2 Cell incubator (ThermoFisher, USA)

[0114] 3 Optical microscope, inverted fluorescence microscope (Leica, Germany)

[0115] 4 Freeze dryer (Millrock, USA)

[0116] 53D bioprinter (Hangzhou Jetnofly Biological Technology Co., Ltd.)

[0117] 6Small animal respirator (Shanghai Orkot Biological Technology Co., Ltd.)

[0118] 7Animal ultrasound machine (Philips, USA)

[0119] 3, Acquisition and separation culture of hMSCs

[0120] (1) Acquisition of hMSCs:

[0121] 1Bone marrow was obtained from 40-50 year-old patients undergoing coronary artery bypass surgery in the Department of Cardiovascular Surgery, the Second Affiliated Hospital of Harbin Medical University.

[0122] ②The needle was inserted obliquely at 0.5-1.0 cm below the sternal angle, and 3-6 ml of bone marrow was slowly extracted and immediately taken back to the laboratory for processing.

[0123] (2) Separation and culture of hMSCs:

[0124] ①The bone marrow fluid was added with an equal amount of human peripheral blood lymphocyte separation medium, and the second layer of ring-shaped white lymphocyte layer was collected after centrifugation.

[0125] ②The cells were washed twice with PBS buffer and inoculated in low-glucose DMEM culture medium containing 10% FBS and cultured at 37℃, 5% CO2.

[0126] ③The medium was changed every 3 days, and the cells were subcultured when the density of adherent cells reached 80-90%.

[0127] (3) Identification of hMSCs:

[0128] The third generation of cells was identified by surface markers using CD34, CD45, CD73, CD90, and CD105 flow cytometry antibodies to ensure their purity and characteristics.

[0129] The results are shown in Figure 1 The results showed that CD34 and CD45 were negatively expressed, and the positive rate was less than 3%, which met the international standard for MSCs identification, and the purity was high, which was suitable for subsequent experiments.

[0130] Example 1 Preparation of heart patch based on 3D bioprinting technology

[0131] 1, Preparation of rhVEGF165-loaded chitosan nanoparticles and GelMA bio-ink

[0132] (1) Preparation of chitosan nanoparticles:

[0133] 1. 200 mg of low molecular weight chitosan powder was dissolved in 100 ml of 1% (v / v) acetic acid solution to prepare a chitosan acetic acid solution at a concentration of 2 mg / mL, and the solution was stirred magnetically overnight.

[0134] 2. The pH of the acetic acid solution was adjusted to 5.0 using a 20 wt% aqueous sodium hydroxide solution, and then the chitosan solution was heated in a 60°C water bath for 10 minutes. The solution was filtered immediately using a 0.45 μm filter to remove insoluble impurities, thereby preparing a chitosan stock solution.

[0135] 3. 45 mg of sodium tripolyphosphate (TPP) powder was weighed and dissolved in water to prepare a 1.5 mg / mL TPP aqueous solution. The solution was filtered using a 0.22 μm filter to remove impurities, thereby preparing a TPP stock solution.

[0136] 4. The chitosan stock solution and the TPP stock solution were sterilized using ultraviolet light in a clean bench for 30 minutes. Then, 7.5 ml of the chitosan stock solution was placed in a 25 ml round-bottomed beaker, and the beaker was placed on a magnetic stirrer at a rotation speed of 900 rpm. Using a Pasteur pipette, 2 ml of the TPP stock solution was added slowly to the beaker, and the solution was stirred for 15 minutes. A light blue opalescence appeared in the solution, thereby preparing a blank chitosan nanoparticle suspension.

[0137] 5. Preparation of rhVEGF165-loaded chitosan nanoparticles

[0138] 10 μg of recombinant human rhVEGF165 powder was dissolved in 100 μl of sterile ultrapure water to prepare a rhVEGF165 protein solution at a concentration of 100 ng / μL. The rhVEGF165 protein solution was aliquoted in EP tubes to which 5 μl of PBS had been added in advance, at a concentration of 50 ng / μL, and the mixture was thoroughly mixed and stored in a -80°C refrigerator. The chitosan stock solution and the TPP stock solution were sterilized using ultraviolet light in a clean bench for 30 minutes. Then, 7.5 ml of the chitosan stock solution was placed in a 25 ml round-bottomed beaker, and 10 μl of the rhVEGF165 protein solution was added. The beaker was placed on a magnetic stirrer at a rotation speed of 900 rpm, and 2 ml of the TPP stock solution was added slowly to the beaker using a Pasteur pipette. The solution was stirred for 15 minutes, and a light blue opalescence appeared in the solution, thereby preparing a rhVEGF165-loaded chitosan nanoparticle suspension.

[0139] Figure 2 A schematic diagram of the chitosan nanoparticles was prepared, and the morphology under an electron microscope was observed. The results showed that the blank and rhVEGF165-loaded chitosan nanoparticles prepared by ion crosslinking were spherical in shape, uniform in size, and consistent in distribution. The size of the rhVEGF165-loaded chitosan nanoparticles was larger than that of the blank chitosan nanoparticles.

[0140] Figure 3The particle size distribution and Zeta potential of the blank and rhVEGF165-loaded chitosan nanoparticles were determined. The results showed that the nanoparticles had good physicochemical properties and were suitable for further experiments.

[0141] (2) Preparation of methacrylated gelatin (GelMA) bio-ink:

[0142] ① Take 20 ml PBS and add it to a brown bottle containing initiator LAP (Lithium phenyl-2,4,6-trimethylbenzoylphosphinate) (containing 0.05 g LAP).

[0143] ② Dissolve in a 40-50°C water bath for 15 minutes, shake several times during the process, and avoid light. After dissolution, wrap in tin paper and store at 4°C.

[0144] ③ Take the required amount of GelMA and place it in a 50 ml centrifuge tube. Add the initiator standard solution to the centrifuge tube and shake to fully soak and swell.

[0145] ④ Heat in a 60-70°C water bath in the dark for 20-30 minutes, shake several times during the process, and fully dissolve to obtain a GelMA solution.

[0146] ⑤ Dissolve the GelMA solution in PBS buffer, then immediately sterilize it with a 0.22 μm sterile filter (to prevent low-temperature gelation), filter into a 15 ml centrifuge tube, and prepare a 5% (w / v) GelMA solution. Wrap it in tin paper and store it at -20°C for the next step of printing.

[0147] (3) Mixing materials:

[0148] Mix the rhVEGF165-loaded chitosan nanoparticle suspension prepared in step ⑤ with 3 ml of GelMA solution and mix thoroughly to form a bio-ink containing therapeutic factors.

[0149] Mix the blank chitosan nanoparticle suspension prepared in step ④ with 3 ml of GelMA solution and mix thoroughly to form a GelMA+blank chitosan nanoparticle bio-ink.

[0150] 2. 3D bioprinting and functional verification

[0151] (1) 3D bioprinting:

[0152] ① Open the air pump valve switch. When the switch position is parallel to the air pipe direction, it is in the open state, ensuring good air pressure. Prepare a 37°C water bath in advance.

[0153] ② Turn on the power supply through the right upper side switch of the 3D bioprinter, wipe it with alcohol for disinfection, and sterilize for 30 minutes.

[0154] ③ After sterilization, turn on the EFU (dust removal), start the computer, open the 3D-Architect software, click "Connect Device", and connect the computer and the device.

[0155] ④ New or import project, set parameters (air pressure 0.1 MPa, printing speed 15 mm / s, spacing 1 mm, layer thickness 200 μm, 3 layers), click "Path Generation", and generate the printing path.

[0156] ⑤ Click the "Manual Load Nozzle" button in "Device Control", mount the low-temperature nozzle, and perform temperature control processing (23℃) and platform temperature control (5℃).

[0157] ⑥ Load the biological ink, place it at 4℃ for 5 minutes, load the 0.21mm conical needle and adapter, remove the nozzle, and load the pre-cooled cartridge into the printing nozzle.

[0158] ⑦ After reaching the material forming temperature, test the material out of the silk, and determine that the material reaches a smooth out-of-silk state.

[0159] ⑧ Click the "Platform Height Measurement" button in "Device Calibration", input the height compensation (-0.35) after calibration, and then perform nozzle height measurement and calibration in turn.

[0160] ⑨ Click "Start Printing" to start the printing work. The layer thickness is 200 μm, and the final size is 1.5 cm x 1.5 cm. After printing, place it under a wavelength of 405 nm blue light source for 30 seconds.

[0161] GelMA epicardial patches (GelMA), GelMA+blank chitosan nanoparticle epicardial patches (GelMA+CNPs), and GelMA+chitosan nanoparticle loaded with rhVEGF165 epicardial patches (GelMA+VEGFCNPs) are prepared by the above method.

[0162] Figure 5 The appearance and microstructure of GelMA and GelMA+blank chitosan nanoparticle epicardial patches are described, showing good network structure and stable three-dimensional scaffold structure.

[0163] (2) In vitro release efficiency test:

[0164] ① At the same time on the 1st, 2nd, 3rd, 4th, 5th, 6th, and 7th days, draw 200 μl of chitosan nanoparticle suspension loaded with rhVEGF165, and add 200 μl of fresh PBS.

[0165] ② The rhVEGF165 content of 200 μl of the rhVEGF165-loaded chitosan nanoparticle suspension taken at different time points was detected by using a human VEGF Elisa Kit reagent box. The rhVEGF165 concentration at each time point was calculated according to the standard curve, and then the cumulative release amount at each time point was calculated, and the release curve was drawn.

[0166] ③ The swelling rate and degradation performance of the printed blank chitosan nanoparticle epicardial patch were detected.

[0167] Figure 4 The release efficiency of rhVEGF165-loaded chitosan nanoparticles at different time points was demonstrated. The results showed that the release rate reached 69.22±3.78% within the first 6 hours, and reached 78.63±2.48% at 48 hours.

[0168] Figure 7 The swelling and degradation of the GelMA / GelMA+blank chitosan nanoparticle epicardial patch were demonstrated. By analyzing the swelling and degradation performance of the GelMA and GelMA+blank chitosan nanoparticle epicardial patch, the excellent physical and chemical properties were demonstrated.

[0169] Figure 8 The in vitro release of the GelMA+rhVEGF165-loaded chitosan nanoparticle epicardial patch was demonstrated. The results showed that the cumulative release of the GelMA+rhVEGF165-loaded chitosan nanoparticle epicardial patch reached 78.88±1.72% within 7 days.

[0170] (3) Compression modulus test

[0171] Since the thickness of the printed epicardial patch is only 600 μm, the general biomechanical detector cannot measure its compression modulus. In order to match the mechanical detector range, and better and more accurately detect the mechanical properties of the bio-ink, the hollow pipe mold was made in sterile conditions, and the above prepared GelMA and GelMA+blank chitosan nanoparticle bio-ink was added into the mold in equal amount, and the regular cylinder with a bottom radius of 5 mm and a height of 8 mm was taken out after photocuring for 30 seconds. It was used for the next compression modulus test, and the steps were as follows:

[0172] Connect the biomechanical tester to the computer, open the software and connect the device; motor zero, place the test sample to the center of the tray and weigh the skin; new test, set parameters (sample cross-sectional area 78.54 mm2, sampling frequency 500 point / min, measurement speed 1 mm / min), select single compression (fixed load 2N, holding time 10 seconds); start test, after the test is completed, collect test data, draw stress-strain curve with origin drawing software, and calculate compression modulus according to the initial segment of the curve. The calculation formula is as follows: compression modulus = stress / strain.

[0173] Figure 6 The compression modulus of the GelMA / GelMA+blank chitosan nanoparticle epicardial patch is shown. By comparing the mechanical properties of GelMA and GelMA+blank chitosan nanoparticle epicardial patches, it is found that the compression modulus after adding chitosan nanoparticles is greater than that of pure GelMA.

[0174] Example 2 Application of autologous bone marrow mesenchymal stem cell cardiac patch based on 3D bioprinting technology in the treatment of myocardial infarction

[0175] 1. Preparation of epicardial patch containing hMSCs

[0176] ①Take 3ml of GelMA, GelMA+blank chitosan nanoparticle, and GelMA+loaded rhVEGF165 chitosan nanoparticle three kinds of bio-ink, and place them in a 37℃ water bath for standby.

[0177] ②Take the third generation hMSCs with 80% to 90% cell fusion, and after trypsinization, count the collected cells using a cell counter.

[0178] ③The hMSCs cell suspension was loaded into 3 15ml centrifuge tubes at a ratio of 3x10 6 cells per tube. After centrifugation at 1000rpm for 5 minutes, the supernatant was discarded.

[0179] ④Add the above three kinds of bio-ink to the centrifuge tubes containing hMSCs cell precipitate, resuspend the cells, mix thoroughly, and avoid air bubbles. Finally, three kinds of bio-ink with a concentration of 1x10 6 cells / mL were prepared, and were placed at 37℃ for storage, for the next step of printing.

[0180] ⑤3D bioprinting, the specific operation method is the same as example 1.

[0181] 2. Cell compatibility and proangiogenic ability detection:

[0182] ①Calcein / PI live dead cell staining kit was used to detect the cell survival of hMSCs-containing GelMA epicardial patches (GelMA), hMSCs-containing GelMA+blank chitosan nanoparticle epicardial patches (GelMA+CNPs), and hMSCs-containing GelMA+rhVEGF165-loaded chitosan nanoparticle epicardial patches (GelMA+VEGFCNPs) in 1, 4, and 7 days, respectively.

[0183] ②First, the to-be-detected patches were cut into 4 equal parts according to the size of 0.75 cm x 0.75 cm, and then transferred to a 12-well plate for subsequent detection.

[0184] ③According to the system of 500 μl of detection working solution per well of the 12-well plate, prepare an appropriate amount of Calcein AM / PI detection working solution and mix thoroughly.

[0185] ④Add 500 μl of the above-prepared detection working solution to each well. Incubate at 37℃ in the dark for 30 minutes.

[0186] ⑤After incubation, observe the cell survival under a laser confocal microscope (Calcein AM is green fluorescence, Ex / Em = 494 / 517 nm; PI is red fluorescence, Ex / Em = 535 / 617 nm).

[0187] ⑥Use Matrigel matrix glue plating to culture HUVECs to detect the pro-angiogenic ability of the supernatant of the patches.

[0188] ⑦Take the culture supernatant of each group from the-80℃ refrigerator and place it in the 4℃ refrigerator for thawing.

[0189] ⑧Place Matrigel matrix glue, EP tubes, 96-well plates, and gun head boxes in the 4℃ refrigerator overnight for pre-cooling.

[0190] ⑨Operate the gel plating on the ice box in the clean bench, add 50 μl per well to avoid air bubbles, and place it in a 37℃ cell incubator for incubation for 30 minutes until the matrix glue solidifies.

[0191] ⑩Use a cell density of 2.5 x 10 4 HUVECs per well, suspend the cells in the culture wells with the matrix glue plated with serum-free medium and culture supernatant at a ratio of 1:1, 50 μl per well, and incubate in a 37℃, 5% CO2 cell incubator for 2 hours. Carefully observe the tubule formation under a light microscope.

[0192] Take the culture supernatant of each group from the-80℃ refrigerator and place it in the 4℃ refrigerator for thawing.

[0193] Put Matrigel, EP tube, 96-well plate, and gun tip box in the 4℃ refrigerator overnight for pre-cooling.

[0194] In the clean bench, put the ice box on the table and operate the gel coating. Add 50μl per well to avoid bubbles. Put it in the 37℃ cell incubator for 30 minutes to solidify the matrix gel.

[0195] Use the cell density of 2.5×10 4 HUVECs per well. Mix the cells with serum-free medium and culture supernatant at a ratio of 1:1, and suspend them in the culture well with matrix gel at 50μl per well. Incubate in a 37℃, 5% CO2 cell incubator for 2 hours.

[0196] Carefully observe the tube formation under the optical microscope, and use Image J to calculate the number of tube lumens.

[0197] Figure 9 The cell survival rate and proliferation of epicardial patches containing hMSCs at different time points are shown, which confirms the good cell compatibility of GelMA+VEGFCNPs as a scaffold material.

[0198] Figure 10 The effects of three different components of epicardial patches on promoting HUVECs tube formation ability are compared, which shows that the GelMA+VEGFCNPs patch has the best effect.

[0199] 3. Animal experiments and efficacy evaluation

[0200] (1) Establishment of rat myocardial infarction model:

[0201] Use the method of ligating the left anterior descending branch to make a rat heart infarction model. Randomly divide into four groups: Control group, GelMA+hMSCs group, GelMA+CNPs (empty chitosan nanoparticles)+hMSCs group, and GelMA+VEGFCNPs (loaded rhVEGF165 chitosan nanoparticles)+hMSCs group. The Control group does not use patch treatment.

[0202] (2) Epicardial patch transplantation:

[0203] ① Immediately after the heart infarction operation, the epicardial patch is fixed to the infarction site, that is, the cut 0.75 cm x 0.75 cm size patch is carefully and evenly covered on the ischemic site below the ligation line, and the patch is ensured to be not bent or folded. A 5-0 non-injury suture line with a needle is used, and the needle is inserted from the right upper corner and the left lower corner of the patch (i.e. 1 o'clock and 7 o'clock directions) respectively, and the depth of the needle is about 0.5 mm. The needle is taken out from the patch, and the knot is gently and carefully tied to fix the patch to the infarction site.

[0204] 2 The curative effects of each group are evaluated from the aspects of cardiac ultrasound, infarction area, pathological morphology, inflammatory cell infiltration, myocardial cell apoptosis, angiogenesis and the like.

[0205] (3) Transplanted cell survival condition monitoring:

[0206] The small animal live imaging instrument is used to monitor the survival condition of the transplanted cells in the body, and the long-term survival rate of the transplanted cells within 4 weeks is evaluated.

[0207] Experimental results:

[0208] Figures 11-17 The cardiac ultrasound, myocardial fibrosis, inflammatory cell infiltration, myocardial cell apoptosis, new vein, new artery and transplanted patch hMSCs survival condition of the four groups of infarction rats after treatment are respectively shown. It can be seen from the results that the patch of the GelMA+VEGFCNPs (loaded rhVEGF165 chitosan nanoparticles) +hMSCs group not only can promote the long-term survival of stem cells and the paracrine effect, but also can significantly increase the angiogenesis of the ischemic myocardial region, improve the ventricular remodeling, and thus improve the recovery effect of cardiac function.

[0209] Through the above specific embodiments, the present application provides a new type of 3D bioprinted epicardial patch, which not only significantly improves the survival rate and implantation efficiency of stem cells, but also promotes the angiogenesis of the ischemic myocardial region, improves the ventricular remodeling, and provides a new effective strategy for the treatment of myocardial infarction.

Claims

1. A 3D bioprinted epicardial patch for treating myocardial infarction, characterized in that, The 3D bioprinted epicardial patch is obtained by 3D bioprinting of a methacrylated gelatin (GelMA) bio-ink containing chitosan nanoparticles loaded with recombinant human vascular endothelial growth factor (rhVEGF165) and human mesenchymal stem cells (hMSCs).

2. The epicardial patch of claim 1, wherein, The chitosan nanoparticles loaded with rhVEGF165 are prepared by ion crosslinking method and uniformly dispersed in the methacrylated gelatin bio-ink.

3. The epicardial patch of claim 1, wherein, The bio-ink containing chitosan nanoparticles loaded with rhVEGF165 and hMSCs is printed into an epicardial patch with a specific structure by 3D bioprinting; after printing, the patch is cured using a blue light source with a wavelength of 405 nm to obtain a methacrylated gelatin (GelMA) epicardial patch containing chitosan nanoparticles loaded with rhVEGF165 and hMSCs, which has a good network structure and a stable three-dimensional scaffold structure.

4. A method of preparing the epicardial patch of any one of claims 1-3, characterized in that, The method comprises the following steps: (1) preparing a GelMA bio-ink and mixing it with chitosan nanoparticles loaded with rhVEGF165 and hMSCs; (2) using a 3D bioprinter to print the mixture into an epicardial patch with a specific structure; (3) performing blue light curing treatment on the printed patch.

5. The method of claim 4, wherein, The chitosan nanoparticles loaded with rhVEGF165 are prepared by the following method: (1) dissolving low molecular weight chitosan in acetic acid solution to form a chitosan solution; (2) under stirring, adding a rhVEGF165 solution and adjusting the pH value to 7.0-7.4 to form chitosan nanoparticles and load rhVEGF165; (3) purifying the obtained chitosan nanoparticles loaded with rhVEGF165 by centrifugation and dialysis.

6. The method of claim 4, wherein, The step of preparing the GelMA bio-ink comprises: (1) dissolving GelMA powder in PBS buffer, adding an initiator LAP, and heating to dissolve under light-proof conditions to obtain a GelMA solution; (2) sterilizing and filtering the obtained GelMA solution and storing it for standby use.

7. The method of claim 4, wherein, The method comprises the following steps: (1) preparing chitosan nanoparticles loaded with rhVEGF165: ① weigh low molecular weight chitosan powder, dissolve it in acetic acid solution, and prepare a chitosan acetic acid solution, and magnetically stir overnight; ② adjust the pH of the acetic acid solution to 5.0 with a sodium hydroxide aqueous solution, then heat the chitosan solution in a 60℃ water bath for 10 minutes, and quickly filter it with a 0.45μm filter to remove insoluble impurities, to obtain a chitosan stock solution; ③ weigh sodium tripolyphosphate (TPP) powder, prepare a 1.5mg / mL TPP aqueous solution, filter the impurities with a 0.22μm filter, and obtain a TPP stock solution; (2) Preparation of methacrylated gelatin (GelMA) bio-ink: ① Take 20 ml PBS and add it to a brown bottle containing 0.05 g initiator LAP (Lithium phenyl-2,4,6-trimethylbenzoylphosphinate); ② Heat to dissolve for 15 minutes in a 40-50°C water bath, shake several times during the process, and avoid light. After dissolving, wrap it in tin paper. A 0.25% w / v initiator standard solution is prepared and stored at 4°C; ③ Put the required amount of GelMA into a 50 ml centrifuge tube, and add the initiator standard solution to the centrifuge tube. Shake it to fully soak and swell; ④ Heat in a 60-70°C water bath for 20-30 minutes, avoiding light. Shake several times during the process to fully dissolve. A GelMA solution is obtained; ⑤ Dissolve the GelMA solution in PBS buffer, then immediately sterilize it with a 0.22 μm sterile filter and filter it into a 15 ml centrifuge tube. A 5% w / v GelMA solution is prepared and stored at -20°C wrapped in tin paper; (3) Mixed materials ① Mix the prepared rhVEGF165-loaded chitosan nanoparticle suspension with 3 ml of GelMA solution, mix thoroughly to form a bio-ink containing rhVEGF165-loaded chitosan nanoparticles, and place it in a 37°C water bath for standby; ② Take the third generation hMSCs with 80%-90% cell fusion, digest them with 0.25% trypsin, and use a cell counter to count the collected cells; (4) 3D bioprinting ​ ​ iii. The hMSCs cell suspension was loaded into three 15ml centrifuge tubes at a ratio of 3x10 6 cells per tube. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded. (4) Take 3 ml of the bio-ink containing rhVEGF165-loaded chitosan nanoparticles, add to the centrifuge tube containing the hMSCs cell pellet, resuspend the cells, mix well, avoid air bubbles, and finally obtain a bio-ink with a concentration of 1 x 10 6 cells / mL, place at 37°C for storage, and use for the next printing step; ​ The bio-ink containing rhVEGF165-loaded chitosan nanoparticles and hMSCs is printed into an epicardial patch with a specific structure by 3D bioprinting; after printing, the patch is cured using a 405 nm blue light source to obtain a methacrylated gelatin (GelMA) epicardial patch containing rhVEGF165-loaded chitosan nanoparticles and human mesenchymal stem cells (hMSCs), which has a good network structure and a stable three-dimensional scaffold structure.

8. Use of the epicardial patch according to any one of claims 1-3 in the preparation of a medical device for treating myocardial infarction.

9. Use according to claim 8, characterized in that, The epicardial patch can effectively promote the recovery of heart function, improve myocardial remodeling, and significantly increase the angiogenesis of the ischemic area.

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