Neutrophils promoting bone formation and angiogenesis, and preparation method and application thereof

Abstract

The present invention discloses neutrophils that promote bone formation and angiogenesis, and a preparation method and application thereof. The repair neutrophils are CD45 + Ly6G + CD54 + CD274 + GLUT1 + The cell subpopulation induced by physical electrical stimulation can play a role in promoting tissue repair. In addition, the preparation process of the repair neutrophils of the present invention is simple and easy to operate, which can achieve mass production and clinical promotion and application.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to application number CN 202310174241.7, filed on February 28, 2023, entitled “A repairing neutrophil and its induced activation method and use”, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to the fields of immunology and regenerative medicine, and in particular to neutrophils for promoting bone formation and angiogenesis, and a preparation method and application thereof. Background Art

[0004] With the deepening of research on the immune system, the regulation of tissue regeneration and repair by immune cells has become a research hotspot in recent years. Studies have shown that the immune response is an important factor in regulating tissue regeneration. The role of immune cells in promoting repair is gradually being recognized. However, current understanding of reparative immune cells mainly focuses on macrophages. Neutrophils are generally considered to be immune cells that clear necrotic tissue and trigger inflammatory responses when tissue damage occurs. However, research on their pro-repair function is relatively limited, resulting in a lack of understanding on how to better utilize the regulatory effects of immune cells to promote tissue repair. Reparative neutrophils may have a significant impact on the clinical repair of damaged tissues.

[0005] Currently, some researchers are using macrophage exosomes to promote the differentiation of bone marrow mesenchymal stem cells into osteoblasts. However, the use of macrophage exosomes to promote the differentiation of bone marrow mesenchymal stem cells into osteoblasts has not been verified in animal studies, and the stability of clinical effects is difficult to guarantee.

[0006] In addition, there are reports using the immunomodulatory effects of human amniotic mesenchymal stem cells to optimize the bone defect microenvironment, promote the aggregation of M2 macrophages that promote vascularized bone regeneration, secrete factors that promote angiogenesis and osteogenesis, and stimulate endogenous bone regeneration. However, the human amniotic mesenchymal stem cells used are derived from fresh placental tissue obtained by cesarean section without infectious diseases. The source is limited and involves the informed consent and ethical requirements of cesarean section patients, thus limiting their clinical promotion and application.

[0007] The information in the background technology is only intended to illustrate the general background of the invention and should not be regarded as an admission or any form of suggestion that this information constitutes the prior art known to a person of ordinary skill in the art. Summary of the Invention

[0008] To solve at least some of the technical problems in the prior art, the present invention provides a method for classifying and inducing the activation of repair-type neutrophils and its use. Specifically, the present invention includes the following contents.

[0009] One aspect of the present invention provides a neutrophil or its isolate that promotes angiogenesis, wherein the neutrophil is CD45 + Ly6G + CD54 + CD274 + GLUT1 + Cell subpopulations that promote angiogenesis following induction by physical electrical stimulation.

[0010] In certain embodiments, the angiogenesis-promoting neutrophils or the isolates thereof according to the present invention, wherein the angiogenesis comprises one of the following properties:

[0011] (1) Increased expression of vascular regulation-related genes or related proteins;

[0012] (2) at least one of the total tube length, number of connection points, and number of lumens increases;

[0013] (3) increased vascular extension length or sprouting;

[0014] (4) The volume of the vascular lumen increases.

[0015] In certain embodiments, according to the angiogenesis-promoting neutrophils or the isolates thereof of the present invention, the vascular regulation-related genes include at least one of CXCL16, MMP9, VEGF, Proliferin and Osteopontin.

[0016] In certain embodiments, the angiogenesis-promoting neutrophils or the isolates thereof according to the present invention, wherein the isolate comprises cell lysates, secretions, or components thereof.

[0017] In one aspect of the present invention, a composition for promoting angiogenesis is provided, comprising the angiogenesis-promoting neutrophils or an isolated substance thereof as described herein.

[0018] In certain embodiments, the composition for promoting angiogenesis according to the present invention comprises in vitro lumen formation, in vivo early vascularization of tissue repair or maturation thereof.

[0019] One aspect of the present invention provides use of the angiogenesis-promoting neutrophils or the isolates thereof described herein in preparing a composition for promoting angiogenesis.

[0020] In one aspect, the present invention provides a method for promoting angiogenesis in vitro, comprising the step of contacting the angiogenesis-promoting neutrophils described herein or the separated substance thereof with cells in vitro.

[0021] In certain embodiments, according to the method for promoting angiogenesis in vitro of the present invention, the angiogenesis includes in vitro lumen formation.

[0022] In certain embodiments, the method for preparing angiogenesis-promoting neutrophils or their isolates according to the present invention comprises separating or screening CD45 + Ly6G + CD54 + CD274 + GLUT1 + The steps of selecting cell subsets or overexpressing CD54, CD274 and GLUT1 in neutrophils by genetic engineering means.

[0023] In certain embodiments, the angiogenesis-promoting neutrophils or the isolate thereof according to the present invention are isolated from bone marrow.

[0024] In certain embodiments, the angiogenesis-promoting neutrophils or the isolate thereof according to the present invention are isolated from peripheral blood.

[0025] In certain embodiments, according to the angiogenesis-promoting neutrophils or separated materials thereof of the present invention, the physical electrical stimulation is 20 μA-70 μA.

[0026] One aspect of the present invention provides a neutrophil or its isolate that promotes bone formation, wherein the neutrophil is CD45 + Ly6G + CD54 + CD274 + GLUT1 + Cell subpopulations that promote bone formation after induction by physical electrical stimulation.

[0027] In certain embodiments, the neutrophils or the isolates thereof according to the present invention, wherein the bone formation comprises one of the following properties:

[0028] (1) Bone regeneration or accelerated bone regeneration;

[0029] (2) New bone formation;

[0030] (3) increased bone volume fraction;

[0031] (4) Increased bone density.

[0032] In certain embodiments, according to the neutrophils or the isolate thereof of the present invention, the bone formation further comprises increasing the expression of vascular regulation-related genes or the amount of related proteins.

[0033] In certain embodiments, according to the neutrophils or the isolate thereof according to the present invention, the vascular regulation-related genes include at least one of CXCL16, MMP9, VEGF, Proliferin and Osteopontin.

[0034] In certain embodiments, the neutrophils or the isolate thereof according to the present invention, wherein the isolate comprises cell lysate, secretion, or a component thereof.

[0035] In certain embodiments, the neutrophils or isolates thereof according to the present invention are characterized in that the electrical stimulation is generated by comprising at least one of the following: a charged matrix, a conductive matrix, direct current, alternating current, pulsed electricity, magnetoelectricity, piezoelectricity or ferroelectric active materials.

[0036] In one aspect of the present invention, a composition for promoting bone formation is provided, comprising the neutrophils or the isolate thereof for promoting bone formation as described herein.

[0037] One aspect of the present invention provides use of the neutrophils or isolates thereof for promoting bone formation according to the present invention in preparing a composition for promoting bone formation.

[0038] In certain embodiments, the method for preparing neutrophils or their isolates that promote bone formation according to the present invention comprises using a reagent to separate or screen CD45 + Ly6G + CD54 + CD274 + GLUT1 + The steps of selecting cell subsets or overexpressing CD54, CD274 and GLUT1 in neutrophils by genetic engineering means.

[0039] In certain embodiments, according to the method for preparing neutrophils or isolates thereof that promote bone formation according to the present invention, the reagent comprises an antibody.

[0040] In certain embodiments, the neutrophils or the isolate thereof for promoting bone formation according to the present invention are isolated from bone marrow.

[0041] In certain embodiments, the neutrophils or the isolate thereof for promoting bone formation according to the present invention are isolated from peripheral blood.

[0042] In certain embodiments, according to the neutrophils or the isolate thereof for promoting bone formation according to the present invention, the physical electrical stimulation is 20 μA-70 μA.

[0043] In one aspect of the present invention, a cell composition for promoting bone repair is provided, which comprises immune cells or isolates thereof and / or endothelial cells or isolates thereof.

[0044] In certain embodiments, according to the cell composition of the present invention, the immune cells are neutrophils.

[0045] In certain embodiments, according to the cell composition of the present invention, the neutrophils are CD45 + Ly6G + CD54 + CD274 + GLUT1 + , and the cell subpopulations activated by physical electrical stimulation.

[0046] In certain embodiments, according to the cell composition of the present invention, neutrophils or their isolates can secrete or produce vascular regulation-related genes and further regulate endothelial cell activation, thereby promoting bone-related cell activation.

[0047] In certain embodiments, according to the cell composition of the present invention, the endothelial cells are activated endothelial cells, and the activated endothelial cells have at least one of the following properties:

[0048] (1) Invasion distance and number of sprouts increased;

[0049] (2) at least one of the total tube length, number of connection points, and number of lumens increases;

[0050] (3) increased vascular extension length or sprouting;

[0051] (4) The volume of the vascular lumen increases.

[0052] In certain embodiments, according to the cell composition of the present invention, the vascular regulation-related gene includes at least one of CXCL16, MMP9, VEGF, Proliferin and Osteopontin.

[0053] In certain embodiments, according to the cell composition of the present invention, the activation of bone-related cells includes initiating, promoting or increasing the secretion of osteocalcin, osteogenic proteins and / or extracellular matrix by osteoblasts, thereby promoting bone formation.

[0054] In certain embodiments, according to the cell composition of the present invention, the bone-related cells include mesenchymal stem cells, osteoblasts and / or osteocytes.

[0055] One aspect of the present invention provides a method for promoting bone formation, comprising the step of regulating endothelial cells to activate bone-related cells by immune cells.

[0056] One aspect of the present invention provides use of a composition for preparing a medicament for promoting bone-related cell activation or bone formation, wherein the composition comprises neutrophils or their isolates and / or endothelial cells or their isolates.

[0057] In certain embodiments, according to the cell composition of the present invention, the neutrophils are isolated from bone marrow.

[0058] In certain embodiments, according to the cell composition of the present invention, the neutrophils are isolated from peripheral blood.

[0059] In certain embodiments, according to the cell composition of the present invention, the physical electrical stimulation is 20 μA-70 μA.

[0060] The present invention also provides an electrical stimulation patch TESP, which can activate CD45 + Ly6G + CD54 + CD274 + GLUT1 + neutrophil subsets, thereby promoting angiogenesis and / or bone healing.

[0061] In certain embodiments, the electrical stimulation patch TESP according to the present invention comprises inorganic ferroelectric particles and piezoelectric polymers.

[0062] In certain embodiments, the electrical stimulation patch TESP according to the present invention comprises barium titanate and P(VDF-TrFE).

[0063] In certain embodiments, the electrical stimulation patch TESP according to the present invention is prepared by the following method: after surface modification of barium titanate nanoparticles with a 0.01-1 mol / L dopamine hydrochloride aqueous solution, the dopamine-modified nanoparticles are embedded in a P(VDF-TrFE) (70 / 30 mol%) matrix, 1-20% BTO nanoparticles and P(VDF-TrFE) solution are dispersed in an N,N-dimethylformamide (DMF) solvent by stirring and ultrasonic treatment to form a stable mixture, and then the mixture is cast into a nanocomposite film, and heated to evaporate the solvent, and the nanocomposite film is treated using corona poling at room temperature.

[0064] The present invention also provides the use of TESP in preparing an electrical stimulation bone healing dressing for activating specific neu1, wherein the specific neu1 is CD45+ Ly6G + CD54 + CD274 + GLUT1 + Neutrophil subsets.

[0065] In certain embodiments, according to the use of the present invention, the TESP is prepared from a piezoelectric or ferroelectric active material.

[0066] The present invention also provides the use of an electrotherapy device in combination with a neutrophil subpopulation in the preparation of a device or system for promoting angiogenesis and / or bone healing, wherein the electrotherapy device includes a module or component capable of providing physical electrical stimulation, wherein the module or component is configured to provide 20μA-70μA of physical electrical stimulation, thereby activating the neutrophil subpopulation and promoting angiogenesis and / or bone healing.

[0067] In certain embodiments, according to the use of the present invention, the neutrophil subpopulation is CD45 + Ly6G + CD54 + CD274 + GLUT1 + neutrophil subsets.

[0068] The technical effects of the present invention include but are not limited to:

[0069] (1) The neutrophil subpopulation with repair function of the present invention is the first immune cell subpopulation discovered that can be induced by electrical stimulation to play a pro-repair function. Under the induction of the electrical microenvironment, the intracellular calcium signaling pathway can be activated, thereby playing a function such as promoting tissue repair.

[0070] (2) The neutrophil subpopulation with repair function of the present invention has a good effect of promoting vascularization and osteogenesis in animals, and can be used for tissue repair treatment, which effectively solves the problem of poor bone defect repair effect in clinical practice.

[0071] (3) The preparation process of the present invention is simple and easy to operate, and can be used for mass production and clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 A cascade reaction is shown for an exemplary "effector" of the invention.

[0073] Figure 2 Representative HE and Masson staining images of the skull defect area of ​​rats 12 weeks after surgery are shown.

[0074] Figure 3-5Therapeutic electrical stimulation patch (TESP) was shown to promote bone regeneration and increase neutrophil infiltration during early bone regeneration.

[0075] Figure 6-7 It was shown that TESP upregulated the expression of chemotaxis-related cytokines by neutrophils.

[0076] Figure 8-14 It was shown that TESP induced the pro-angiogenic function of neutrophils and promoted bone regeneration.

[0077] Figure 15-16 It shows that Neu1 is a unique electrically responsive neutrophil subpopulation that is induced to exhibit vascularization characteristics under the action of electrical signals.

[0078] Figure 17 It shows that the Neu1 neutrophil subset induced by TESP promotes angiogenesis and bone regeneration in vivo, wherein the first two columns in Figures B and D are the control group CON, and the last two columns are the TESP group.

[0079] Figure 18 shows that TESP through Ca 2+ / CaMKII signaling pathway activates the angiogenic function of Neu1.

[0080] Figure 19 It was shown that human- or mouse-derived Neu1 can promote angiogenesis and osteogenesis in response to TESP or direct current (DC).

[0081] Figure 20 It was shown that direct current of different magnitudes induced human Neu1 or mouse Neu1 to promote angiogenesis. DETAILED DESCRIPTION

[0082] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0083] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0084] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related thereto. In the event of any conflict with any incorporated document, the present specification shall prevail. Unless otherwise specified, "%" refers to percentages based on weight.

[0085] Unless otherwise specified, the neutrophil subsets described herein refer to those expressing CD45 + Ly6G + CD54 + CD274 + GLUT1 + Neu1 / Neu2 / 3 / 4 refers to the cell subsets expressing CD45 + Ly6G + CD54 + CD274 + GLUT1 + Other cell subsets, such as but not limited to those expressing CD45 + Ly6G + CD54 - CD274 - GLUT1 - and other cell subpopulations.

[0086] In the present invention, the term "effector" refers to a cascade reaction between a specific subset of activated neutrophils, induced endothelial cells (particularly vascular endothelial cells), and osteoblasts, and this cascade reaction ultimately leads to or promotes angiogenesis and bone formation, thereby playing an important role in tissue repair. This cascade reaction has a certain order or occurrence order, that is, the activated neutrophil subset first secretes or produces vascular regulation-related proteins and / or genes, and then activates vascular endothelial cells, so that the vascular endothelial cell invasion distance and the number of sprouts increase, the total tube length, the number of connection points, and the number of lumens increase at least one, the blood vessel extension length increases or the sprouting increases, the blood vessel lumen volume increases, and the vascular endothelial cell activation further activates bone-related cells, that is, causes bone-related cells including mesenchymal stem cells, osteoblasts and / or osteocytes to start, promote or increase osteoblasts to secrete osteocalcin, osteogenic proteins and / or extracellular matrix, and finally the above-mentioned components are mineralized and deposited to cause bone formation. In certain embodiments, osteoblasts include those cells that secrete osteocalcin, sometimes also referred to herein as "OCN+ cells," wherein osteocalcin is secreted during the differentiation of bone marrow mesenchymal stem cells into osteoblasts.

[0087] Repair neutrophils

[0088] One aspect of the present invention provides an isolated repair neutrophil. The term "isolated" when used to describe one or more neutrophils refers to one or more cells that have been separated from their natural environment, including separation from the object of cell origin (e.g., subject), and / or separation from one or more other components in the natural environment (e.g., fragments, tissues, tissue aggregates, and other cells). Subject refers to a vertebrate, preferably a mammal, including but not limited to rodents, apes, livestock, humans, etc., preferably humans. Neutrophils and their progeny of biological entities obtained in vitro or cultured in vitro are also covered within the scope of protection of the present invention.

[0089] In the present invention, the source of neutrophils is not particularly limited, and includes, but is not limited to, bone marrow, blood (such as peripheral blood), tissue (such as connective tissue), etc. The present invention also discovered that specific neutrophil subpopulations derived from peripheral blood can also serve as reparative neutrophils, thereby promoting angiogenesis and bone formation.

[0090] In certain embodiments, physical electrical stimulation is used to induce specific neutrophil cell subpopulations of the present invention to promote angiogenesis, and it is found that stimulation or activation of such neutrophils can activate intracellular calcium signaling pathways. The inventors found that only the isolated specific neutrophil cell subpopulations (Neu1, State) does not have the function of promoting angiogenesis, but after extraction and separation, it has the function of promoting angiogenesis after electrical stimulation induction. Those skilled in the art will understand that any known materials and methods can also be used to stimulate or activate neutrophils. The elevated calcium in such cells after activation further binds to CaMK II to activate downstream pathways, thereby increasing the expression of vascular regulation-related genes, which include at least one of CXCL16, MMP9, VEGF, Proliferin and Osteopontin, and preferably the expression of all of the above genes or the amount of related proteins. Those skilled in the art are familiar with how to determine the expression of related genes or the amount of related proteins, such as using antibodies or probes that specifically bind to at least one of the above CXCL16, MMP9, VEGF, Proliferin and Osteopontin, or primers designed to amplify the vascular regulation-related genes. In addition, the angiogenesis-promoting effect of the neutrophils of the present invention is also reflected in an increase in at least one of the total tube length, the number of connection points and the number of lumens, an increase in the invasion distance or extension length of new blood vessel sprouts or an increase in sprouting, and an increase in the volume of the vascular lumen.

[0091] In the present invention, activated neutrophils not only promote angiogenesis, but also further promote bone formation. Preferably, bone formation includes but is not limited to the following: (1) bone regeneration or accelerated bone regeneration; (2) formation of new bones; (3) increase in bone volume fraction; and (4) increase in bone density.

[0092] The neutrophils of the present invention further include isolates thereof. Examples of isolates include, but are not limited to, cell lysates (e.g., nucleic acid substances such as RNA), secretions (e.g., polypeptides, proteins, lipids, cytokines, exosomes, etc.), or components thereof. In certain embodiments, further studies of secretions conducted by the present invention have revealed that neutrophil secretions can promote the expression of pro-angiogenic cytokines (including VEGF), promote angiogenesis of endothelial cells in vitro, and promote bone formation.

[0093] In the present invention, examples of physical electrical stimulation include but are not limited to electrical stimulation signals provided by piezoelectric materials, ferroelectric materials, or direct current, alternating current, electromagnetic fields, etc. In certain embodiments, direct physical electrical stimulation can be provided by an electrotherapy device, which can adopt an electrotherapy instrument known in the art, which at least includes a module or component capable of providing a stable current, thereby outputting 20μA-70μA, preferably 20μA-60μA, also preferably 20μA-55μA, further preferably 20μA-50μA, for example, 20μA, 22μA, 24μA, 26μA, 28μA, 30μA, 32μA, 34μA, 36μA, 38μA, 40μA, 42μA, 44μA, 46μA, 48μA, 50μA or any value within the range. The present invention has experimentally demonstrated that the use of the above-mentioned electrotherapy device can activate neutrophil subsets and promote angiogenesis and / or bone healing. It is understood that such an electrotherapy device can be implanted, semi-implanted, or non-semi-implanted for electrical stimulation. Furthermore, the electrotherapy device can have a specific frequency, amplitude, or pulse width to achieve the purpose of combined therapy with activated cell subpopulations and / or activated endothelial cells. The electrotherapy device can stimulate the activated cell subpopulations and / or activated endothelial cells herein to jointly perform repair functions, or promote, enhance, or improve the bone repair or angiogenesis functions that the electrotherapy device itself can achieve.

[0094] In certain embodiments, the electrotherapy device includes an electrical stimulation providing device that provides a stimulation signal having an amplitude and duration, and a stimulation control device electrically connected to an operating element, the stimulation control device including a power supply and a stimulation signal generating circuit. Components comprising the electrotherapy device include, but are not limited to, a single-chip microcomputer, a D / A converter, a programmable timer, a stimulation pulse synthesis device, a power amplifier, a transformer, a stimulation current detection and display unit, and an overload protection device.

[0095] In certain embodiments, the electrotherapy device may include a pulsed electrical signal generating device that generates a pulsed electromagnetic field of a certain frequency, voltage, or pulse.

[0096] In certain embodiments, the electrotherapy device can utilize chemical reactions to convert chemical energy into electrical energy, examples of which include, but are not limited to, dry cell batteries and storage batteries. In certain embodiments, the electrotherapy device can include a DC generator to convert mechanical energy into DC electrical energy through mechanical motion or other forms of energy conversion, including, but not limited to, motors and generators. In certain embodiments, the electrotherapy device can include a rectifier to convert alternating current (AC) into unidirectional DC power after rectification via diodes or capacitors.

[0097] In certain embodiments, physical electrical stimulation based on piezoelectric materials is used to stimulate and activate a subpopulation of cells, comprising the following steps:

[0098] (1) preparing a composite film material comprising barium titanate nanoparticles or modified barium titanate nanoparticles and a ferroelectric polymer P(VDF-TrFE);

[0099] (2) Screening for specific neutrophil subsets: First, mouse bone marrow cells were incubated with specific antibodies at low temperature, wherein the specific antibodies carried fluorescent groups, and then the cells expressing CD45 were sorted by flow cytometry. + Ly6G + CD54 + CD274 + GLUT1 + Neutrophil subsets;

[0100] (3) contacting and culturing the composite membrane material in step (1) with the neutrophils in step (2), thereby activating the intracellular calcium signaling pathway;

[0101] In another exemplary embodiment, neutrophils from mouse bone marrow can be stimulated or activated first, and then cells expressing CD45 can be obtained by flow cytometry sorting. + Ly6G + CD54 + CD274 + GLUT1 + Neutrophil subpopulation, wherein the neutrophils can activate intracellular calcium signaling pathways and further promote angiogenesis.

[0102] In step (1) of the present invention, barium titanate nanoparticles or modified barium titanate nanoparticles and a ferroelectric piezoelectric polymer P(VDF-TrFE) are dispersed in an organic solution and then cast into a film, which is then corona polarized at room temperature to obtain the composite film material of the present invention. The organic solvent is not particularly limited, but is preferably an aprotic polar solvent. Examples thereof include, but are not limited to, one or more of N,N-dimethylformamide, toluene, chloroform, dichloromethane, methanol, and ethyl acetate, with N,N-dimethylformamide being particularly preferred.

[0103] It is understood that the prepared composite membrane material can be used as a culture substrate and / or screening substrate specifically for culturing and / or screening neutrophils, and the substrate can also include necessary components required for culturing or screening neutrophils (such as antibodies, buffers, antibiotics, etc.) and culture devices, such as culture dishes.

[0104] In step (2) of the present invention, bone marrow cells are incubated with screening antibodies at low temperature. The screening antibodies are all commercially available products. The antibodies can bind to the following proteins on the surface or inside of neutrophils: CD45, Ly6G, CD54, CD274 and GLUT1. Examples of the above antibody products include but are not limited to: FITC-CD54, PE-CD274, APC / Cy7-CD45, PE / Cy7-Ly-6G, GLUT1. After incubation, it further includes low-temperature incubation with a secondary antibody. Low temperature refers to a temperature of 0-10°C, preferably 4°C. Subsequently, cells expressing CD45 are sorted by flow cytometry. + Ly6G + CD54 + CD274 + GLUT1 + Neutrophil subsets.

[0105] In step (3) of the present invention, the composite membrane material is contacted with the neutrophil subpopulation and cultured for 1-24 hours, preferably 2-20 hours, more preferably 4-10 hours, and even more preferably 6 hours. The culture conditions are RPMI-1640 culture medium containing 5-15% fetal bovine serum and 0.5-1.2% penicillin-streptomycin, in 5% CO2, at 30-40°C, and preferably at 35-37°C.

[0106] The above method of the present invention further uses direct current to induce neutrophil subpopulations for 0.2-5 hours, preferably 0.5-4 hours, more preferably 0.8-2 hours, and most preferably 1 hour. It is understood that those skilled in the art can choose to induce by physical electrical stimulation based on piezoelectric materials or direct current induction, or the two can be induced successively, for example, direct current induction occurs after induction by physical electrical stimulation based on piezoelectric materials, or direct current induction and induction by physical electrical stimulation based on piezoelectric materials are performed simultaneously.

[0107] Preparation method of repair neutrophils

[0108] One aspect of the present invention provides a method for preparing repair neutrophils, comprising at least one of the following:

[0109] (1) a step of inducing neutrophils to express CD54, CD274, and GLUT1. How to induce neutrophils to express CD54, CD274, and GLUT1 is known in the art, for example, by directly inducing using an inducer or an inducer; or

[0110] (2) a step of causing neutrophils to overexpress CD54, CD274, and GLUT1, for example, introducing a nucleic acid construct carrying the coding genes of the above proteins into a vector, and overexpressing CD54, CD274, and GLUT1 after transformation; or

[0111] (3) A step of isolating repair neutrophils from, for example, peripheral blood or bone marrow cells, for example, using antibodies that specifically bind to CD54, CD274, and GLUT1 as described herein, and then isolating them using separation techniques known in the art, such as sorting using a flow cytometer.

[0112] For the classification method of neutrophils, reagents can be used to detect neutrophil surface or internal marker proteins or their mRNA, and the reagents include biological macromolecules and / or small molecule reagents. Macromolecule reagents include but are not limited to antibodies that can specifically bind to CD54, CD274 and GLUT1, and small molecule reagents include but are not limited to probes that can specifically bind to CD54, CD274 and GLUT1 genes, and / or primers that can amplify them.

[0113] The neutrophils can be further identified by identifying markers CD45 and Ly6G.

[0114] The present invention also provides a method for activating repair neutrophils, comprising the step of stimulating the repair neutrophils described herein using an electrical material. The type of electrical material is not particularly limited, as long as it has electrical properties or piezoelectric activity. Such materials can be found, for example, in CN115252872A, CN114904054A, CN104208754A, CN115286883A, and CN115282345A, which are incorporated herein by reference in their entirety.

[0115] Preparation method of composition for promoting angiogenesis and bone formation

[0116] The present invention also provides a method for preparing a composition for promoting angiogenesis and bone formation, comprising the steps of isolating or screening specific neutrophils, or overexpressing CD54, CD274 and GLUT1 in neutrophils by genetic engineering means, wherein the separation or screening includes the step of using a reagent to detect a marker protein on the surface or inside of the neutrophils or its mRNA, the reagent comprising a biomacromolecule and / or a small molecule reagent, the macromolecular reagent including but not limited to antibodies that can specifically bind to CD54, CD274 and GLUT1, the small molecule reagent including but not limited to probes that can specifically bind to CD54, CD274 and GLUT1 genes, and / or primers that can amplify them. The method of genetic engineering is not particularly limited, and those skilled in the art know how to overexpress CD54, CD274 and GLUT1, for example, by introducing the above-mentioned gene fragment into an expression vector, and overexpressing CD54, CD274 and GLUT1 after transformation into cells. The neutrophils can be further identified by identifying the markers CD45 and Ly6G.

[0117] Methods for promoting angiogenesis and bone formation

[0118] The present invention also provides a method for promoting angiogenesis and osteogenesis, comprising contacting neutrophils or an isolate with bone tissue. The neutrophils described herein can be stimulated or activated using electrical materials or genetic engineering techniques. In certain embodiments, electrical materials are used to provide physical electrical stimulation. The type of electrical material is not particularly limited, as long as it has electrical properties or piezoelectric activity.

[0119] In an exemplary embodiment, the electrical material of the present invention includes a ferroelectric polymer and an inorganic ferroelectric material. In the present invention, examples of the inorganic ferroelectric material include, but are not limited to, one or more of barium titanate, barium strontium titanate, strontium titanate, bismuth ferrite, potassium sodium niobate, and lithium niobate.

[0120] In the present invention, the ferroelectric polymer is not particularly limited, and includes polyvinylidene fluoride or its copolymer, examples of which include but are not limited to polyvinylidene fluoride, polyvinylidene fluoride-hexafluoropropylene, polyvinylidene fluoride-trifluoroethylene, and polylactic acid.

[0121] Example 1

[0122] 1. Experimental Methods

[0123] 1. Electrical materials

[0124] In the examples, a therapeutic electrical stimulation patch (abbreviated herein as "TESP") was used, and a control material without electrical stimulation signal was referred to as CON.

[0125] 2. Animal experiments

[0126] The present invention used 7-week-old male Sprague-Dawley rats and C57BL / 6 mice. The experimental protocol was approved by the Peking University Animal Care and Use Committee (LA2020290). To study the therapeutic effect of TESP on bone defects, a rat skull defect model was established. Rats were anesthetized via intraperitoneal injection, and the dorsal skull was exposed. Two critical-sized full-thickness bone defects (5 mm in diameter) were then created in the center of the skull of each rat. The bone defects were then covered with either the nanocomposite membrane (TESP group) or the non-polarized nanocomposite membrane (CON group).

[0127] To investigate the effects of neutrophils on angiogenesis and osteogenesis in vivo, a specific neutrophil inhibitor, paquinimod, was used. Following skull defect surgery, the TESP group received intraperitoneal injections of paquinimod, while the control group received intraperitoneal injections of saline for four days. At 1, 2, 4, 8, and 12 weeks after implantation, rat skull samples were collected and fixed in paraformaldehyde.

[0128] CT scan

[0129] Bone tissue samples were scanned using a micro-CT scanner and bone volume was analyzed. Images were processed and 3D reconstructions were created using Scanco software.

[0130] 3. Flow cytometry and neutrophil subset sorting

[0131] Bone marrow cells from C57BL / 6 mice were collected and incubated with the following primary antibodies: FITC-CD54 (BioLegend, YN1 / 1.7.4), PE-CD274 (BioLegend, MIH7), APC / Cy7-CD45 (BioLegend, 30-F11), PE / Cy7-Ly-6G (BioLegend, 1A8), Glut (HUABIO, SA0377). The cells were then washed and incubated with the secondary antibody Alexa Fluor 647 (Abcam). Subsequently, dead cells were excluded with 7-AAD (7-AAD; eBioscience). Cells were sorted and analyzed by flow cytometry, and the data were analyzed using FlowJo software (Tree Star).

[0132] Peripheral blood (PB) was collected from healthy volunteers (PKUSSIRB-202388084). After red blood cell (RBC) lysis, PB were incubated for 30 min with the following primary antibodies: APC-eFluor-780-CD11b (eBioscience; ICRF44), PE-Cyanine7-CD66b (eBiocience; G10F5), FITC-CD54 (eBioscience, RR1 / 1), APC-CD274 (eBioscience, MIH1), and GLUT1 (Invitrogen, SA0377). Cells were then washed with PBS and incubated with the secondary antibody Alexa Fluor 594 (Abcam) for 30 min at 4°C. Subsequently, cells were treated with 7-amino-actinomycin D (7-AAD; eBioscience) for 5 min at room temperature to exclude dead cells. Cells were sorted and analyzed by flow cytometry using an Aria 2 or Aria Sorp FACS system, and data were analyzed using FlowJo software.

[0133] To investigate the angiogenic and osteogenic functions of Neu1 (the neutrophil subset of the present invention) in vivo, a mouse calvarial defect model was established. Two critical-size, full-thickness bone defects with a diameter of 3 mm were created to investigate angiogenic functions, and one full-thickness bone defect with a diameter of 1 or 3 mm was created to investigate osteogenic functions. To assess neovascularization, microvascular perfusion was performed with Microfil (MV-112, Flow Tech, Inc., Carver, MA) at 2 or 4 weeks post-surgery.

[0134] 4. Histological Analysis

[0135] Rat skulls (2, 4, 8, and 12 weeks post-surgery) were decalcified, paraffin-embedded, and subjected to CT analysis and histomorphometric analysis. Tissue sections were prepared and stained with hematoxylin-eosin (H&E) and Masson's trichrome. Images were captured using a camera.

[0136] 5. Cell Culture and Conditioned Medium Sample Collection

[0137] HUVECs were purchased from ScienCell Research Laboratories and cultured in endothelial cell medium (ECM; 1001; ScienCell Research Laboratories).

[0138] HL-60 cells were differentiated into granulocyte-like cells (designated HL-60-derived neutrophils, abbreviated as H-neutrophils) by treatment with ATRA (Sigma, R2625). H-neutrophils or flow cytometry-sorted neutrophils were cultured in RPMI-1640 (proce cell). All cells were cultured in a humidified incubator at 37°C with 5% CO2.

[0139] H-neutrophils or sorted neutrophil subsets were cultured on TESP or CON membranes, and the cell culture supernatants were collected for subsequent experiments.

[0140] To investigate the role of calcium influx in the pro-angiogenic function of TESP-Neu1, the specific CaMKII inhibitor KN93 (MedChemExpress) was used. Sorted Neu1 cells were cultured on TESP in RPMI-1640 (proce cell) for 6 hours with or without the addition of KN93. The culture medium was then replaced with RPMI-1640. The cell culture supernatant was collected for subsequent experiments.

[0141] 6. In vitro tube formation test

[0142] The experiments were performed in 24-well plates. Each well was coated with 250 μL of Matrigel. The plates were then placed in an incubator to allow the Matrigel to solidify. Next, HUVECs were plated on the Matrigel. Finally, the 24-well plates were incubated at 37°C in a 5% CO2 air incubator. Tubular structures were observed under a confocal microscope after 4 and 8 hours. Image Pro Plus software was used to quantify the relative total tubule length, number of junctions, and number of tubules per field of view to assess tubule formation.

[0143] 7. In vitro spheroid budding assay

[0144] Gelatin methacryloyl gel was used to embed HUVEC spheroids generated overnight. These were then light-cured and conditioned medium was added. After 12 hours, the spheroids had fully sprouted. The distance of cell invasion and the number of sprouts were digitally quantified using Image Pro Plus software. Approximately 10 spheroids were analyzed for each experimental group.

[0145] 8. Single-cell (10X) RNA sequencing and preprocessing data

[0146] Single-cell RNA-seq libraries were prepared and analyzed using the Chromium platform (10X Genomics). Raw data were aligned to the rat reference genome using Cellranger Count (version 2.0). Single-cell data analysis (including quality control, data normalization, dimensionality reduction, and cluster detection) was performed using Seurat 3 in R.

[0147] 9. Smart-seq2 RNA sequencing and data preprocessing

[0148] Neu1 and Neu2 / 3 / 4 were sorted separately and pooled, then incubated with lysis fractions and RNase inhibitors. Libraries were then prepared using the Smart-Seq2 method. PE150 sequencing was then performed using qualified libraries loaded on the Illumina HiSeq platform.

[0149] A useful Perl script was used to remove adapter-containing reads from the raw data to obtain clean data. Reference gene and genome annotation files were then downloaded from UCSC (http: / / Hg download.SOE.ucsc.edu / golden path / gal gal 4). A reference genome library was constructed using Bowtie2 v2.2.3, and the clean data were mapped to the reference genome using TopHat v2.0.12. Transcriptome data analysis was performed using TopHat software. Read fragments were split into map references to identify exon-exon splice sites.

[0150] 10. Differentially expressed gene analysis and surface protein expression analysis

[0151] Differentially expressed genes were identified by running the "FindAllMarkers" function in Seurat using the "Wilcox test." The surface marker list was downloaded from the Cell Surface Protein Atlas (http: / / wlab.ethz.ch / cspa / ).

[0152] 11. Functional enrichment analysis (GO) analysis

[0153] To clarify the biological significance of the characteristic genes of each cluster, GO analysis was performed on the differential genes of each cluster using the ClusterProfiler package in R.

[0154] 12. Material co-culture, tail vein reinfusion transplantation test and in situ implantation experiment

[0155] To investigate the electrical response potential and angiogenic capacity of Neu1 populations, CD45.2 mice were used as donors for Neu1 and Neu2 / 3 / 4 cells (CD45.2 background). FACS-sorted Neu1 cells or Neu2 / 3 / 4 cells were plated on TESP or CON membranes in RPMI1640 (proce cell) culture medium. After co-culture, cells were collected and then injected into 8-week-old male recipient mice (CD45.2 background) via the tail vein. The recipients were pre-treated with two critical-sized full-thickness bone defects (1 mm diameter), and the defect area was covered with CON membrane.

[0156] To investigate whether the pro-angiogenic and pro-osteogenic functions of Neu1 could be induced by direct current (DC), DC- or TESP-induced Neu1 cells were collected and embedded in Gelma (Corning) before orthotopic implantation into the skull defect of mice. Equal numbers of cells were implanted in each group.

[0157] Angiogenesis and bone regeneration were examined 2 and 12 weeks after transplantation, respectively, as described previously.

[0158] 13. Enzyme-linked immunosorbent assay (ELISA)

[0159] Cell culture supernatants were collected as described previously. ELISA kits for CXCL1 (Solarbio, SEKH-0065), CXCL3 (FineTest, EH3179), CCL3 (Solarbio, SEKH-0246), MMP9 (FineTest, EH0238), and VEGF (Solarbio, SEKH-0052, SEKM-0039) and a mouse angiogenesis array kit (R&D Systems, ARY015) were purchased and ELISA assays were performed according to the instructions of their respective manufacturers.

[0160] 14. Immunohistochemistry

[0161] After CT analysis, the rat skull was decalcified for three weeks. Tissue samples were then embedded in paraffin (bulk histology) and sample sections were prepared. Subsequently, the samples were dewaxed, dehydrated, and subjected to antigen retrieval in a microwave oven. The slides were then washed with PBS, blocked, and incubated with the primary antibody. Subsequently, the slides were washed with PBS and incubated with the secondary antibody at room temperature. The buffer was heated to thoroughly elute the primary and secondary antibodies. In the same manner, the two antigens were labeled with different fluorophores. Cell nuclei were finally stained with DAPI. Images were taken under a confocal laser scanning microscope (Leica). The primary antibodies used were as follows: CD34 (Abcam) and OCN (Proteintech).

[0162] 15. Calcium influx detection

[0163] To investigate whether calcium influx in Neu1 cells is caused by TESP, cells were stained with the fluorescent calcium probe Fluo-4, and intracellular calcium signals were monitored. Briefly, sorted Neu1 cells were cultured on TESP membranes, stained with Fluo-4 at 37°C in the dark, washed, and then suspended in RPMI1640 medium. Fluorescence was then measured using an Aria Sorp (BD Biosciences). Images were captured under a confocal microscope and quantitatively analyzed using Image J software.

[0164] 16. Statistical analysis

[0165] Results are expressed as mean ± SD or mean ± SEM. Unpaired two-tailed t-tests were used for two samples, Mann-Whitney test for non-normally distributed data, and one-way ANOVA for groups with more than two samples. P < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0166] 2. Results

[0167] Figure 1 The cascade reaction of an exemplary "effector" of the present invention is shown. A therapeutic electrical stimulation patch (TESP) is used to cover a critical-sized bone defect model. In the early stages of bone regeneration, TESP attracts more neutrophils (Neu1, neutrophil CD45) through electrotaxis. + Ly6G + CD54 + CD274 + GLUT1 + These recruited neutrophils highly express chemokines such as CXCL3, CCL3, and CXCL1, and accelerate bone regeneration by secreting cytokines such as VEGFA to promote angiogenesis.

[0168] Figure 2 Representative HE (a) and Masson's staining (b) images of the rat skull defect area 12 weeks after surgery show that TESP promotes bone regeneration. (MC: medullary cavity. FT: fibrous tissue. NB: newly formed bone. OT: osteoid tissue. MT: mineralized tissue)

[0169] Figure 3-5 It was shown that TESP promoted bone regeneration and increased neutrophil infiltration during the early stages of bone regeneration. Figure 3(a) Representative CT images of rat bone defects 12 weeks after TESP implantation. The dotted line represents the defect boundary. (b) The relative bone volume fraction (BV / TV) and bone mineral density (BMD) in the TESP group were significantly higher than those in the control group. Figure 4 The t-SNE plots in the upper part show the immune cell populations in the CON group (left) and the TESP group (right). Figure 4 The figure below shows the relative distribution ratio of each cell population at different time points. Figure 5 The figure above shows the relative distribution ratio of myeloid cells and lymphoid cells at different time points. Figure 5 The figure below shows the percentage of each cell subset in myeloid cells, indicating that TESP promotes bone regeneration and increases the proportion of neutrophils in the early stage (day 3) of bone regeneration.

[0170] Figure 6-8 It was shown that TESP-recruited neutrophils promoted bone regeneration. Figure 6 (a) Heat map of the top 20 differentially expressed genes in neutrophils on day 3 after bone defect. (b) Violin plot of chemotaxis-related genes highly expressed in neutrophils in the TESP group on day 3 after bone defect. (c) GSEA plot showing the enriched chemotaxis-related signaling pathways in neutrophils in the TESP group on day 3 after bone defect. Figure 7 ELISA experiments showed that the levels of CXCL3, CCL3 and CXCL1 in the culture supernatant of neutrophils in the TESP group were significantly higher than those in the control group (n=3). Figure 8 (a) Representative CT images of rat calvarias 12 weeks after surgery. The dotted line represents the defect boundary. (b) At 12 weeks after surgery, the volume of newly formed bone was significantly lower in the inhibitor group compared with the TESP group (n = 7). (c) Representative Masson-stained images of rat calvarial defects 12 weeks after surgery show that the bone defect area in the inhibitor group exhibited incomplete, immature bone tissue repair, while the bone defect area in the TESP group was completely repaired by mature bone tissue. (MC: medullary cavity; FT: fibrous tissue; NB: newly formed bone; OT: osteoid tissue; MT: mineralized tissue).

[0171] Figure 9-14 It was shown that TESP induces the pro-angiogenic and osteogenic functions of neutrophils. Figure 9 (a) Violin plots show that angiogenesis-related genes were upregulated in neutrophils of the TESP group on day 3 after surgery. (b) ELISA analysis showed that the expression levels of MMP9 and VEGF were higher in TESP compared with the control group, n = 3. Figure 10 and Figure 11 The upper panel shows 2D and 3D spheroid sprouting experiments of HUVECs in neutrophil-conditioned medium induced by the material. Figure 10The lower panel shows quantitative analysis of tubule formation, showing that the relative total length of blood vessels, the number of junctions, and the number of tubules increased in the TESP group (n=3). Figure 11 The lower panel shows quantitative analysis of 3D sprouting angiogenesis, showing that the TESP group increased the invasion distance and number of sprouts (n=10). Figure 12 Representative immunohistochemical staining at day 7 after surgery (left panel) shows a decrease in the number of CD34+ vascular endothelial cells after administration of a neutrophil inhibitor. The dotted line indicates the bone defect area. Figure 12 The right graph in the upper column shows that the relative fluorescence intensity of CD34 was significantly decreased in the neutrophil inhibitor-administered group compared with the TESP group (n=20). Figure 12 The lower panel shows representative CT images of a neutrophil inhibitor-induced reduction in neovascularization. The dotted line indicates the defect boundary. Figure 13 Quantitative analysis showed that treatment with neutrophil inhibitors in i resulted in a decrease in vascular volume. Figure 14 The left panel shows representative immunohistochemical staining of rat calvarial defects 8 weeks after surgery. Neutrophil inhibitors significantly reduced the number of CD34+ and OCN+ cells. The dotted lines indicate the indicated tissues. Figure 14 In the right figure, neutrophil inhibitors significantly reduced the relative fluorescence intensity of CD34 and OCN (n = 20), suggesting that neutrophil inhibitors significantly inhibited the angiogenic and osteogenic effects of TESP.

[0172] Figure 15-16 Neu1 is shown to be a unique electrically responsive neutrophil subset capable of promoting angiogenesis. Figure 15 (a) t-SNE plot showing four neutrophil subsets (Neu1, Neu2, Neu3, and Neu4). (b) Histogram of angiogenesis (GO:BP) pathway enrichment, showing that the angiogenesis pathway is more enriched in Neu1 in the TESP group than in other subsets. (c) Boxplot of angiogenesis scores for each subset, showing that the angiogenesis-related score of Neu1 in the TESP group is significantly higher than that of other subsets, suggesting that Neu1 in the TESP group has pro-angiogenic capacity. (d) Violin plot showing that the angiogenesis score of Neu1 is higher in the TESP group compared to the control group. (e) TESP increased the proportion of the Neu1 subset on day 3 after surgery. Figure 16 (a) Bubble plot showing highly expressed cell surface marker genes in each neutrophil subpopulation. (b) t-SNE shows that CD274, Icam1, and Slc2a1 are enriched in the Neu1 subpopulation. (c) ELISA assay demonstrates that TESP significantly promotes VEGF secretion by Neu1.

[0173] Figure 17Figure 3: Neu1 neutrophils induced by TESP promote angiogenesis and bone regeneration in vivo. (A) Representative microCT images of the microfiber-perfused vasculature of mouse skulls 2 weeks after surgery. The dotted line indicates the bone defect area (diameter: 3 mm). (B) Quantitative analysis of the volume of new blood vessels in A, n = 9. (C) Representative microCT images of mouse skulls 12 weeks after surgery. The dotted line indicates the bone defect area (diameter: 1 mm). (D) Quantitative analysis of new bone volume (BV) and bone mineral density (BMD) (n ≥ 6) in C (**p < 0.01, ***p < 0.001, ****p < 0.001).

[0174] Figure 18 shows that TESP through Ca 2+ / CaMKII signaling pathway activates Neu1 to promote angiogenesis. Figure 18 (a) Heatmap of differentially expressed genes between naive Neu1 and Neu2 / 3 / 4, indicating that naive Neu1 overexpresses genes involved in voltage-dependent ion channels. (b) Representative immunofluorescence images of Fluo-4 (calcium probe) in each subpopulation. Dashed lines indicate cells. (c) Quantitative analysis of Fluo-4 fluorescence intensity in b, indicating a significant increase in intracellular calcium concentration in Neu1 cells after electrical induction (n = 50). (d) Representative flow cytometry analysis of Fluo-4 (calcium probe) in each subpopulation. (e) Quantitative analysis of Fluo-4 fluorescence intensity in d, indicating a significant increase in intracellular calcium concentration in Neu1 cells after electrical induction (n = 3). (f) ELISA assay for angiogenesis-related proteins, performed on culture supernatants of TESP-neu1 cells treated or not with the calcium signaling pathway inhibitor KN93. (g) Quantitative analysis of the expression levels of the indicated vascular regulation-related genes, indicating that the calcium signaling pathway inhibitor significantly inhibited the angiogenesis-promoting ability of TESP-neu1 cells after induction (n = 4). (h) Schematic diagram of the tube formation assay and (j) 3D spheroid sprouting angiogenesis assay. (i) Quantitative analysis of total tube length, number of junctions, and number of tubules in h (n = 3) indicates that calcium signaling inhibitors significantly inhibit the angiogenic capacity of neu1 after TESP induction. (k) Quantitative analysis of invasion distance and number of newly formed vascular sprouts in j (n ≥ 9) indicates that calcium signaling inhibitors significantly inhibit the angiogenic capacity of neu1 after TESP induction.

[0175] Example 2

[0176] HUVECs were cultured in endothelial cell culture medium. HL-60 cells were incubated with ATRA (Sigma, R2625, 1 μmol / L) for 5 days to differentiate into granulocyte-like cells (designated HL-60-derived neutrophils). HL-60-derived neutrophils (H-neutrophils) and sorted mouse-Neu1 (mNeu1), mouse-Neu2 / 3 / 4 (mNeu2 / 3 / 4), human-Neu1, and human-Neu2 / 3 / 4 [hNeu2 / 3 / 4] were cultured in RPMI-1640 (Procell) supplemented with FBS and penicillin / streptomycin solution. All cells were cultured in a humidified incubator at 37°C with 5% CO2.

[0177] H-neutrophils or sorted mouse Neu1 (mNeu1), mouse Neu2 / 3 / 4 (mNeu2 / 3 / 4), human Neu1 (hNeu1), and human Neu2 / 3 / 4 [hNeu2 / 3 / 4] were cultured on TESP or CON membranes for 6 hours. Isolated neutrophil subsets from mice or humans were induced with DC for 1 hour to characterize the general response of Neu1. Cell culture supernatants were collected for subsequent experiments. Human bone marrow mesenchymal stem cells (hBMSCs) were purchased from Cyagen Biosciences and cultured in α-MEM (Procell) containing 10% (v / v) FBS and 1% (v / v) penicillin / streptomycin.

[0178] The results are as follows Figure 19(A) Schematic diagram of DC stimulation. (B) HUVEC angiogenesis in mouse-Neu1 conditioned medium. Arrows indicate junctions and lumens. (C) Quantitative analysis of angiogenesis in B shows increased relative total vessel length, number of junctions, and number of lumens in the mNeu1 group treated with TESP or DCs, n = 3. (D) Representative microCT images of mouse calvarias 2 weeks after surgery are shown. The dotted line indicates the defect boundary (diameter: 3 mm). (E) Quantitative analysis of neovascularization volume is shown, indicated by D, n = 10. (F) Representative microCT images of mouse calvarias 12 weeks after surgery are shown. The dotted line indicates the defect boundary (diameter: 3 mm). (G) Quantitative analysis of new bone volume (BV) (n = 10) and bone mineral density (BMD) (n = 10) are shown in F. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (H) Schematic diagram of the human Neu1 enrichment strategy. (I) Shows angiogenesis of HUVECs in human-Neu1-conditioned medium. Arrows indicate junctions and lumens. (J) Quantitative analysis of angiogenesis in Figure 1 shows increased relative total tube length, number of junctions, and number of lumens in the hNeu1 group treated with TESP or DCs (n = 3). (K) Representative immunofluorescence images of hBMSCs. (L) Shows that the mean RUNX2 intensity in the hNeu1 group treated with TESP or DCs was significantly higher than that in the CON-hNeu1 group (n = 12). (****p < 0.0001).

[0179] Figure 20 This figure shows that direct currents of varying magnitude can induce angiogenesis in human or mouse Neu1. (A) HUVEC angiogenesis in mouse Neu1-conditioned medium. Arrows indicate junctions and lumens. (B) Quantitative analysis of angiogenesis in A shows increased relative total tube length, number of junctions, and number of lumens in the TESP group. n = 3.

[0180] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various modifications and variations may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims is to be given the broadest interpretation so as to encompass all such modifications and equivalent structures and functions.

Claims

1. A neutrophil that promotes angiogenesis and / or osteogenesis, characterized in that: The neutrophils are CD45 + Ly6G + CD54 + CD274 + GLUT1 + The cell subpopulation is treated with 20 μA-30 μA of physical electrical stimulation, and promotes angiogenesis and / or bone formation after physical electrical stimulation.

2. The neutrophil for promoting angiogenesis and / or osteogenesis according to claim 1, characterized in that The electrical stimulation is generated by at least one selected from the group consisting of a charged substrate, a conductive substrate, a direct current, an alternating current, a pulsed current, a magnetoelectric, a piezoelectric, and a ferroelectric active material.

3. A composition for promoting angiogenesis and / or osteogenesis, characterized in that: It comprises the neutrophils according to claim 1 or 2.

4. A method for promoting angiogenesis in vitro, characterized in that: A step of contacting the neutrophils according to claim 1 or 2 with cells in vitro.

5. The method for promoting angiogenesis in vitro according to claim 4, characterized in that: The angiogenesis includes in vitro tube formation.

6. The method for preparing neutrophils according to claim 1 or 2, characterized in that: Including the use of reagents to isolate or screen CD45 + Ly6G + CD54 + CD274 + GLUT1 + The steps of treating the cell subpopulation with physical electrical stimulation of 20 μA-30 μA, or overexpressing CD54, CD274 and GLUT1 in neutrophils by genetic engineering means and treating the cell subpopulation with physical electrical stimulation of 20 μA-30 μA.

7. The preparation method according to claim 6, characterized in that The neutrophils are isolated from bone marrow.

8. The preparation method according to claim 6, characterized in that The neutrophils are isolated from peripheral blood.

9. The preparation method according to claim 6, characterized in that The reagents include antibodies.

10. Use of the neutrophils according to any one of claims 1 or 2 in preparing a composition for treating bone defects.

11. Use of an electrotherapy device in combination with a neutrophil subset in the preparation of a device or system for promoting bone healing, characterized in that: The electrotherapy device includes a module or component capable of providing physical electrical stimulation, wherein the module or component is configured to provide 20μA-30μA of physical electrical stimulation, thereby activating a neutrophil subpopulation and promoting bone healing, and the neutrophil subpopulation is the neutrophil described in claim 1 or 2.

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