Tissue repair biomaterials, preparation methods and applications, and rotator cuff injury repair products

Abstract

The present invention provides a tissue repair biomaterial, preparation method, and application, as well as a rotator cuff injury repair product, relating to the field of biotechnology. The tissue repair biomaterial comprises a ROS-responsive hydrogel with immunomodulatory effects and a nanofiber membrane that promotes bone and cartilage repair. The ROS-responsive hydrogel comprises a hydrogel and M2 macrophage exosomes dispersed within the hydrogel. The fibers comprising the nanofiber membrane have a core-shell structure, with the nuclear layer containing exosomes from bone marrow mesenchymal stem cells. The ROS-responsive hydrogel coats the surface of the nanofiber membrane. This tissue repair biomaterial has the advantages of low immunogenicity and good targeting, enabling the spatiotemporal therapeutic purpose of first modulating the immune-inflammatory microenvironment and then continuously promoting bone and cartilage regeneration.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a tissue repair biomaterial, a preparation method and application thereof, and a rotator cuff injury repair product. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Perfectly repairing a rotator cuff tear remains a challenge in the field of sports medicine. Arthroscopic rotator cuff repair is currently the primary treatment for rotator cuff tears, but the high rate of postoperative re-tear remains a clinical pain point. Commonly used rotator cuff repair patches can provide mechanical support to the damaged rotator cuff tissue, and improved surgical techniques can also help restore the anatomical structure of the rotator cuff injury area. However, at the microscopic level, it remains difficult to repair damage to the tendon-bone interface after a rotator cuff tear, particularly damage to the fibrocartilage zone, the transition zone between bone and cartilage.

[0004] The difficulty in healing the tendon-bone interface after rotator cuff tear is due to a complex pathological process. Studies have found that the tendon-bone interface after injury shows manifestations of immune imbalance such as persistent chronic inflammatory infiltration and increased oxygen free radical pressure, while endogenous bone and cartilage cells are difficult to regenerate and repair under the inflammatory microenvironment. In view of such a complex pathological process, the patches currently available on the market cannot solve it well. With the development of tissue engineering and biomaterials, the combination of new biomaterials and bioactive substances (cells, drugs or organelles) has become a research hotspot. New materials provide a good delivery medium for therapeutic media. However, the application of bioactive substances also has problems such as high immunogenicity, poor targeting, and short duration of action.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The present invention aims to provide a tissue repair biomaterial with low immunogenicity and good targeting, capable of achieving the spatiotemporal therapeutic goal of first modulating the immune-inflammatory microenvironment and then continuously promoting bone and cartilage regeneration. A second objective of the present invention is to provide a rotator cuff injury repair product based on the aforementioned tissue repair biomaterial, capable of continuously promoting bone and cartilage regeneration.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] In a first aspect, a tissue repair biomaterial is provided, comprising a ROS-responsive hydrogel and a nanofiber membrane, wherein the ROS-responsive hydrogel is coated on the surface of the nanofiber membrane;

[0009] The ROS-responsive hydrogel comprises a hydrogel and first nanoparticles dispersed in the hydrogel; the first nanoparticles are microspheres containing second nanoparticles and peroxidase encapsulated by liposomes; the second nanoparticles are microspheres containing M2 macrophage exosomes and a perfluorinated compound, and the shell of the second nanoparticles comprises a lactic acid-glycolic acid copolymer and a polysulfide;

[0010] The fibers constituting the nanofiber membrane are of a core-shell structure, and the nuclear layer contains exosomes derived from bone marrow mesenchymal stem cells.

[0011] In a second aspect, a method for preparing the tissue repair biomaterial according to the first aspect is provided, comprising:

[0012] (A) mixing a perfluorochemical solution containing M2 macrophage exosomes with a lactic acid-glycolic acid copolymer and a polysulfide, emulsifying and ultrasonicating the mixture to form a water-in-oil-in-water double emulsion, separating and collecting the second nanoparticles formed in the system; preparing liposomes containing catalase by a thin film dispersion method, and then mixing and incubating the liposomes with the second nanoparticles to obtain the first nanoparticles;

[0013] (B) loading exosomes onto hydrosol nanoparticles, dispersing the exosome-loaded hydrosol nanoparticles into an electrospinning solution, and obtaining a nanofiber membrane with a core-shell structure by electrospinning, wherein the hydrosol nanoparticles are composed of the water-soluble polymer in an aqueous medium;

[0014] (C) mixing the first nanoparticles prepared in step (A) with the hydrogel, and then placing the nanofiber membrane in the hydrogel to obtain a nanofiber membrane coated with the ROS-responsive hydrogel.

[0015] In a third aspect, there is provided the application of the tissue repair biomaterial of the first aspect or the preparation method of the second aspect in the preparation of tissue or organ damage repair products.

[0016] In a fourth aspect, a rotator cuff injury repair product is further provided, wherein the rotator cuff injury repair product comprises (i) or (ii):

[0017] (i) the tissue repair biomaterial of the first aspect;

[0018] (ii) The ROS-responsive hydrogel described in the first aspect, and the nanofiber membrane described in the first aspect.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The ROS-responsive hydrogel loaded with M2 macrophage exosomes in the tissue repair biomaterial provided by the present invention responds to the high-pressure oxygen free radical environment after implantation at the injury interface, acting as a switch to release the M2 macrophage exosomes within the hydrogel. On the one hand, the ROS-responsive hydrogel reduces the oxygen free radical pressure at the injury interface by binding ROS. The M2 macrophage exosomes regulate immunity by inducing the transformation of M1 macrophages into M2 macrophages. Simultaneously, the M2 macrophage exosomes can inhibit the secretion of inflammatory factors by M1 macrophages, suppressing inflammatory infiltration. Subsequently, the nanofiber membrane slowly releases exosomes derived from bone marrow mesenchymal stem cells, which continuously promotes cell regeneration at the injury site. This tissue repair biomaterial can target the pathophysiological characteristics of local immune imbalance, increased oxygen free radical pressure, and inflammatory factor infiltration after rotator cuff injury. It can effectively target the time sequence of the pathological process of rotator cuff injury, innovatively achieve the priority reduction of oxygen free radical pressure, regulate the immune imbalance environment, and inhibit the infiltration of inflammatory factors to create an environment for subsequent bone and cartilage repair. As a rotator cuff injury repair product, it can continuously promote bone and cartilage regeneration.

[0021] The rotator cuff injury repair product using the above-mentioned tissue repair biomaterials firmly grasps the fundamental factor of immune imbalance in the injured area (mainly manifested as increased oxygen free radical pressure and inflammatory infiltration), and focuses on the fundamental problem of endogenous cell repair dysfunction. Through the treatment concept of first regulating the immune system to suppress inflammation and then continuously promoting cartilage regeneration, the two new biomaterials are combined with exosomes with low immunogenicity, good targeting, and multi-phase therapeutic functions, so as to regulate the immune inflammatory microenvironment and continuously promote cartilage regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 、 Figure 2 and Figure 3 This is a photo of the ROS-responsive hydrogel prepared in Example;

[0024] Figure 4 This is an electron microscope photo of the rice fiber membrane prepared in Example;

[0025] Figure 5 The expression levels of RUX2, ALP, COL1A1 and OCN genes in C3H10 cells at different time points after the rotator cuff patch prepared in the example was co-cultured with C3H10 cells. DETAILED DESCRIPTION

[0026] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0027] In a first aspect, a tissue repair biomaterial is provided. The tissue repair biomaterial includes a ROS-responsive hydrogel and a nanofiber membrane, wherein the ROS-responsive hydrogel is coated on the surface of the nanofiber membrane.

[0028] The ROS-responsive hydrogel includes a hydrogel and first nanoparticles dispersed in the hydrogel; the first nanoparticles are microspheres containing second nanoparticles and peroxidase encapsulated by liposomes; the second nanoparticles are microspheres containing M2 macrophage exosomes (M2-exo) and perfluorochemicals (PFCs), and the shell of the second nanoparticles includes lactic acid-glycolic acid copolymer (PLGA) and polysulfide (PPS).

[0029] The perfluorinated compound referred to herein is Perfluoro-15-crown-5-ether (CAS: 97571-69-2), abbreviated as PFC. PFC has high oxygen solubility and good biocompatibility, and can act as an oxygen carrier. Within hydrogels, it can deliver oxygen to bone defects and responsively release ROS. Nanoparticles containing PFC possess ROS scavenging and sustained oxygen generation capabilities, improving the hypoxic microenvironment and promoting tissue repair and regeneration.

[0030] In an optional embodiment, the M2 macrophage exosomes are derived from the macrophage cell line Thp-1 (human monocytic leukemia) or RAW264.7 (mouse monocytic macrophage leukemia cells).

[0031] In an optional embodiment, the particle size of the second nanoparticles is 150 to 200 nm.

[0032] In an alternative embodiment, the peroxidase comprises catalase (CAT).

[0033] In an alternative embodiment, the hydrogel comprises gelatin methacryloyl hydrogel (GelMA).

[0034] In an optional embodiment, the liposome is composed of phosphatidylcholine (SPC), cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000).

[0035] In an optional embodiment, the first nanoparticles are prepared according to the following method:

[0036] A perfluorinated compound solution containing M2 macrophage exosomes is mixed with lactic acid-glycolic acid copolymer and polysulfide, emulsified and ultrasonically treated to form a water-in-oil-in-water double emulsion, and the second nanoparticles formed in the system are separated and collected; liposomes containing catalase are prepared by a thin film dispersion method, and then mixed and incubated with the second nanoparticles to obtain the first nanoparticles.

[0037] The fibers in the nanofiber membrane of the tissue repair biomaterial are of a core-shell structure, and the core layer of the fibers contains exosomes derived from bone marrow mesenchymal stem cells (BMSCs-exo).

[0038] In an optional embodiment, the nanofiber membrane is an electrospun nanofiber membrane.

[0039] In an alternative embodiment, the exosomes derived from bone marrow mesenchymal stem cells are modified with cholesterol and a chondrocyte-targeting peptide. Cholesterol, due to its lipophilicity, can be embedded in the phospholipid membrane of the exosomes, while the chondrocyte-targeting peptide has the ability to specifically bind to chondrocytes. By attaching these molecules to the exosome surface, the exosomes can be made to target chondrocytes in in vivo or in vitro experiments.

[0040] In an optional embodiment, the targeting of bone marrow mesenchymal stem cell exosomes is increased by using a CAP lipid anchor kit.

[0041] In an optional embodiment, the core layer of the electrospun nanofiber membrane contains a water-soluble polymer loaded with mesenchymal stem cell-derived exosomes.

[0042] In an optional embodiment, the water-soluble polymer includes hyaluronic acid.

[0043] In an alternative embodiment, the shell layer of the electrospun nanofiber membrane contains poly (L-lactic acid) (PLLA) and N,N-dimethylformamide (DMF).

[0044] In an optional embodiment, the electrospun nanofiber membrane is prepared according to the following method: exosomes are loaded on hydrosol nanoparticles, and the hydrosol nanoparticles loaded with exosomes are dispersed in an electrospinning solution, and an electrospun fiber membrane with a core-shell structure is obtained by electrospinning; the hydrosol nanoparticles are composed of the water-soluble polymer in the aqueous medium.

[0045] The ROS-responsive hydrogel portion loaded with M2 macrophage exosomes in the tissue repair biomaterial provided by the present invention responds to the high-pressure environment of oxygen free radicals after implantation at the damaged interface, acting as a switch to release the M2 macrophage exosomes within the hydrogel. A high concentration of oxygen free radicals exists at the damaged interface. When the oxygen free radicals come into contact with the ROS-responsive hydrogel, the peroxidase in the first nanoparticle catalyzes the ROS, triggering a change in the hydrophilicity of the PPS. In the ROS environment, the sulfide portion of the PPS core block is solidified, ultimately destroying the morphology of the second nanoparticle, causing the M2 macrophage exosomes and perfluorinated compounds therein to be released. The ROS-responsive hydrogel reduces the oxygen free radical pressure at the damaged interface by binding to ROS. The M2 macrophage exosomes induce the transformation of M1 macrophages into M2 macrophages, thereby regulating immunity. At the same time, the M2 macrophage exosomes can inhibit the secretion of inflammatory factors by M1 macrophages, thereby suppressing inflammatory infiltration. The nanofiber membrane then slowly releases exosomes derived from mesenchymal stem cells, which continuously promote cell regeneration at the injured site. The nanofiber membrane also provides mechanical strength to the tissue repair biomaterial. The tissue repair biomaterial provided by this invention is particularly suitable for rotator cuff injuries and can effectively target the chronological progression of rotator cuff injury pathology.

[0046] In a second aspect, a method for preparing the tissue repair biomaterial according to the first aspect is also provided, comprising:

[0047] (A) mixing a perfluorochemical solution containing M2 macrophage exosomes with a lactic acid-glycolic acid copolymer and a polysulfide, emulsifying and ultrasonicating the mixture to form a water-in-oil-in-water double emulsion, separating and collecting the second nanoparticles formed in the system; preparing liposomes containing catalase by a thin film dispersion method, and then mixing and incubating the liposomes with the second nanoparticles to obtain the first nanoparticles;

[0048] (B) loading exosomes onto hydrosol nanoparticles, dispersing the exosome-loaded hydrosol nanoparticles into an electrospinning solution, and obtaining a nanofiber membrane with a core-shell structure by electrospinning, wherein the hydrosol nanoparticles are composed of the water-soluble polymer in an aqueous medium;

[0049] (C) mixing the first nanoparticles prepared in step (A) with the hydrogel, and then placing the nanofiber membrane in the hydrogel to obtain a nanofiber membrane coated with the ROS-responsive hydrogel.

[0050] In an optional embodiment, step (B) comprises emulsifying hyaluronic acid and exosomes from bone marrow mesenchymal stem cells into an oil-in-water emulsion, and adding poly-L-lactic acid and N,N-dimethylformamide to the emulsion to obtain an electrospinning solution. During electrospinning, the exosome-loaded hydrosol nanoparticles dispersed in the electrospinning solution aggregate to form a core layer, thereby obtaining the nanofiber membrane having a core-shell structure.

[0051] In an optional embodiment, the electrospinning conditions include: a spinning voltage of 12 kV, which can produce a stable jet stream; a spinning needle diameter of 0.9 mm, a spinning solution flow rate of 0.8 mL / h, which can ensure the continuity and stability of the spinning solution; and a receiving distance of 15 cm, which helps to form uniform fiber deposition.

[0052] In an optional embodiment, the nozzle rotation speed is 200 to 1600 rpm.

[0053] In a third aspect, the present invention provides the application of the tissue repair biomaterial of the first aspect or the preparation method of the second aspect in the preparation of tissue or organ damage repair products.

[0054] In a fourth aspect, a rotator cuff injury repair product is provided, wherein the rotator cuff injury repair product comprises (i) or (ii):

[0055] (i) The tissue repair biomaterial of the first aspect.

[0056] (ii) The hydrogel containing the first nanoparticles of the first aspect and the nanofiber membrane of the first aspect are applied to the affected area by coating the hydrogel containing the nanoparticles on the surface of the nanofiber membrane.

[0057] In an optional embodiment, the rotator cuff injury repair product includes a rotator cuff patch.

[0058] The rotator cuff injury repair product using the above-mentioned tissue repair biomaterials firmly grasps the fundamental factor of immune imbalance in the injured area (mainly manifested as increased oxygen free radical pressure and inflammatory infiltration), and focuses on the fundamental problem of endogenous cell repair dysfunction. Through the treatment concept of first regulating the immune system to suppress inflammation and then continuously promoting cartilage regeneration, the two new biomaterials are combined with exosomes with low immunogenicity, good targeting, and multi-phase therapeutic functions, so as to regulate the immune inflammatory microenvironment and continuously promote cartilage regeneration.

[0059] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be understood as limiting the present invention in any form.

[0060] Example

[0061] Main reagents:

[0062] RAW264.7: Acmec Catalog No.: AC15883-bottle; Phorbol Myristate Acetate (PMA): Acmec Catalog No.: PMA-P33390-1mg; IFN-γ: Sigma-Aldrich 02-0320-00; LPS: Acmec LPS-AC12037-100mg; IL-4: Acmec AC13064-10μg; FBS: Acmec AS9010-100ml; 1640: Acmec 1640-A10491-500ml; PBS: Acmec PBS-AC13317-500ml; DMEM: Acmec DMEM-A12100S-500ml; SD rats: Spafford; PLGA: Acmec P39480-1g; PFC330: Miaoling Bio P13291; PPS: Acmec X67155-1ml.

[0063] Preparation of CAT@Lip:

[0064] 1. Soybean phosphatidylcholine: Acmec; 2. L51990-100mg; 3. Cholesterol: Acmec C70092-200mg; DSPE-PEG2000: ruixibio R-1028-2K; PFC330: Miaoling Bio P13291.

[0065] CAP lipid anchor kit: Lipid Anchor Kit Catalog No. EA-17-1.

[0066] 1. Preparation and Extraction of Macrophage Exosomes

[0067] (1) Preparation of macrophage-derived exosomes: Macrophage culture and differentiation: RAW cell lines were used and treated with 200 nM Phorbol Myristate Acetate (PMA) for 6 hours to generate M0 macrophages. Interferon-γ (IFN-γ) and lipopolysaccharide (LPS) or interleukin-4 (IL-4) / IL-13 were then used to induce the differentiation of M1 and M2 macrophages, respectively.

[0068] (2) Collection of exosomes: After macrophage differentiation, the culture medium was replaced with RPMI 1640 medium supplemented with fetal bovine serum (FBS) to remove exosomes, and the culture was continued for 48 h.

[0069] (3) Initial centrifugation: The culture medium was centrifuged at 2000 g for 20 min to remove debris and dead cells, and then ultracentrifuged at 100,000 g for 90 min to collect exosomes.

[0070] (4) Washing and re-ultracentrifugation: The exosome pellet was washed with phosphate-buffered saline (PBS), then ultracentrifuged again at 100,000 g for 90 min and resuspended in PBS.

[0071] 2. Preparation and Extraction of Exosomes from Bone Marrow Mesenchymal Stem Cells

[0072] (1) Extraction and culture of bone marrow mesenchymal stem cells: BMSCs were extracted from the femur and tibia of 5-week-old Sprague-Dawley (SD) rats and then cultured in DMEM medium containing 10% FBS.

[0073] (2) Exosome collection: When the BMSCs confluency reached 80%, the medium was replaced with exosome-depleted FBS and cultured for another 48 hours. Afterwards, the medium was collected and subjected to a series of centrifugation steps, including 300 × g and 2000 × g, to remove cells and cell debris.

[0074] (3) Ultrafiltration and ultracentrifugation: Filter using a 0.22 μm filter, then centrifuge at 4000 × g in an Amicon ultrafiltration device to a volume of approximately 200 μL. Wash twice with PBS and ultrafilter to a volume of 200 μL.

[0075] (4) Density gradient centrifugation: The filtrate was placed on 30% sucrose / D2O buffer and centrifuged at 100,000×g for 60 minutes using an ultracentrifuge.

[0076] (5) Collection and storage of exosomes: The precipitate containing BMSC-Exos was collected using an 18-G needle, diluted with PBS, and centrifuged at 4000 × g in a centrifugal filter unit to a final volume of 200 μL. BMSC-Exos were stored at −80°C.

[0077] (6) Use the CAP lipid anchor kit to increase the targeting of bone marrow mesenchymal stem cell exosomes and follow the kit instructions. The CAP lipid anchor kit achieves cartilage targeting of exosomes by using specific lipid anchor molecules. These lipid anchor molecules contain cholesterol and fluorescein (FITC)-labeled chondrocyte targeting peptide (CAP). Due to its lipophilicity, cholesterol molecules can be embedded in the phospholipid membrane of exosomes, while CAP peptides have the ability to specifically bind to chondrocytes. By attaching these molecules to the surface of exosomes, exosomes can be made to have the ability to target chondrocytes in in vivo or in vitro experiments.

[0078] 3. Preparation of ROS-responsive hydrogels containing M2 macrophage exosomes (M2-exo) (Part 1)

[0079] (1) Synthesis of M2-exo / PFC@PLGA / PPS nanoparticles: Nanoparticles containing M2-exo and PFC were prepared using a double emulsion (water / oil / water) solvent evaporation method. 11 A 20 μL PFC solution of M2-exo (M2 macrophage-derived exosomes prepared in the above steps) was mixed with PLGA / PPS (2.4 mg, w / w = 1:1) and dissolved in dichloromethane (100 μL). Then, 1 mL of 5% w / v PVA (polyvinyl alcohol) solution was added. The mixture was sonicated in an ice-water bath for 5 minutes using a microtip-equipped ultrasonic processor. An additional 0.6 mL of PVA solution was then added to homogenize the emulsion. The product was then centrifuged at 14,800 rpm at 4°C for 10 minutes and washed several times with purified water to remove the PVA. The M2-exo / PFC@PLGA / PPS nanoparticles were collected and stored in deionized water.

[0080] (2) Synthesis of CAT-PFC@PLGA / PPS@Lip nanoparticles (CPP-L): CAT-loaded liposomes containing SPC, cholesterol, and DSPE-PEG2000 were prepared using a thin film dispersion method. Each component was dissolved in chloroform at a molar ratio of 10:1:3, and the chloroform was removed by drying to obtain a thin lipid film. The film was dispersed in 2 mL of phosphate-buffered saline (PBS) containing 3 mg of CAT and hydrated to form CAT-liposomes. M2-exo / PFC@PLGA / PPS was incubated with CAT-liposomes at 4°C overnight, and CPP-L nanoparticles were collected by centrifugation.

[0081] (3) CPP-L nanoparticles were added to the GelMA solution containing a photoinitiator, and the hydrogel solution was cross-linked by ultraviolet light to form a ROS-responsive hydrogel. The prepared hydrogel was as follows Figure 1 、 Figure 2 and Figure 3 shown.

[0082] Preparation of electrospinning fibers loaded with bone marrow mesenchymal stem cell exosomes (BMSCs-exo) (part 2)

[0083] (1) Preparation of hydrosol solution: First, drug-containing hyaluronic acid (HA) hydrosol nanoparticles were prepared by ultrasonic emulsification.

[0084] Take 100 mg of HA and BMSCs-exo (concentration of 10×10 11 particles / ml).

[0085] HA and BMSCs-exo were added to a solution containing 3 ml of dichloromethane (DCM) with 1% Span-80 as a surfactant (1 mg for every 100 mg of PLLA). Stir vigorously for 25 minutes to form a uniform oil-in-water (W / O) emulsion. Ensure that the stirring speed is high enough to form fine emulsion droplets. PLLA (200 mg) and N,N-dimethylformamide (4 mL) were added to the emulsion until completely dissolved. The amounts of PLLA and DMF should be adjusted based on the viscosity and conductivity of the final electrospinning solution to ensure good electrospinning performance.

[0086] (2) Electrospinning process: Using an electrospinning device, the electrospinning solution was transferred to a 10 mL syringe, the voltage was set to 12 kV, and the solution was applied through a needle with a diameter of 0.9 mm. The flow rate of the electrospinning solution was set to 0.9 mL / h, and the electrospun fibers were collected on a grounded aluminum foil 15 cm away from the needle. Nanofiber membranes were prepared at nozzle speeds of 200 rpm, 400 rpm, 600 rpm, 800 rpm, 1000 rpm, 1200 rpm, 1400 rpm, and 1600 rpm.

[0087] (3) Characterization of fiber membrane: Field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) were used to observe the morphology and internal structure of the fiber membrane. Figure 4 shown. Figure 4 1 to 8 are nanofiber membranes prepared at nozzle speeds of 200 rpm, 400 rpm, 600 rpm, 800 rpm, 1000 rpm, 1200 rpm, 1400 rpm and 1600 rpm, respectively.

[0088] 5. Preparation of rotator cuff patch:

[0089] CPP-L nanoparticles were added to a GelMA solution containing a photoinitiator, the hydrogel solution was combined with the nanofiber membrane by coating, and then cross-linked by ultraviolet light, finally forming a nanofiber membrane wrapped with ROS-responsive hydrogel.

[0090] Effect Examples

[0091] The rotator cuff patch prepared in the above example was co-cultured with mouse mesenchymal stem cells (C3H10), comprising the following steps:

[0092] (1) Trim the rotator cuff patch to 1 cm 2 Big small pieces.

[0093] (2) The materials were cultured together in an incubator (humidity above 95%, temperature 37°C, carbon dioxide concentration 5%).

[0094] (3) Detection time points: 0d, 7d, and 14d. The cells and rotator cuff patch co-culture samples were sent for testing after culturing for 0d, 7d, and 14d. The expression of RUX2, ALP, COL1A1, and OCN genes at 0d, 7d, and 14d were detected by qRT-PCR. The results are shown in Tables 1 to 4 and Figure 5 As shown, it can be seen that the RUX2, ALP, COL1A1 and OCN gene expression levels of C3H10 cells not co-cultured with the rotator cuff patch (0D) are lower than those of C3H10 cells co-cultured with the rotator cuff patch (7D and 14D), and the RUX2, ALP, COL1A1 and OCN gene expression levels increase with the increase of co-culture time, indicating that the rotator cuff patch prepared in the example can promote the expression of RUX2, ALP, COL1A1 and OCN genes related to osteogenic and chondrogenic differentiation.

[0095] Table 1 RUX2 gene expression

[0096]

[0097] Table 2 OCN gene expression

[0098]

[0099] Table 3 COL1A1 gene expression

[0100]

[0101] Table 4 ALP gene expression

[0102]

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A tissue repair biomaterial, characterized in that: It comprises a ROS-responsive hydrogel and a nanofiber membrane, wherein the ROS-responsive hydrogel is coated on the surface of the nanofiber membrane; The ROS-responsive hydrogel comprises a hydrogel and first nanoparticles dispersed in the hydrogel; the first nanoparticles are microspheres containing second nanoparticles and peroxidase encapsulated by liposomes; the second nanoparticles are microspheres containing M2 macrophage exosomes and a perfluorinated compound, and the shell of the second nanoparticles comprises a lactic acid-glycolic acid copolymer and a polysulfide; The fibers constituting the nanofiber membrane are of a core-shell structure, and the nuclear layer contains exosomes derived from bone marrow mesenchymal stem cells.

2. The tissue repair biomaterial according to claim 1, characterized in that: The peroxidase includes catalase.

3. The tissue repair biomaterial according to claim 2, characterized in that: The first nanoparticles are prepared according to the following method: A perfluorinated compound solution containing M2 macrophage exosomes is mixed with lactic acid-glycolic acid copolymer and polysulfide, emulsified and ultrasonically treated to form a water-in-oil-in-water double emulsion, and the second nanoparticles formed in the system are separated and collected; The liposomes containing catalase are prepared by a thin film dispersion method, and then mixed with the second nanoparticles and incubated to obtain the first nanoparticles.

4. The tissue repair biomaterial according to claim 1, characterized in that: The hydrogel includes methacryloyl gelatin hydrogel.

5. The tissue repair biomaterial according to claim 1, characterized in that: The liposomes include phosphatidylcholine, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000.

6. The tissue repair biomaterial according to claim 1, characterized in that: The nanofiber membrane is an electrostatically spun nanofiber membrane.

7. The tissue repair biomaterial according to claim 6, characterized in that: The core layer of the electrospun nanofiber membrane contains a water-soluble polymer loaded with mesenchymal stem cell-derived exosomes.

8. The tissue repair biomaterial according to claim 7, characterized in that: The water-soluble polymer includes hyaluronic acid.

9. The tissue repair biomaterial according to claim 7, characterized in that: The electrospun nanofiber membrane is prepared according to the following method: exosomes are loaded on hydrosol nanoparticles, and then the hydrosol nanoparticles loaded with exosomes are dispersed in an electrospinning solution, and an electrospun fiber membrane with a core-shell structure is obtained by electrospinning; the hydrosol nanoparticles are composed of the water-soluble polymer in the aqueous medium.

10. The tissue repair biomaterial according to claim 6, characterized in that: The shell layer of the electrospun nanofiber membrane contains poly (L-lactic acid) and N,N-dimethylformamide.

11. The tissue repair biomaterial according to claim 1, characterized in that: The exosomes of the bone marrow mesenchymal stem cells are modified with cholesterol and a chondrocyte-targeting peptide.

12. The method for preparing a tissue repair biomaterial according to any one of claims 1 to 11, characterized in that: include: (A) A perfluorinated compound solution containing M2 macrophage exosomes is mixed with lactic acid-glycolic acid copolymer and polysulfide, emulsified and ultrasonically treated to form a water-in-oil-in-water double emulsion, and the second nanoparticles formed in the system are separated and collected; liposomes containing catalase are prepared by a thin film dispersion method, and then mixed and incubated with the second nanoparticles to obtain the first nanoparticles; (B) loading exosomes onto hydrosol nanoparticles, dispersing the exosome-loaded hydrosol nanoparticles into an electrospinning solution, and obtaining a nanofiber membrane having a core-shell structure by electrospinning, wherein the hydrosol nanoparticles are composed of the water-soluble polymer in an aqueous medium; (C) The first nanoparticles prepared in step (A) are mixed with the hydrogel, and then the nanofiber membrane is placed in the hydrogel to obtain a nanofiber membrane coated with the ROS-responsive hydrogel.

13. The preparation method according to claim 12, characterized in that The step (B) comprises emulsifying hyaluronic acid and exosomes of bone marrow mesenchymal stem cells to form an oil-in-water emulsion, and adding poly-L-lactic acid and N,N-dimethylformamide to the emulsion to obtain an electrospinning solution.

14. The preparation method according to claim 13, wherein The electrospinning conditions included: spinning voltage of 12 kV, spinning needle diameter of 0.9 mm, spinning solution flow rate of 0.8 mL / h, and receiving distance of 15 cm.

15. The preparation method according to claim 12, characterized in that: The hydrogel is a methacryloyl gelatin hydrogel, and the preparation method comprises adding the first nanoparticles into a methacryloyl gelatin hydrogel solution containing a photoinitiator, and then performing cross-linking by ultraviolet light irradiation.

16. Use of the tissue repair biomaterial according to any one of claims 1 to 11, or the preparation method according to any one of claims 12 to 15, in the preparation of tissue or organ damage repair products.

17. Rotator cuff injury repair product, characterized in that: The tissue repair biomaterial comprises the tissue repair biomaterial according to any one of claims 1 to 11.

18. The rotator cuff injury repair product according to claim 17, characterized in that: The rotator cuff injury repair product includes a rotator cuff patch.

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