Micro-nano dual-phase structure scaffold for rotator cuff tissue regeneration, preparation method and application thereof

Abstract

The application discloses a micro-nano dual-phase structure scaffold for shoulder rotator cuff tissue regeneration, a preparation method and application. The micro-nano dual-phase structure scaffold comprises an oriented nanofiber membrane in an upper layer and a porous micrometer fiber scaffold in a lower layer of the oriented nanofiber membrane; the oriented nanofiber membrane comprises a first high-molecular polymer-small-molecule drug; the porous micrometer fiber scaffold comprises a second high-molecular polymer, and the porous micrometer fiber scaffold is loaded with one or more of a small-molecule compound, an inorganic mineral and a bioactive ion. The micro-nano dual-phase structure scaffold is particularly suitable for tissue regeneration and repair under a complex interface environment at a rotator cuff insertion. Through the synergistic effect of a biomimetic hierarchical design and biochemical regulation, the micro-nano dual-phase structure scaffold can realize the gradient heterogeneity regulation of an immune microenvironment and induce a biomechanics-biology adaptive response while realizing the differentiation repair function of a tendon and a bone, and finally realizes the biomimetic reconstruction of a rotator cuff injury.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of tissue engineering materials, and particularly relates to a micro-nano dual-phase structure scaffold for rotator cuff tissue regeneration, a preparation method and application thereof. BACKGROUND

[0002] Rotator cuff (RCT) injury is a major component of the family of tendon diseases. Rotator cuff lesions are the main cause of shoulder pain. At present, the most commonly used method for treating rotator cuff injury is arthroscopic rotator cuff repair. Unfortunately, the traditional rotator cuff repair surgery method cannot achieve satisfactory results, especially after large-area rotator cuff injury repair, the incidence of rotator cuff tissue non-healing and rotator cuff re-tear is high, and a considerable proportion of patients may have a tear, resulting in a serious impairment of shoulder joint activity. Therefore, exploring an effective RCT graft has an urgent need to improve the quality of life of patients after surgery and to reduce the burden on families and society.

[0003] At present, many techniques have been studied for repairing torn rotator cuffs. These include various suture techniques, such as single-row and double-row, the use of tissue grafts (allografts, xenografts, autografts, acellular structures), and have shown potential for the reintegration of damaged rotator cuff function. However, due to various reasons, there are still serious complications in these reconstruction schemes at present, such as tissue immune rejection, disease transmission, etc. Moreover, the long-term postoperative immune inflammatory response often leads to the production of scar tissue, and this defective tissue is difficult to bear force load like natural tissue, and cannot restore the function, structure and biomechanical properties of healthy tendons. Therefore, it is of extremely important clinical significance to find a new method that can promote tendon-bone regeneration and repair.

[0004] Tissue engineering is a promising approach to repair damaged tissues or organs due to trauma, injury, disease or aging. By reconstructing regenerative tissues similar to the biomechanics, biochemistry and histology of the natural rotator cuff, it is possible to solve the problem of repairing defective tendons. Electrospinning can produce fibers and porous structures similar to the extracellular matrix of natural tendons. Other advantages include the ability to produce a relatively large surface-to-volume ratio, the ability to control fiber size from the micro to the nanoscale, and the flexibility of material selection. Previous studies have shown that nanofibers with a certain orientation arrangement can mimic the collagen fiber bundles in natural tendons, and the ordered arrangement of nanofibers can increase the tensile strength in the axial direction. Compared with non-oriented scaffolds, oriented electrospun polymer scaffolds have greater anisotropy and are more effective in promoting tendon regeneration. Electrospun nanofiber structures have been shown to promote cell adhesion, proliferation and differentiation in ligament tissue engineering applications. However, this dense fiber structure limits cell migration and penetration, and is not conducive to the transport of oxygen and nutrients and the removal of metabolic waste. On the contrary, 3D printing technology provides a powerful tool for the preparation of tissue engineering porous scaffolds, which can spatially organize different types of materials into predetermined structures for tissue regeneration. 3D printed scaffolds are widely used in tissue repair due to their structural stability and sufficient mechanical strength, good stretchability and flexibility, and the ability to retain the properties of the constituent units. SUMMARY

[0005] The present application provides a micro-nano dual-phase structure scaffold for rotator cuff tissue regeneration, a preparation method and applications thereof. The micro-nano dual-phase structure scaffold of the present application is composed of two functional structure layers, the upper layer is an oriented nanofiber membrane layer, and the lower layer is a micrometer fiber scaffold layer with a three-dimensional porous network structure. Through the synergistic effect of biomimetic hierarchical design and biochemical regulation, the micro-nano dual-phase structure scaffold can realize the differential repair function of tendons and bones, gradient heterogeneity regulate the immune microenvironment, and induce biomechanical-biological adaptive response, and finally realize the biomimetic reconstruction of rotator cuff injury.

[0006] Technical solution: The embodiment of the present application provides a micro-nano dual-phase structure scaffold for rotator cuff tissue regeneration, which comprises an oriented nanofiber membrane in the upper layer and a porous micrometer fiber scaffold in the lower layer of the oriented nanofiber membrane; the oriented nanofiber membrane comprises a first high polymer-small molecule drug; the porous micrometer fiber scaffold comprises a second high polymer, and the porous micrometer fiber scaffold is loaded with one or more of a small molecule compound, an inorganic mineral and a bioactive ion.

[0007] In some embodiments, the micro-nano dual-phase structure scaffold provided by the present application uses an oriented nanofiber membrane structure on the upper layer to promote tendon regeneration, and a porous micron fiber scaffold on the lower layer to promote bone tissue regeneration, thereby promoting rotator cuff tissue regeneration. In some specific embodiments, the upper tendon scaffold uses an oriented nanofiber membrane loaded with PLA-small molecule natural drug grafts, which plays a drug release role, can continuously release anti-inflammatory active ingredients, and long-acting regulates the inflammatory microenvironment of the tendon. At the same time, the oriented fiber structure simulates the fiber arrangement of the natural tendon, can guide the directional growth of tendon cells, and promote the ordered regeneration of tendon tissue. The lower porous micron fiber scaffold is constructed by a melt electrostatic direct writing 3D printing technology, and is a three-dimensional scaffold loaded with bioactive ions, which can provide good bone integration capability.

[0008] The micro-nano dual-phase structure scaffold of the present application forms a biomimetic gradient structure between the layers, realizes the cooperative matching and adaptability reconstruction of the mechanical and biological functions of the tendon-bone interface, and forms a three-dimensional regeneration microenvironment of "bone integration-interface transition-tendon reconstruction" in the gradient interface (interface transition layer thickness 200 μm-800 μm). It is especially suitable for tissue regeneration and repair in the complex interface environment at the rotator cuff stop point. Through the combination of the porous micron fiber scaffold and the nanofiber structure, the defects of the dense accumulation of electrospun nanofiber structure which limits cell migration and penetration, and is not conducive to the transportation of oxygen and nutrients and the removal of metabolic waste are overcome.

[0009] In some embodiments, the diameter of the porous micron fiber scaffold is 15 μm-25 μm. For example, the diameter of the porous micron fiber scaffold is any value or a range consisting of any two values selected from 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 21 μm, 22 μm, 23 μm, 24 μm and 25 μm.

[0010] In some embodiments, the diameter of the oriented nanofiber membrane is 250 nm-350 nm. For example, the diameter of the oriented nanofiber membrane is any value or a range consisting of any two values selected from 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm and 350 nm.

[0011] In some embodiments, the first high molecular polymer in the first high molecular polymer-small molecule drug includes one or more of polyethylene terephthalate (PET), polylactic acid (PLA), polycaprolactone (PCL), lactic acid-glycolic acid copolymer (PLGA), silk fibroin (SF), methacrylated gelatin (GelMA), lactic acid-caprolactone copolymer (PLCL), gelatin, sodium alginate (SA), carboxymethyl cellulose (CMC), chitosan (CS), and hyaluronic acid (HA).

[0012] In some embodiments, the small molecule drug in the first high-molecular polymer-small molecule drug comprises one or more of curcumin, quercetin, resveratrol, and honokiol.

[0013] In some embodiments, the second high-molecular polymer comprises one or more of polyethylene terephthalate (PET), polylactic acid (PLA), polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), silk fibroin (SF), and methacrylated gelatin (GelMA).

[0014] In some embodiments, the small molecule compound comprises one or more of a growth factor, an anti-inflammatory substance, an antioxidant substance, and an anti-adhesion substance.

[0015] In some embodiments, the inorganic mineral comprises one or more of bioactive glass, hydroxyapatite, silicon dioxide, magnesium oxide, zinc oxide, and manganese oxide.

[0016] In some embodiments, the bioactive ion comprises one or more of silicon, phosphorus, calcium, iron, zinc, magnesium, strontium, and manganese ions.

[0017] In some embodiments, the first high-molecular polymer-small molecule drug is prepared by the following method: dissolving the first high-molecular polymer in an organic solvent under nitrogen protection, adding EDCI and DMAP, stirring at room temperature, then adding the small molecule drug, and reacting at room temperature to obtain the first high-molecular polymer-small molecule drug.

[0018] In some embodiments, the first high-molecular polymer-small molecule drug is a PLA-small molecule drug, and the second high-molecular polymer is PLGA. In this application, the PLA oriented nanofiber membrane can simulate the parallel collagen fiber structure of the tendon, provide initial mechanical support and guide the directional arrangement of cells. After the PLGA is combined with bioactive glass, an active surface for calcium and phosphorus deposition is provided, bone integration is accelerated, and the degradation rate of PLA is slow, matching the tendon regeneration period. The degradation rate of PLGA is controllable and can match the speed of bone integration, achieving ideal tendon-bone interface reconstruction and repair. Moreover, the polymers and natural polymers selected in this application are biodegradable materials, which can gradually degrade within a certain period of time, which is conducive to the formation of regenerated rotator cuff tissue. The degradation products can be excreted through normal metabolism of the human body, and there is no any side effect on the human body, achieving ideal tendon-bone interface reconstruction and repair.

[0019] In some embodiments, the porous microfiber scaffold is loaded with the inorganic mineral, and the mass percentage of the inorganic mineral loaded in the total mass of the porous microfiber scaffold is 0-5%.

[0020] In some embodiments, the inorganic mineral is manganese mesoporous bioactive glass.

[0021] In some embodiments, the mass percentage of manganese ions is 1% to 5% based on the total mass of the manganese mesoporous bioactive glass, such as any value or a range between any two values selected from 1%, 2%, 3%, 4%, and 5%.

[0022] In some embodiments, the manganese mesoporous bioactive glass is prepared by the following method: adding tetraethyl orthosilicate, triethyl phosphate, calcium chloride, and manganous chloride in water with CTAB as a template agent, stirring at room temperature, and calcining to obtain the manganese mesoporous bioactive glass.

[0023] In some embodiments, the molar ratio of tetraethyl orthosilicate, triethyl phosphate, calcium chloride, and manganous chloride is 10:2:7:1.

[0024] In some embodiments, the bioactive ions are one or more of calcium ions, iron ions, zinc ions, magnesium ions, strontium ions, and manganese ions.

[0025] In some embodiments, the micro-nano dual-phase structure scaffold has an appearance including but not limited to a long strip, a flat shape, a ring, and a cylinder.

[0026] In some embodiments, the micro-nano dual-phase structure scaffold has a rectangular structure, and in some specific embodiments, the length of the micro-nano dual-phase structure scaffold is 5 mm to 50 mm, and the width of the micro-nano dual-phase structure scaffold is 5 mm to 20 mm.

[0027] In some embodiments, the oriented nanofiber membrane has a thickness of 50 μm to 500 μm, such as any value or a range between any two values selected from 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, and 500 μm.

[0028] In some embodiments, the porous microfiber scaffold has a thickness of 300 μm to 1 mm, such as any value or a range between any two values selected from 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, and 1 mm.

[0029] In some specific embodiments, the upper oriented nanofiber membrane is a nanofiber of a PLA-small molecule natural drug graft prepared by electrospinning, and the lower porous microfiber scaffold is a microfiber of a high molecular polymer loaded with bioactive ions prepared by melt electrostatic direct writing 3D printing.

[0030] In some embodiments, the upper layer of oriented nanofiber membrane and the lower layer of porous microfiber scaffold are assembled by interlayer gradient through bioadhesive, and the mechanical interlocking interface of the biphasic structure is realized through the chemical cross-linking (such as amide bond formation) and physical interlocking (fiber interlocking) of the bioadhesive.

[0031] The embodiment of the present application also provides a preparation method of a micro-nano biphasic structure scaffold for rotator cuff tissue regeneration, comprising the following steps:

[0032] The first high molecular polymer with the added coupling small molecule drug is dissolved in a solvent to obtain a spinning solution, the spinning solution is transferred to a syringe, a high voltage electric field is applied between a spinneret and a high-speed rotating roller, and nanofibers are deposited on the high-speed rotating roller to form an oriented nanofiber membrane.

[0033] The second high molecular polymer and one or more inorganic minerals of the loaded small molecule compound, inorganic mineral and bioactive ion are melted, a negative high voltage electric field is applied between a nozzle and a collection plate, and the melted material is stretched into a porous microfiber scaffold under the action of the electric field.

[0034] The oriented nanofiber membrane and the porous microfiber scaffold are assembled to obtain a micro-nano biphasic structure scaffold for rotator cuff tissue regeneration.

[0035] In some embodiments, the upper layer of tendon scaffold part is prepared by the following method: an oriented nanofiber membrane loaded with a PLA-small molecule drug graft is prepared by using an electrospinning technology combined with a high-speed receiver.

[0036] In some embodiments, the lower layer of bone scaffold part is prepared by the following method: a microfiber scaffold loaded with bioactive ions is prepared by a melt electrostatic direct writing 3D printing technology.

[0037] The upper layer of oriented nanofiber membrane and the lower layer of porous microfiber scaffold are combined to form a micro-nano biphasic structure scaffold, and the micro-nano biphasic structure scaffold realizes functional reconstruction of the tendon-bone interface through the synergistic effect of the upper and lower layers.

[0038] In some specific embodiments, the following preparation steps are included:

[0039] (1) Preparation of polymer spinning solution: a spinning solution loaded with a PLA-small molecule drug graft is prepared; and the second high molecular polymer is preheated and blended with inorganic minerals into a mixture.

[0040] (2) Preparation of oriented nanofiber membrane: the spinning solution of the PLA-small molecule drug graft is loaded into a syringe and installed on a syringe pump, a high voltage electric field is applied, and nanofibers are deposited on a collection plate to form an oriented nanofiber membrane arranged in a certain direction.

[0041] (3) Preparation of three-dimensional printing porous micro-fiber scaffold: the mixture in step (1) is transferred to a 30CC stainless steel dispensing needle cylinder, heated to 155-165℃ to melt, and the molten material is made to enter the screw valve pipe by applying 15-20kPa air pressure, uniformly extruded to the needle tip by screw extrusion, and the fibers are precisely deposited on the collection plate under a negative high voltage electric field of 2.5-3.0kV.

[0042] In some embodiments, in step (1), the mass fraction concentration of the spinning solution is 5%-20%. Further, the mass fraction concentration of the spinning solution is 6%, 8%, 9%, 10.5%, 15%, 20%, and more preferably, the mass fraction concentration of the spinning solution is 10%.

[0043] In some embodiments, in step (1), the mass fraction of inorganic minerals added to the mixture is 0-5%; such as any value or range composed of any two values in 1%, 2%, 3%, 4%, 5% of the mass percentage content of inorganic minerals.

[0044] In some embodiments, in step (1), the solvent for preparing the spinning solution is one or more of hexafluoroisopropanol, trifluoroethanol, dichloromethane, and trifluoroacetic acid.

[0045] In some embodiments, in step (2), the oriented nanofiber membrane process is: the spinning solution is added to a syringe, then a spinning nozzle is connected, a positive (or negative) high voltage electric field of 16kV is applied, the pump speed is 1.6mL / h, the receiving distance is 17cm, and the high-speed receiver is 2500r / min.

[0046] In some embodiments, in step (3), the three-dimensional printing porous micro-fiber scaffold process is: the material is added to a 30CC stainless steel dispensing needle cylinder, then heated to a molten state at 160℃, a negative pressure of 2.7kV is applied, a gas pressure of 15kPa, and the needle tip is received at a distance of 3mm from the conductive glass on the collection plate.

[0047] The application also provides the use of the micro-nano dual-phase structure scaffold for shoulder ligament tissue regeneration or the micro-nano dual-phase structure scaffold prepared by the preparation method of the micro-nano dual-phase structure scaffold for shoulder ligament tissue regeneration in the preparation of shoulder ligament scaffold materials for repairing shoulder ligament damage.

[0048] Beneficial effects: The application provides a micro-nano dual-phase structure scaffold for rotator cuff tissue regeneration, a preparation method and application thereof. The micro-nano dual-phase structure scaffold for rotator cuff tissue regeneration comprises an oriented nanofiber membrane located at an upper layer and a porous micrometer fiber scaffold located at a lower layer of the oriented nanofiber membrane; the oriented nanofiber membrane comprises a first high polymer-small molecule drug; the porous micrometer fiber scaffold comprises a second high polymer, and the porous micrometer fiber scaffold is loaded with one or more of a small molecule compound, an inorganic mineral and a bioactive ion. The micro-nano dual-phase structure scaffold of the application realizes the synergistic effect of the upper anti-inflammatory tendon repair and the lower osteogenic active scaffold, optimizes the regeneration microenvironment of the tendon and bone tissue, reduces postoperative fibrous scar formation, and improves the long-term stability of rotator cuff injury repair. BRIEF DESCRIPTION OF DRAWINGS

[0049] In order to more clearly illustrate the technical solutions in the embodiments of the application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0050] Figure 1 Preparation process schematic diagram of the oriented nanofiber membrane and the porous micrometer fiber scaffold of the application;

[0051] Figure 2 Structure schematic diagram of the micro-nano dual-phase structure scaffold prepared by the application;

[0052] Figure 3 Actual object diagram of the micro-nano dual-phase structure scaffold prepared in Example 1 of the application; wherein, A is the oriented nanofiber membrane, and B is the porous micrometer fiber scaffold;

[0053] Figure 4 Scanning electron microscope schematic diagram of the oriented nanofiber membrane prepared in Example 1 of the application; wherein, the left diagram is the scanning electron microscope schematic diagram of PLA, the middle diagram is the scanning electron microscope schematic diagram of PLA-Que grafting, and the right diagram is the scanning electron microscope schematic diagram of PLA / Que mixing;

[0054] Figure 5 Scanning electron microscope schematic diagram of the porous micrometer fiber scaffold prepared in Example 1 of the application; wherein, the left diagram is the scanning electron microscope schematic diagram of PLGA, the middle diagram is the scanning electron microscope schematic diagram of PLGA@MBG grafting, and the right diagram is the scanning electron microscope schematic diagram of PLGA@Mn-MBG mixing;

[0055] Figure 6Results of cell compatibility of the mesoporous bioactive glass porous micron fiber scaffolds loaded with manganese prepared in Example 1 of the present application to MC3T3-E1 cells using live and dead cell staining test;

[0056] Figure 7 Results of proliferative activity of the mesoporous bioactive glass porous micron fiber scaffolds loaded with manganese prepared in Example 1 of the present application to MC3T3-E1 cells using CCK-8 method;

[0057] Figure 8 Results of cell compatibility of the oriented nanofiber membranes before and after grafting of PLA and quercetin to BMSCs cells using live and dead cell staining test in the present application;

[0058] Figure 9 Results of proliferative activity of the oriented nanofiber membranes before and after grafting of PLA and quercetin to BMSCs cells using CCK-8 method in the present application;

[0059] Figure 10 Results of the ability of the mesoporous bioactive glass porous micron fiber scaffolds loaded with manganese prepared in Example 1 of the present application to induce differentiation of HUVEC in vitro using in vitro tube formation experiment, wherein, A is a schematic diagram of in vitro tube formation experiment, B is total length of blood vessels, and C is number of blood vessel intersections;

[0060] Figure 11 Results of the ability of the mesoporous bioactive glass porous micron fiber scaffolds loaded with manganese prepared in Example 1 of the present application to induce bone differentiation of MC3T3-E1 using BCIP / NBT alkaline phosphatase color developing kit detection;

[0061] Figure 12 Statistical results of the ability of the mesoporous bioactive glass porous micron fiber scaffolds loaded with manganese prepared in Example 1 of the present application to induce bone differentiation of MC3T3-E1 using alkaline phosphatase detection kit;

[0062] Figure 13 Antibacterial rate results of the mesoporous bioactive glass porous micron fiber scaffolds loaded with manganese prepared in Example 1 of the present application, wherein, A is a photograph of colonies on agar plates after the mesoporous bioactive glass porous micron fiber scaffolds loaded with manganese in Example 1 of the present application were applied to Escherichia coli and Staphylococcus aureus, B is the antibacterial rate of the mesoporous bioactive glass porous micron fiber scaffolds loaded with manganese in Example 1 of the present application to Escherichia coli, and C is the antibacterial rate of the mesoporous bioactive glass three-dimensional printed porous micron fiber scaffolds loaded with manganese in Example 1 of the present application to Staphylococcus aureus;

[0063] Figure 14Results of ROS scavenging ability of oriented nanofiber membrane prepared in Example 1 before and after grafting with quercetin evaluated by ROS reactive oxygen species assay kit for this application;

[0064] Figure 15 Results of antioxidant ability of oriented nanofiber membrane prepared in Example 1 before and after grafting with quercetin evaluated by DPPH radical scavenging experiment for this application;

[0065] Figure 16 NMR and infrared spectra of PLA-quercetin graft prepared in Example 1 for this application, wherein the left graph is the NMR spectrum and the right graph is the infrared spectrum. DETAILED DESCRIPTION

[0066] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. In addition, in the description of the present application, the term "comprising" means "including but not limited to". The terms first, second, third, etc. are only used as labels and do not impose numerical requirements or establish sequences. Various embodiments of the present application can exist in a range of forms; it should be understood that the description in a range of forms is only for the convenience and brevity, and should not be understood as a hard limit to the scope of the present application; therefore, it should be considered that the described range has been specifically disclosed all possible sub-ranges and single values in the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers in the range, such as 1, 2, 3, 4, 5 and 6, regardless of the range. In addition, whenever a numerical range is indicated in this text, it means that any cited number (fraction or integer) in the indicated range is included.

[0067] Example 1: As shown in Figure 1 , prepare the tendon scaffold part according to A in Figure 1 : oriented nanofiber membrane loaded with PLA-quercetin graft; prepare the bone scaffold part according to B in Figure 1 : 3D printed porous microfiber scaffold of PLGA (85:15) loaded with manganese mesoporous bioactive glass. The specific preparation process is as follows:

[0068] (1) Preparation of PLA-quercetin graft (PLA-Que): Under nitrogen protection, 3g PLA was dissolved in 40mL dichloromethane, and 0.204g EDCI (CAS: 25952-53-8) and 0.086g DMAP (CAS: 1122-58-3) were added. The mixture was stirred at room temperature for 30min. 0.3g quercetin was dissolved in 10mL anhydrous ethanol and then slowly added to the PLA solution. The reaction was carried out at room temperature for 6h. The reaction solution was precipitated in deionized water overnight. After extraction with dichloromethane, the solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by repeated dissolution and purification with dichloromethane three times and then dried under vacuum to obtain the PLA-quercetin graft. Figure 16 As shown, the NMR and IR spectra of the PLA-quercetin graft are displayed. It can be seen from the figures that the characteristic hydrogen atom on the phenolic hydroxyl group of quercetin disappears, and the NMR spectrum is between 3400 and 3200 cm⁻¹. -1 The stretching vibration of the phenolic hydroxyl group (-OH) disappears at 1582 cm⁻¹. -1 The presence of a double bond characteristic absorption peak indicates successful esterification. The structural formula of the PLA-quercetin graft is as follows:

[0069]

[0070] (2) Preparation of spinning solution: Weigh 1g of PLA with a molecular weight of about 80,000 and dissolve it in 10mL of hexafluoroisopropanol (purchased from Shanghai Darui Fine Chemicals Co., Ltd.) to prepare a PLA spinning solution with a mass concentration of 10%.

[0071] (3) Preparation of spinning solution loaded with PLA-quercetin graft: Weigh 1g of PLA-quercetin graft and dissolve it in 10mL of hexafluoroisopropanol to prepare a PLA-quercetin graft spinning solution with a mass concentration of 10%.

[0072] (4) Preparation of PLA oriented nanofiber membrane: Place the PLA spinning solution from step (2) into a 10 mL syringe and install it on the injection pump. Apply a positive (or negative) high voltage electric field between the spinneret and the collecting plate. The nanofibers are deposited on the high-speed collecting plate to form an oriented nanofiber membrane.

[0073] (5) Preparation of PLA-quercetin grafted nanofiber membrane: The PLA-quercetin grafted spinning solution from step (3) was placed in a 10 mL syringe and installed on an injection pump. A positive (or negative) high voltage electric field was applied between the spinneret and the collecting plate. The nanofibers were deposited on the high-speed collecting plate to form an oriented nanofiber membrane. The actual image is shown below. Figure 3 As shown in Figure A.

[0074] (6) The electrospinning nanofiber process of the above steps (4), (5) is: the spinning solution is added into a syringe, then a spinning nozzle is connected, a positive (or negative) high voltage electric field of 16 kV is applied, the pump speed is 1.6 mL / h, the receiving distance is 17 cm, and the high-speed receiver is 2500 r / min.

[0075] (7) Preparation of Mn bioactive glass (Mn-MBG): CTAB (cetyltrimethylammonium bromide) is used as a template agent to synthesize Mn-MBG by a sol-gel method. The specific experimental steps are: CTAB is added to a beaker, and after adding deionized water, it is stirred at 30°C until it dissolves. Ethyl acetate is added and continues to stir for 30 min, then ammonia water (1 mol / L) is added and stirred for 15 min, then tetraethyl silicate, triethyl phosphate, calcium chloride, and manganous chloride (n / n=10:2:7:1) are added every 30 min, stirred at room temperature for 4 hours, centrifuged at 7800 r / min for 10 min, and the supernatant is discarded. The precipitate is washed with ethanol and deionized water alternately for 3 times, and dried in a constant temperature drying box at 65°C for 24 hours. Mn-MBG is obtained by calcining in a muffle furnace at 650°C for 3 hours by the method of programmed temperature, and then sieved through a 300 mesh sieve and stored in a sealed container. Similarly, mesoporous bioactive glass (MBG) without manganese is prepared.

[0076] (8) Preparation of porous micron fiber scaffold: PLGA (87000-106000 Da) and manganese mesoporous bioactive glass (Mn-MBG) are preheated and blended at a mass ratio of 100:1, then transferred to a stainless steel dispensing needle cylinder, and heated to a molten state; air pressure is applied to the top of the cylinder, and a negative voltage electric field is applied between the needle tip and the conductive glass collection plate, and the receiving distance is controlled to accurately deposit the molten fiber on the conductive glass plate to obtain a porous micron fiber scaffold; the actual object diagram is shown in Figure 3 B of the figure.

[0077] (9) The porous micron fiber scaffold process of the above step (8) is: the material is added to a 30CC stainless steel dispensing needle cylinder, then heated to a molten state at 160°C, a negative voltage of 2.7 kV is applied, and the air pressure is 15 kPa, the receiving distance between the needle tip and the conductive glass on the collection plate is 3 mm.

[0078] (10) Preparation of bioadhesive: PVA solution and PAA solution are obtained as turbid liquid at room temperature by peristaltic pump. The liquid is further centrifuged to collect the gel adhesive, and the pH of the adhesive should be adjusted to 2.0.

[0079] (11) After printing is completed, the prepared scaffold is bonded with the bioadhesive as shown in Figure 2 , then is subjected to ultrasonic cleaning, freeze-drying, and then ethylene oxide sterilization treatment for 24 hours, and finally a micro-nano bidirectional structure scaffold is obtained, which can be used for the rotator cuff tendon-bone interface.

[0080] The scaffold prepared in Example 1 of this application is an oriented nanofiber membrane prepared by electrospinning and a porous microfiber scaffold prepared by three-dimensional printing. The processing is relatively simple, and the length and width of the scaffold can be adjusted according to actual needs.

[0081] like Figure 4 As shown, the scanning electron microscope images of the oriented nanofiber membranes prepared in Example 1 of this application are as follows: the left image is an electrospun fiber membrane prepared by PLA, the middle image is an oriented nanofiber membrane prepared by PLA-quercetin graft (PLA-Que), and the right image is an oriented nanofiber membrane prepared by a mixture of PLA and quercetin. It can be seen from the images that the diameter of the oriented nanofiber membrane prepared by PLA-quercetin graft (PLA-Que) is approximately 300 ± 0.03 nm.

[0082] like Figure 5 As shown, the left image is a scanning electron microscope (SEM) schematic diagram of PLGA, the middle image is a scanning electron microscope schematic diagram of PLGA@MBG grafted PLGA, and the right image is a scanning electron microscope schematic diagram of PLGA@Mn-MBG hybrid PLGA. The diameter of the PLGA@Mn-MBG prepared in this embodiment is 18.2 ± 0.03 μm. Figure 5 As can be seen, the addition of bioactive glass to the 3D porous micro-scaffold has virtually no impact on fiber deposition.

[0083] Example 2: In this example, the tendon scaffold is an oriented nanofiber membrane loaded with PLA-PDA@ZnO; the bone scaffold is a three-dimensional printed porous microfiber scaffold loaded with PCL and hydroxyapatite.

[0084] The specific preparation process is as follows:

[0085] (1) PDA-modified ZnO: ZnO nanoparticles were added to a Tris buffer solution at pH 8.5 and then ultrasonically stirred for 1 hour to obtain a uniform dispersion. Subsequently, DA was added to the above solution and then magnetically stirred at 37°C for 24 hours. The obtained modified nanoparticles PDA@ZnO were centrifuged and repeatedly washed with deionized water and ethanol, and then vacuum dried at 60°C for 24 hours.

[0086] (2) Preparation of spinning solution: Weigh 1g of PLA with a molecular weight of about 80,000 and dissolve it in 10mL of hexafluoroisopropanol (purchased from Shanghai Darui Fine Chemicals Co., Ltd.) to prepare a PLA spinning solution with a mass concentration of 10%.

[0087] (3) Preparation of PLA-PDA@ZnO spinning solution: PDA@ZnO was ultrasonically dispersed in a mixture of 5 mL LCM and DMF. Then, PLA particles were added to the above dispersion and stirred for 12 hours to prepare a PLA-PDA@ZnO spinning solution with a mass concentration of 10%.

[0088] (4) Preparation of PLA oriented nanofiber membrane: PLA spinning solution of step (2) is placed in a 10 mL syringe and installed on a syringe pump, and a positive (or negative) high voltage electric field is applied between the spinneret and the collection plate, and the nanofiber is deposited on the high-speed collection plate to form an oriented nanofiber membrane.

[0089] (5) Preparation of PLA-PDA@ZnO oriented nanofiber membrane: PLA-PDA@ZnO spinning solution of step (3) is placed in a 10 mL syringe and installed on a syringe pump, and a positive (or negative) high voltage electric field is applied between the spinneret and the collection plate, and the nanofiber is deposited on the high-speed collection plate to form an oriented nanofiber membrane.

[0090] (6) Electrospinning nanofiber process of the above steps (4), (5): the spinning solution is added to the syringe, then the spinning nozzle is connected, a positive (or negative) high voltage electric field of 15 kV is applied, the pump speed is 2 mL / h, the receiving distance is 18 cm, and the high-speed receiver is 2000 r / min.

[0091] (7) Preparation of porous microfiber scaffold: PCL and hydroxyapatite are preheated and blended at a mass ratio of 100:1, then transferred to a stainless steel dispensing needle cylinder, and heated to a molten state; a gas pressure is applied at the top of the cylinder, and a negative voltage electric field is applied between the needle tip and the conductive glass collection plate, and the receiving distance is controlled to accurately deposit the molten fiber on the conductive glass plate to obtain a porous microfiber scaffold.

[0092] (8) Process of the above step (7) porous microfiber scaffold: the material is added to a 30 CC stainless steel dispensing needle cylinder, then heated to a molten state at 95°C, a negative voltage of 3 kV is applied, a gas pressure of 17 kPa is applied, and the receiving distance between the needle tip and the conductive glass on the collection plate is 1.5 mm.

[0093] (9) Preparation of bioadhesive: PVA solution (pH 2.0) and PAA solution (pH 2.0) are obtained as a turbid liquid at room temperature by peristaltic pump. The liquid is further centrifuged to collect the gel adhesive, and the pH of the adhesive should be adjusted to 2.0.

[0094] (10) After printing, the prepared scaffold is bonded with bioadhesive, then ultrasonic cleaned and freeze-dried, then subjected to ethylene oxide sterilization for 24 hours, to obtain a micro-nano bidirectional structure scaffold which can be used for shoulder rotator cuff tendon-bone interface. The scaffold in the above example 2 is an oriented nanofiber membrane prepared by electrospinning and a three-dimensional printed porous microfiber scaffold, which is relatively simple to process, and the length and width of the scaffold can be adjusted according to actual needs.

[0095] Calcein-AM / PI cell viability staining method was used to determine the cell compatibility of the oriented nanofiber membrane before and after grafting of PLA and quercetin on BMSCs cells. PLA, PLA-Que, and PLA / Que were placed in a 24-well plate and sterilized by ultraviolet irradiation (wavelength 254 nm) for 30 minutes. 15,000 BMSCs cells were planted in each well and placed in a carbon dioxide incubator for culture. The cell culture plate was taken out at 1, 3, and 5 days, respectively, and the staining working solution was prepared according to the Calcein-AM / PI cell viability staining kit instructions. The staining working solution was added to the sample, incubated at 37°C for 30 min in the dark, and then the staining working solution was discarded. The sample was washed with PBS for three times, and the staining was observed under a fluorescence microscope. The results are shown in Figure 6 The oriented nanofiber membrane had no obvious cytotoxicity to BMSCs cells.

[0096] CCK-8 method was used to test the proliferation activity of the oriented nanofiber membrane before and after grafting of PLA and quercetin on BMSCs cells. PLA, PLA-Que, and PLA / Que were placed in a 24-well plate and sterilized by ultraviolet irradiation (wavelength 254 nm) for 30 minutes. 15,000 BMSCs cells were planted in each well and placed in a carbon dioxide incubator for culture. The absorbance value of each well was measured at 450 nm using CCK-8 reagent at 1, 3, and 5 days, respectively. The results are shown in Figure 7 The oriented nanofiber membrane had no obvious cytotoxicity to BMSCs cells.

[0097] Calcein-AM / PI cell viability staining method was used to determine the cell compatibility of the oriented nanofiber membrane before and after grafting of PLA and quercetin on BMSCs cells. PLA, PLA-Que, and PLA / Que were placed in a 24-well plate and sterilized by ultraviolet irradiation (wavelength 254 nm) for 30 minutes. 15,000 BMSCs cells were planted in each well and placed in a carbon dioxide incubator for culture. The cell culture plate was taken out at 1, 3, and 5 days, respectively, and the staining working solution was prepared according to the Calcein-AM / PI cell viability staining kit instructions. The staining working solution was added to the sample, incubated at 37°C for 30 min in the dark, and then the staining working solution was discarded. The sample was washed with PBS for three times, and the staining was observed under a fluorescence microscope. The results are shown in Figure 8 The results showed that the scaffold had no obvious cytotoxicity to MC3T3-E1 cells.

[0098] CCK-8 method was used to test the proliferation activity of MC3T3-E1 cells on the three-dimensional printing porous microfiber scaffold of manganese-loaded mesoporous bioactive glass. PLGA, PLGA@MBG and PLGA@Mn-MBG were placed in a 24-well plate and treated with ultraviolet irradiation (wavelength 254 nm) for 30 minutes. 15,000 MC3T3-E1 cells were planted in each well and placed in a carbon dioxide incubator. The absorbance value of each well was measured at 450 nm using CCK-8 reagent at 1, 3 and 5 days. The results are shown in Figure 9 The results show that the scaffold has no obvious cytotoxicity to MC3T3-E1 cells.

[0099] The in vitro tube formation experiment was used to evaluate the induction ability of the manganese-loaded mesoporous bioactive glass porous microfiber scaffold on the differentiation of HUVEC. HUVEC was starved for 24 hours using 0.2% FBS-containing 1640 medium before seeding the cells. Matrigel was evenly spread on a pre-cooled 24-well plate, 100 uL of matrigel was added to each well, and the matrigel was solidified in a carbon dioxide incubator at 37°C for 1 hour. HUVEC was seeded on the surface of the matrigel, and the cell density was 1×10 3 After 4 and 8 hours of incubation at 37°C, the tube formation was observed under a microscope and photographed, and Image J was used for quantitative analysis. The results are shown in Figure 10 The total length of the blood vessels in the Control group was 5762.5±294.6 pixels, and the number of blood vessel intersections was 132.3±3.0; the total length of the blood vessels in the PLGA group was 6705.3±284.7 pixels, and the number of blood vessel intersections was 192.8±7.5; the total length of the blood vessels in the PLGA@MBG group was 7267.5±9.2 pixels, and the number of blood vessel intersections was 228.5±128.8; the total length of the blood vessels in the PLGA@Mn-MBG group was 8625.3±299.2 pixels, and the number of blood vessel intersections was 267.5±15.3. The total length of the blood vessels and the number of blood vessel intersections in the PLGA@Mn-MBG group were more than those in the other groups, indicating that the manganese-loaded mesoporous bioactive glass porous microfiber scaffold has a role in promoting angiogenesis.

[0100] The BCIP / NBT alkaline phosphatase color reagent kit was used to detect the bone differentiation ability of the manganese-loaded mesoporous bioactive glass porous micron fiber scaffold to induce MC3T3-E1; PLGA, PLGA@MBG, and PLGA@Mn-MBG were placed in a 24-well plate and sterilized by ultraviolet irradiation (wavelength 254 nm) for 30 minutes. 15,000 MC3T3-E1 cells were planted in each well and placed in a carbon dioxide incubator for culture. After 7 days of cell culture, the cell culture plate was taken out, the old culture solution was discarded, and the cells were washed with PBS three times. 4% paraformaldehyde solution was added to each well to fix the cells for 15 minutes, and then the cells were washed with PBS buffer three times. The staining working solution was prepared according to the BCIP / NBT alkaline phosphatase color reagent kit instructions, and the staining working solution was added to the sample. The sample was incubated at room temperature for 2 hours in the dark until the color developed to the expected depth. Then the staining working solution was removed, and the color development reaction was terminated by washing with distilled water for 1-2 times. Finally, the sample was photographed under a microscope. The results are shown in Figure 11 Fig. 6. The alkaline phosphatase expression of the PLGA@Mn-MBG group was higher than that of the other groups, indicating that the manganese-loaded mesoporous bioactive glass porous micron fiber scaffold had stronger bone differentiation ability to induce MC3T3-E1.

[0101] The alkaline phosphatase detection kit was used to detect the bone differentiation ability of the manganese-loaded mesoporous bioactive glass porous micron fiber scaffold to induce MC3T3-E1. PLGA, PLGA@MBG, and PLGA@Mn-MBG were placed in a 24-well plate and sterilized by ultraviolet irradiation (wavelength 254 nm) for 30 minutes. 15,000 MC3T3-E1 cells were planted in each well and placed in a carbon dioxide incubator for culture. After 7 days of cell culture, the cell culture plate was taken out, the old culture solution was discarded, and the cells were washed with PBS three times. 200 μL of cell lysis solution was added to each well to lyse the cells, and the supernatant was collected by centrifugation (4°C, 5 min, 10,000 rpm). The supernatant was incubated with p-nitrophenyl phosphate at 37°C for 30 min, and the reaction was terminated by adding reaction termination solution. The absorbance of the solution was measured at 405 nm wavelength by an enzyme marker. Finally, the ALP level standard was converted to the total protein amount of the supernatant using the BCA protein determination method. The results are shown in Figure 12 Fig. 7. The specific activity of the Control group (control group) was 157.2±5.1 U / mg, the specific activity of the PLGA group was 268.5±13.6 U / mg, the specific activity of the PLGA@MBG group was 342.7±19.7 U / mg, the specific activity of the PLGA@MBG group was 403.0±11.7 U / mg, and the specific activity of the PLGA@Mn-MBG group was higher than that of the other groups, indicating that the manganese-loaded mesoporous bioactive glass porous micron fiber scaffold had stronger bone differentiation ability to induce MC3T3-E1.

[0102] The in vitro antibacterial effect of the porous micron fiber scaffold loaded with manganese mesoporous bioactive glass was tested by plate coating method. The PLGA, PLGA@MBG and PLGA@Mn-MBG were sterilized by ultraviolet irradiation (wavelength 254 nm) for 30 minutes in a 24-well plate. 1 mL of 1x10 6 CFU / mL bacterial suspension was added to the 24-well plate, and cultured at 37°C and 1000 rpm for 24 hours. After the culture was completed, the bacterial solution was diluted to 1x10 3 CFU / mL, and 100 μL of the diluted bacterial solution was evenly spread on an LB culture dish, which was placed in a 37°C biochemical incubator for overnight culture. After the appropriate size of single colonies were grown in each group, the number of bacteria was counted under a bacterial counter. The results are shown in Figure 13 The E. coli inhibition rate of the PLGA group was 31.7±7.9%, and the S. aureus inhibition rate was 39.3±3.1%; the E. coli inhibition rate of the PLGA@MBG group was 86.5±3.3%, and the S. aureus inhibition rate was 79.1±1.2%; the E. coli inhibition rate of the PLGA@Mn-MBG group was 90.1±3.3%, and the S. aureus inhibition rate was 92.6±1.2%. The porous micron fiber scaffold loaded with manganese mesoporous bioactive glass prepared in Example 1 has good antibacterial effect on E. coli and S. aureus, and the antibacterial effect is better after adding manganese ions.

[0103] The in vivo ROS scavenging capacity of the oriented nanofiber membrane before and after grafting with quercetin was evaluated by ROS active oxygen detection kit; PLA, PLA-Que and PLA / Que were sterilized by ultraviolet irradiation (wavelength 254 nm) for 30 minutes in a 24-well plate, 1 mL of culture solution was added to each well for extraction, 30,000 RAW264.7 cells were planted in each well of a 24-well plate, and the plate was placed in a carbon dioxide incubator for culture. After 24 hours, the old culture medium was discarded, and each group of scaffold extract was replaced and co-cultured with LPS induction. After a certain period of time, the extract was discarded, the DCFH-DA fluorescent probe was diluted according to the ROS active oxygen detection kit instructions, and the staining working solution was configured. An appropriate amount of staining working solution was added to each well, and incubated at 37°C in the dark for 30 min. The fluorescence intensity was observed by fluorescence microscope. The results are shown in Figure 14 The average fluorescence intensity of the control group was 64.66±5.6 RFU, the average fluorescence intensity of the PLA group was 55.7±3.2 RFU, the average fluorescence intensity of the PLA-Que group was 30.6±5.7 RFU, and the average fluorescence intensity of the PLA / Que group was 23.2±5.0 RFU. It is shown that the oriented nanofiber membrane loaded with quercetin has stronger ROS scavenging capacity.

[0104] The DPPH free radical scavenging experiment was used to evaluate the in vitro antioxidant capacity of the oriented nanofiber membrane before and after grafting of PLA and quercetin. 30 mg of PLA, PLA-Que, and PLA / Que oriented nanofiber membranes were sterilized by 3 h of ultraviolet irradiation. The antioxidant capacity of the oriented nanofiber membranes was detected by DPPH. According to the kit instructions, each was immersed in 2 mL of a solution containing 0.039 mg / mL-DPPH and incubated in the dark for 30 min. The OD value at 517 nm was measured by ultraviolet spectrophotometry. The DPPH scavenging rate formula is:

[0105] Scavenging rate(%)=(A0-A) / A0*100%

[0106] A0 is the blank OD value at 517 mm, and A is the sample OD value at 517 nm

[0107] The results are shown in Figure 15 The scavenging rate of the PLA group was 18.9 ± 3.1%, the scavenging rate of the PLA-Que group was 97.1 ± 0.2%, and the scavenging rate of the PLA / Que group was 96.3 ± 0.6%. The oriented nanofiber membrane loaded with quercetin has a higher scavenging rate, indicating that the oriented nanofiber membrane loaded with quercetin has higher antioxidant capacity.

[0108] From the above results, it can be seen that the micro-nano dual-phase structure scaffold of the application can optimize the regeneration microenvironment of the tendon and bone tissue by the synergistic effect of the upper anti-inflammatory tendon repair and the lower osteogenic active scaffold, reduce postoperative fibrous scar formation, and improve the long-term stability of rotator cuff injury repair.

[0109] The above provides a micro-nano dual-phase structure scaffold for shoulder muscle tissue regeneration, a preparation method thereof, and applications thereof. The principles and implementation modes of the application are described using specific examples. The above examples are only used to help understand the method and core idea of the application. For those skilled in the art, the specific implementation modes and application ranges will be changed according to the idea of the application. In summary, the content of the specification should not be understood as a limitation of the application.

Claims

1. A micro-nano biphasic structure scaffold for rotator cuff tissue regeneration, characterized in that, The micro-nano bi-phasic structure scaffold for shoulder rotator cuff tissue regeneration comprises an oriented nanofiber membrane on an upper layer and a porous microfiber scaffold on a lower layer of the oriented nanofiber membrane; the oriented nanofiber membrane comprises a first high polymer-small molecule drug; the porous microfiber scaffold comprises a second high polymer, and the porous microfiber scaffold is loaded with one or more of a small molecule compound, an inorganic mineral and a bioactive ion.

2. The micro-nano bi-phasic structure scaffold for rotator cuff tissue regeneration according to claim 1, wherein, The diameter of the oriented nanofiber membrane is 250-350 nm; and the diameter of the porous microfiber scaffold is 15-25 μm.

3. The micro-nano bi-phasic structure scaffold for rotator cuff tissue regeneration according to claim 1, wherein, The first high polymer in the first high polymer-small molecule drug comprises one or more of polyethylene terephthalate, polylactic acid, polycaprolactone, poly(lactic-co-glycolic acid), silk fibroin, methacrylated gelatin, poly(lactic-co-caprolactone), gelatin, sodium alginate, carboxymethyl cellulose, chitosan and hyaluronic acid. The small molecule drug in the first high polymer-small molecule drug comprises one or more of curcumin, quercetin, resveratrol and magnolol.

4. The micro-nano bi-phasic structure scaffold for rotator cuff tissue regeneration according to claim 1, wherein, The second high polymer comprises one or more of polyethylene terephthalate, polylactic acid, polycaprolactone, poly(lactic-co-glycolic acid), silk fibroin and methacrylated gelatin.

5. The micro-nano bi-phasic structure scaffold for rotator cuff tissue regeneration according to claim 1, wherein, The small molecule compound comprises one or more of a growth factor, an anti-inflammatory substance, an antioxidant substance and an anti-adhesion substance; and / or The inorganic mineral comprises one or more of bioactive glass, hydroxyapatite, silicon dioxide, magnesium oxide, zinc oxide and manganese oxide; and / or The bioactive ion comprises one or more of silicon, phosphorus, calcium, iron, zinc, magnesium, strontium and manganese ions.

6. The micro-nano bi-phasic structure scaffold for rotator cuff tissue regeneration according to claim 1, wherein The first high polymer-small molecule drug is prepared by the following method: The first high polymer is dissolved in an organic solvent under nitrogen protection, EDCI and DMAP are added, stirring is performed at room temperature, then the small molecule drug is added, and reaction is performed at room temperature to obtain the first high polymer-small molecule drug.

7. The micro-nano bi-phasic structure scaffold for rotator cuff tissue regeneration according to claim 1, wherein, The porous microfiber scaffold is loaded with the inorganic mineral, and the mass percentage of the inorganic mineral in the total mass of the porous microfiber scaffold is 0-5%.

8. The micro-nano bi-phasic structure scaffold for rotator cuff tissue regeneration according to claim 1, wherein, The thickness of the oriented nanofiber membrane is 50-500 μm; and the thickness of the porous microfiber scaffold is 300-1 mm.

9. A method for preparing a micro-nano dual-phase structure scaffold for rotator cuff tissue regeneration, characterized in that, The method comprises the following steps: The first high polymer to which the coupling small molecule drug is added is dissolved in a solvent to obtain a spinning solution, the spinning solution is transferred to a syringe, a high voltage electric field is applied between a spinneret and a high-speed rotating roller, nanofibers are deposited on the high-speed rotating roller to form the oriented nanofiber membrane; The second high polymer and one or more of the small molecule compound, the inorganic mineral and the bioactive ion are melted, a negative high voltage electric field is applied between a nozzle and a collection plate, and the melted material is stretched into the porous microfiber scaffold under the action of the electric field; The oriented nanofiber membrane and the porous microfiber scaffold are assembled to obtain the micro-nano bi-phasic structure scaffold for shoulder rotator cuff tissue regeneration.

10. Use of the micro-nano biphase structure scaffold for rotator cuff tissue regeneration according to any one of claims 1-8 or the micro-nano biphase structure scaffold prepared by the method for preparing the micro-nano biphase structure scaffold for rotator cuff tissue regeneration according to claim 9 in the preparation of a rotator cuff scaffold for repairing rotator cuff injury.

Images