Tissue engineering nerve conduit as well as preparation method and application thereof

Abstract

The invention relates to a tissue engineering nerve conduit and a preparation method and application thereof. The tissue engineering nerve conduit comprises polyhydroxyalkanoate porous microspheres and a conduit, the polyhydroxyalkanoate porous microspheres are loaded in the conduit, and the conduit is made of a polyhydroxyalkanoate fibrous membrane. The porous microspheres and the fiber membrane are combined to form the composite nerve conduit, the composite nerve conduit can simulate the structure and function of autologous nerves, an ideal microenvironment is provided for growth and regeneration of nerve cells, the nerve conduit has the capacity of promoting growth and regeneration of the nerve cells, and the preparation method is simple, easy to operate and suitable for industrial production. Wide application prospects are realized in the fields of tissue engineering, neural restoration and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of tissue engineering and regenerative medicine, and relates to a tissue engineering nerve conduit and its preparation method and application, and in particular to a long-distance tissue engineering nerve conduit based on polyhydroxyalkanoate three-dimensional porous microspheres and nerve conduits. Background Art

[0002] Peripheral nerve injury is a common complication of various diseases such as trauma and diabetes. The incidence rate is increasing every year, seriously affecting the quality of life of patients. At present, the main methods used in clinical repair of peripheral nerve injury are autologous nerve transplantation and nerve conduit bridging. However, autologous nerve transplantation has problems such as limited donor nerve sources and impaired donor site function, which limits its widespread application. Although nerve conduit bridging has solved the problem of donor nerve sources to a certain extent, it still has shortcomings in the efficiency and quality of guiding nerve regeneration, especially for long-distance and complex nerve injuries. The repair effect is not ideal.

[0003] Porous microsphere carriers, with their large specific surface area and small volume, provide a broad platform for seed cell loading and promote cell attachment and proliferation. At the same time, the porous structure is conducive to the penetration of nutrients and the excretion of metabolites, creating more space for cell proliferation and tissue regeneration. The nerve guide catheter prepared by ordered electrospinning membrane has an orderly arranged surface topology, which can simulate the extracellular matrix of nerve cells, provide a natural growth environment for nerve cells, and promote the orderly arrangement and regeneration of nerve axons. However, existing nerve catheter materials such as poly(lactic acid) glycolide copolymer (PLGA) and polylactic acid (PLA) will produce an acidic microenvironment during the degradation process, aggravating myelin disintegration and triggering a series of complications related to degradation products. Existing nerve catheters have deficiencies in cell delivery, nutrient supply and metabolite excretion, and generally cannot meet the needs of long-distance nerve repair.

[0004] In summary, the development of new tissue engineering nerve conduits that can be effectively used for long-distance nerve repair is of great significance for solving the problem of long-distance peripheral nerve injury repair. Summary of the Invention

[0005] In response to the deficiencies of the existing technology and actual needs, the present invention provides a tissue engineering nerve conduit and its preparation method and application, designs a new tissue engineering nerve conduit, and opens up a new treatment approach for patients with long-distance peripheral nerve injuries.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a tissue engineering nerve conduit, comprising polyhydroxyalkanoate porous microspheres and a conduit, wherein the polyhydroxyalkanoate porous microspheres are loaded inside the conduit, and the conduit is made of a polyhydroxyalkanoate fiber membrane.

[0008] The present invention designs a tissue engineering nerve conduit with a completely new structure. Polyhydroxyalkanoate is used as the main material. The conduit and porous microspheres are designed and assembled. The advantages of three-dimensional porous microspheres and nerve conduits are combined. The porous microspheres have a large specific surface area and abundant internal space, which can provide abundant attachment sites for cells, facilitate the exchange of nutrients and metabolites, and thus provide a good microenvironment for cell growth and tissue construction. It effectively simulates the structure and function of autologous nerves and provides an ideal microenvironment for the growth and regeneration of nerve cells.

[0009] Preferably, the catheter is obtained by winding the polyhydroxyalkanoate fiber membrane on a mold.

[0010] In the present invention, the polyhydroxyalkanoate fiber membrane is used to prepare the conduit, and the mechanical properties thereof meet the requirements of neural tissue engineering. The conduit can effectively guide cells to arrange in an orderly manner and has good cell compatibility and biological functions.

[0011] Preferably, the polyhydroxyalkanoate in the polyhydroxyalkanoate porous microspheres and the polyhydroxyalkanoate fiber membrane independently includes at least one of poly 3-hydroxybutyrate-3-hydroxyvalerate-3-hydroxyhexanoate copolymer (P3HB3HV3HHx), poly 3-hydroxybutyrate-4-hydroxybutyrate copolymer (P34HB) or poly 3-hydroxybutyrate-3-hydroxyhexanoate copolymer (PHBHHx).

[0012] Preferably, the polyhydroxyalkanoate fiber membrane comprises a polyhydroxyalkanoate ordered fiber membrane or a polyhydroxyalkanoate disordered fiber membrane.

[0013] Preferably, the average diameter of the polyhydroxyalkanoate ordered fiber membrane is 3.54±1.19 μm.

[0014] Preferably, the average diameter of the polyhydroxyalkanoate random fiber membrane is 6.43±1.97 μm.

[0015] Preferably, the diameter of the polyhydroxyalkanoate porous microspheres is 362.1±127.4 μm.

[0016] Preferably, the polyhydroxyalkanoate porous microspheres are also loaded with Schwann cells.

[0017] In the present invention, polyhydroxyalkanoate porous microspheres are used to load Schwann cells, so that the Schwann cells can be arranged in an orderly manner in the nerve conduit, thereby promoting the directional growth and migration of nerve cells.

[0018] In a second aspect, the present invention provides a method for preparing the tissue engineering nerve conduit according to the first aspect, the method comprising:

[0019] The polyhydroxyalkanoate porous microspheres and the conduit are prepared, and the polyhydroxyalkanoate porous microspheres are placed in the conduit to obtain the tissue engineering nerve conduit.

[0020] Preferably, the method for preparing the polyhydroxyalkanoate porous microspheres comprises:

[0021] The polyhydroxyalkanoate solution is mixed with the gelatin solution to obtain an emulsion; the stabilizer is mixed with water to obtain an aqueous phase;

[0022] A syringe pump is used for injection, so that the emulsion and the aqueous phase are mixed in a T-shaped microfluidic device, polymer droplets are generated in a T-shaped tube, and the polymer droplets are collected; the solvent and gelatin in the polymer droplets are removed to obtain the polyhydroxyalkanoate porous microspheres.

[0023] Preferably, the solvent of the polyhydroxyalkanoate solution comprises any one of dichloromethane, chloroform or acetonitrile, or a combination of at least two thereof;

[0024] Preferably, the mass concentration of polyhydroxyalkanoate in the polyhydroxyalkanoate solution is 1% to 30%, for example, it can be 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 26%, 27%, 28% or 29%, etc.

[0025] Preferably, the solvent of the gelatin solution includes water.

[0026] Preferably, the gelatin solution further contains polyvinyl alcohol.

[0027] Preferably, the mass concentration of gelatin in the gelatin solution is 1% to 10%, for example, it can be 2%, 3%, 4%, 5%, 6%, 7%, 8% or 9%.

[0028] Preferably, the mass concentration of polyvinyl alcohol in the gelatin solution is 0.1% to 2%, for example, it can be 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 1.6%, 1.7%, 1.8% or 1.9%, etc.

[0029] In the present invention, by controlling the specific concentrations of the polyhydroxyalkanoate solution and the gelatin solution, the pore size and pore size distribution of the microspheres can be effectively regulated, thereby obtaining porous microspheres with high porosity and regular morphology, which have a highly open and interconnected porous surface and provide abundant attachment sites for cells.

[0030] Preferably, the stabilizer comprises polyvinyl alcohol.

[0031] Preferably, the mass concentration of polyvinyl alcohol in the aqueous phase is 0.1% to 2%, for example, it can be 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 1.6%, 1.7%, 1.8% or 1.9%, etc.

[0032] Specifically, the preparation method of the polyhydroxyalkanoate porous microspheres includes:

[0033] (1) preparing a polyhydroxyalkanoate solution and a gelatin solution, mixing the polyhydroxyalkanoate solution and the gelatin solution in a volume ratio of 1:(2-5), and emulsifying the mixture to obtain an emulsion;

[0034] mixing a stabilizer with water to obtain an aqueous phase;

[0035] (2) preparing microsphere droplets by passing the emulsion and the aqueous phase through the inner aqueous phase and the outer aqueous phase of the coaxial needle, respectively, and dropping them into the polyvinyl alcohol solution to form porous microspheres under stirring conditions (e.g., a stirring speed of 500 to 2000 rpm);

[0036] (3) Post-treatment: Excess solvent and unreacted substances are removed by centrifugation (for example, at a centrifugal speed of 3000 to 5000 rpm), washing, and other treatment steps to obtain polyhydroxyalkanoate porous microspheres.

[0037] Preferably, the emulsification treatment speed is 6000-18000 rpm, for example, 6000 rpm, 9000 rpm, 12000 rpm, 15000 rpm or 18000 rpm, etc., the time is 3-20 min, for example, 3 min, 5 min, 10 min, 15 min or 20 min, and the temperature is 10°C-30°C, for example, 10°C, 15°C, 20°C, 25°C or 30°C, etc.

[0038] Preferably, the method for preparing the catheter comprises:

[0039] A polyhydroxyalkanoate fiber membrane is prepared by an electrospinning method, and the polyhydroxyalkanoate fiber membrane is wound on a mold to assemble and prepare a catheter.

[0040] In the present invention, polyhydroxyalkanoate is dissolved in a solvent (such as chloroform) and nanofibers are prepared by electrospinning technology. By changing the speed of the collecting drum, ordered (1000-3000 rpm, for example, 1100, 1200, 1500, 2000, 2200, 2500, 2600, 2700, 2800 or 2900 rpm, etc.) and disordered (50-300 rpm, for example, 60, 70, 80, 90, 100, 150, 200, 220, 250, 260, 270, 280 or 290 rpm, etc.) electrospun fibers are prepared.

[0041] Preferably, the preparation method further comprises the step of mixing the polyhydroxyalkanoate porous microspheres with Schwann cells to obtain Schwann cell-loaded polyhydroxyalkanoate porous microspheres.

[0042] Preferably, the method for preparing Schwann cells comprises:

[0043] Adipose-derived mesenchymal stem cells were induced to differentiate into Schwann cells.

[0044] Compared with the prior art, the present invention has at least the following beneficial effects:

[0045] The present invention designs a long-distance tissue-engineered nerve conduit based on three-dimensional porous polyhydroxyalkanoate microspheres and nerve conduits. This conduit can mimic the body's own nerve conduits and promote the growth and regeneration of nerve cells. Its preparation method is simple and easy to use, and it has broad application prospects in fields such as tissue engineering and nerve repair. Furthermore, the invention utilizes Schwann cells induced by differentiation of adipose-derived mesenchymal stem cells as seed cells, addressing the limited supply of nerve cell donors and reducing immune risks, thus promising broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Diagram of the preparation process of P3HB3HV3HHx porous microspheres. P3HB3HV3HHx OPMs were prepared by double emulsion method (gelatin porogen).

[0047] Figure 2 This is a diagram of P3HB3HV3HHx porous microsphere cell seeding.

[0048] Figure 3 This is the SEM morphology characterization result of P3HB3HV3HHx porous microspheres.

[0049] Figure 4Figures 3 and 4 are the results of morphology and particle size analysis of P3HB3HV3HHx porous microspheres. Figure A is the surface SEM image of P3HB3HV3HHx porous microspheres, Figure B is the cross-sectional SEM image of porous microspheres, Figure C is the bright field image of P3HB3HV3HHx porous microspheres, and Figure D is the particle size distribution results of porous microspheres.

[0050] Figure 5 Figure 3 is the morphology and particle size analysis results of P3HB3HV3HHx non-porous microspheres, where Figure A is the surface SEM image of P3HB3HV3HHx non-porous microspheres, and Figure B is the cross-sectional SEM image of the microspheres; Figure C is the bright field image of P3HB3HV3HHx non-porous microspheres, and Figure D is the particle size distribution results of the microspheres.

[0051] Figure 6 Figure 1 is the result of rADSCs identification. Figure A is flow cytometry identification. From left to right are rADSCs positive cell markers CD29. + 、CD90 + and negative cell marker CD86 - 、CD34 - 、CD45 - ; Figure B shows the calcium nodules stained with Alizarin red during osteogenic differentiation of rADSCs; Figure C shows the fat droplets stained with Oil Red O during adipogenic differentiation of rADSCs; Figure D shows the Alcian blue staining during chondrogenic differentiation of rADSCs.

[0052] Figure 7 Figure 1 is the result of rADSCs inducing differentiation into SCs, among which Figure A is the timeline of rADSC-induced differentiation into SCs and the schematic diagram of the process of adding induction factors; Figure B is the schematic diagram of rADSCs-induced differentiation into SCs; Figure C is the cell morphology during the induction stage of rADSCs differentiation into SCs; Figure D is the result of immunofluorescence staining identification of Schwann cells.

[0053] Figure 8 Schematic diagram of the synthesis of P3HB3HV3HHx nanofibers.

[0054] Figure 9 Figures 2 and 3 show the results of characterization of autologous nerve structure and electrospun fiber membrane morphology. Figure A shows the immunofluorescence staining of NF200 and S100β in the cross-section and longitudinal section of rat sciatic nerve (red: S100β; green: NF200; blue: DAPI); Figure B shows the average diameter distribution of rat sciatic nerve axons; Figure C shows the directional distribution of rat sciatic nerve axons; Figure D shows the SEM image and contact angle of P3HB3HV3HHx ordered fibers and disordered fiber membranes; Figure E shows the average diameter distribution of P3HB3HV3HHx ordered fibers and disordered fibers.

[0055] Figure 10These are the characterization results of P3HB3HV3HHx electrospun fibers, where Figure A is the tensile stress detection image of the P3HB3HV3HHx electrospun membrane; Figure B is the quantitative analysis of the contact angle of the P3HB3HV3HHx electrospun membrane; Figure C is the maximum tensile strength of the P3HB3HV3HHx electrospun membrane; and Figure D is the Young's modulus of the P3HB3HV3HHx electrospun membrane.

[0056] Figure 11 Schematic diagram of P3HB3HV3HHx nanofiber cell seeding.

[0057] Figure 12 This is the cell compatibility result diagram of P3HB3HV3HHx porous microspheres.

[0058] Figure 13 Figure 2 is the result of nanofiber cell compatibility, where Figure A is the immunofluorescence image of the SCs cytoskeleton cultured on ordered and disordered fiber membranes for 7 days (green: βⅢ-tubulin; blue: DAPI); Figure B is the coordinate histogram of SCs tentacles extending on nanofibers.

[0059] Figure 14 These are the characterization results of the P3HB3HV3HHx electrospinning catheter, where Figure A is the tensile stress detection image of the P3HB3HV3HHx electrospinning catheter; Figure B is the elongation of the P3HB3HV3HHx electrospinning catheter; Figure C is the maximum tensile strength of the P3HB3HV3HHx electrospinning catheter; and Figure D is the Young's modulus of the P3HB3HV3HHx electrospinning catheter.

[0060] Figure 15 Schematic diagram of nerve conduit assembly.

[0061] Figure 16 Figure 1 shows the microscopic structure of the nerve catheter and the surgical implantation process. Figure A is an electron microscope scan of the nerve catheter showing the microscopic structure of the catheter; Figure B is a photo of the nerve catheter and the implantation process.

[0062] Figure 17 Figure 3: Evaluation results of nerve conduit-promoting sciatic nerve regeneration in rats. Figure A shows the timeline of AMS NGCs repairing long-distance sciatic nerve defects in rats; Figure B shows representative footprint images of rats 12 weeks after surgery; Figure C shows the real-time paw area of ​​rat footprints 12 weeks after repair; Figure D shows the quantitative analysis of paw area of ​​rat footprints 12 weeks after repair; Figure E shows the quantitative analysis of angles of rat footprints 12 weeks after repair; and Figure F shows the sciatic nerve function index (SFI) values ​​of each group 12 weeks after surgery.

[0063] Figure 18Figure 3 is an evaluation result of the effect of nerve conduit on promoting sciatic nerve repair in rats. Figure A is a representative compound muscle action potential (CMAP) curve for promoting sciatic nerve regeneration in rats; Figure B is the CMAP latency of the sciatic nerves of rats after conduit repair in each group; Figure C is the CMAP peak amplitude of the sciatic nerves of rats after conduit repair in each group.

[0064] Figure 19 The figure shows the evaluation results of nerve conduit promoting sciatic nerve repair in rats.

[0065] Figure 20 Figure 3: Evaluation results of the nerve conduit promoting sciatic nerve repair in rats. Figure A is a photograph of the conduit after 12 weeks of repair, Figure B is a transmission electron microscopy (TEM) image of the nerve myelin sheath, Figure C is the wet weight of the gastrocnemius muscle in each group, Figure D is the average cross-sectional area of ​​the gastrocnemius muscle fibers in each group, and Figure E is the myelin sheath thickness of the myelinated axons in each group. DETAILED DESCRIPTION

[0066] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0067] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0068] In this context, polyhydroxyalkanoate (PHA) refers to a bacterially produced biopolyester. Its excellent biocompatibility, biodegradability, and non-toxic degradation products offer significant advantages in long-term implant applications and biodegradable materials. P3HB3HV3HHx, a PHA with excellent biocompatibility, can be formulated into various forms, including microspheres, nanoparticles, and nanofilms.

[0069] In a specific embodiment of the present invention, the main reagents include:

[0070] Polyvinyl alcohol (PVA): purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number P815725-500g;

[0071] Agarose: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number A434537-100g;

[0072] Ammonium bicarbonate: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number 1066-33-7;

[0073] Dichloromethane: purchased from China National Pharmaceutical Group, product number u1601441;

[0074] P3HB3HV3HHx was purchased from Blue Crystal Biotechnology Co., Ltd. with a molecular weight of 25,000 Da;

[0075] Mechanical property characterization: The mechanical properties of the electrospun fiber membrane and nerve conduit were characterized, including the determination of parameters such as tensile stress, maximum tensile strength, Young's modulus, and elongation at break. The specific methods include: First, sample preparation must strictly follow the specifications: the fiber membrane is cut into 30mm×10mm rectangular strips to ensure uniform fiber orientation (if anisotropy needs to be evaluated, it is necessary to rotate 90° and repeat the test); the nerve conduit is rolled into 1.5mm thin strips from the electrospun membrane to avoid fiber tearing or wrinkling at the cut. The wall thickness is accurately measured using a non-contact laser thickness gauge (range 0.5-2mm). During the test, the sample is clamped at both ends in the pneumatic clamp of the electronic universal testing machine to ensure that the gauge section (such as 20mm) is intact and the clamping force is moderate. It is stretched at a constant rate of 5mm / min, and the force-displacement curve is recorded simultaneously. Tensile stress is calculated by dividing the actual load by the initial cross-sectional area. The maximum tensile strength is calculated as the peak stress of the curve. Young's modulus is obtained by linearly fitting the slope of the stress-strain curve in the elastic phase. Elongation at break is the ratio of the elongation of the gauge length at break to the initial gauge length. Each test is repeated at least three times, and the average and standard deviation are calculated after removing outliers. The data must be annotated with the test environment and equipment parameters to ensure repeatability.

[0076] Example 1

[0077] In this example, P3HB3HV3HHx porous microspheres (P3HB3HV3HHx OPMs) were prepared.

[0078] Figure 1 The preparation process of P3HB3HV3HHx porous microspheres was as follows: P3HB3HV3HHxOPMs were prepared by double emulsion method (gelatin porogen), and the microspheres were received in an ice bath.

[0079] Preparation of P3HB3HV3HHx porous microspheres:

[0080] Solution Preparation: Dissolve 0.2g of P3HB3HV3HHx in 10mL of dichloromethane to prepare a 2% (w / v) P3HB3HV3HHx solution. Dissolve gelatin and PVA in deionized water to prepare a solution containing 7.5% (w / v) gelatin and 1% (w / v) PVA. Dissolve PVA alone in deionized water to prepare a 1% (w / v) PVA solution.

[0081] Ultrasonic emulsification: 1 g of a solution containing 7.5% gelatin and 1% PVA was added to 3 g of P3HB3HV3HHx solution, and ultrasonic treatment was performed (power 10%, ultrasonication for 2 s, pause for 1 s).

[0082] Connection of coaxial electrospinning device: The emulsified liquid and 1% (w / v) PVA solution were transferred into 10 mL glass syringes, 50 mL syringes, respectively, and connected with coaxial needle (25G+18G), polyvinyl chloride tube, transparent silicone hose, etc. in turn after loading in the syringe pump.

[0083] Flow rate ratio setting: The syringe containing emulsion was connected to the inner diameter of the device, representing the discontinuous phase. The 1% PVA solution was connected to the outer diameter as the continuous phase. The flow rate ratio between the two phases was set to 40:1.

[0084] Microsphere collection and treatment: The microspheres were collected in a 500 mL beaker containing 1% PVA and placed in an ice bath for gentle stirring overnight. Then transferred to warm water at 45°C, stirred for 3h, and washed the microspheres four times with deionized water to completely remove the residual PVA.

[0085] Preparation of P3HB3HV3HHx non-porous microspheres:

[0086] Solution preparation: 0.2g P3HB3HV3HHx was dissolved in 10 mL dichloromethane to prepare a 2% (w / v) P3HB3HV3HHx solution. Gelatin and PVA were dissolved in deionized water to prepare a solution containing 7.5% (w / v) gelatin and 1% (w / v) PVA. PVA was dissolved in deionized water alone to prepare a 1% (w / v) PVA solution.

[0087] Ultrasonic emulsification: 1g of 1% PVA solution was added to 3g of P3HB3HV3HHx solution and ultrasonicated (power 10%, ultrasonic 2s, pause 1s).

[0088] Connection of coaxial electrospinning device: The emulsified liquid and 1% (w / v) PVA solution were transferred into 10 mL glass syringes, 50 mL syringes, respectively, and connected with coaxial needle (25G+18G), polyvinyl chloride tube, transparent silicone hose, etc. in turn after loading in the syringe pump.

[0089] Flow rate ratio setting: The syringe containing emulsion was connected to the inner diameter of the device, representing the discontinuous phase. The 1% PVA solution was connected to the outer diameter as the continuous phase. The flow rate ratio between the two phases was set to 40:1.

[0090] Microsphere collection and treatment: The microspheres were collected in a 500 mL beaker containing 1% PVA and placed in an ice bath for gentle stirring overnight. Then transferred to warm water at 45°C, stirred for 3h, and washed the microspheres four times with deionized water to completely remove the residual PVA.

[0091] Example 2

[0092] In this example, the particle size and surface pore size of the porous microspheres prepared in Example 1 were measured.

[0093] Measurement of microsphere particle size using an optical microscope: 100 microspheres were randomly selected from each sample group as samples. After taking pictures under a microscope, the particle size of the microspheres was quantified to obtain a preliminary understanding of the approximate distribution range of the microsphere particle size. Scanning electron microscopy (SEM) was used to observe the surface morphology and particle size of the microspheres.

[0094] The air-dried porous microspheres were fixed to double-sided conductive adhesive and gold-sprayed to enhance their conductivity. Subsequently, the surface morphology of the microspheres in the porous microsphere scaffold was photographed under SEM magnification to obtain a more accurate microsphere particle size.

[0095] The SEM morphology characterization results of P3HB3HV3HHx porous microspheres are as follows Figure 3 As shown in the figure, the P3HB3HV3HHx porous microspheres have obvious porous morphology, and their cross-section presents a honeycomb network structure, indicating that controlling the specific concentration of polyhydroxyalkanoate solution and gelatin solution can effectively regulate the pore size and pore size distribution of the microspheres, and obtain porous microspheres with high porosity and regular morphology.

[0096] Comprehensive data analysis and average particle size calculation: The average particle size and surface pore size distribution range of porous microspheres are calculated.

[0097] Example 3

[0098] In this example, the cryosection method was used to observe the cross section of the porous microspheres.

[0099] Sample preparation: The porous microspheres were spread on a glass slide, dispersed with ultrapure water, and then frozen in a -80°C refrigerator to freeze the sample.

[0100] Cross-section processing: Use a scalpel to gently scrape off the upper layer of ice, retaining the microspheres at the bottom. Air-dry at room temperature to remove moisture, obtaining a cross-section sample of the microspheres with a clear internal structure for subsequent SEM observation and imaging.

[0101] Example 4

[0102] This example measures the pore size of the internal structure of porous microspheres.

[0103] Sample Fixation and Gold Sputtering: Use a toothpick to gently stick a cross-section of the cryosectioned porous microspheres and secure them to the double-sided conductive adhesive on the SEM sample stage. After gold spraying, observe the cross-sectional morphology using an SEM.

[0104] Photography and Measurement: Ten randomly selected porous microsphere cross sections from the same sample were photographed. The internal pore sizes of all porous microsphere scaffolds were recorded by measuring the cross sections of the porous microspheres in the photographs.

[0105] The morphology and particle size analysis results of P3HB3HV3HHx porous microspheres are shown in Figure 4 As shown, Figure A is the SEM image of the surface of P3HB3HV3HHx porous microspheres, Figure B is the SEM image of the cross-section of the porous microspheres, Figure C is the bright field image of the P3HB3HV3HHx porous microspheres, and Figure D is the particle size distribution results of the porous microspheres. The results show that PHAOPMs have a highly open and interconnected porous surface and contain a macroporous structure with a diameter range of 362.1±127.4μm.

[0106] P3HB3HV3HHx nonporous microspheres were prepared according to the preparation method of Example 1, with the following main differences: During the ultrasonic emulsification stage, 1 g of a 1% PVA solution (without the porogen gelatin) was added to 3 g of the P3HB3HV3HHx solution, and ultrasonic treatment was performed (power 10%, ultrasonication for 2 s, pause for 1 s).

[0107] The morphology and particle size analysis results of P3HB3HV3HHx non-porous microspheres are as follows Figure 5 As shown, Figure A is the SEM image of the surface of P3HB3HV3HHx non-porous microspheres, and Figure B is the SEM image of the cross-section of the microspheres; Figure C is the bright field image of P3HB3HV3HHx non-porous microspheres, and Figure D is the particle size distribution result of the microspheres, with a diameter range of 343.2±86.71μm.

[0108] Example 5

[0109] In this example, microspheres were stained with Nile red.

[0110] Nile Red Dye Preparation: Dissolve Nile Red Dye in methanol to prepare a 1 mg / mL stock solution. Dilute the stock solution to a working concentration of 1 μg / mL with PBS before use.

[0111] Porous microspheres were cleaned by washing the porous microspheres twice with sterile phosphate buffered saline (PBS, 0.1 M NaCl, pH 7.0), soaking for 8 min each time to remove impurities.

[0112] Staining and Washing: Soak the cleaned porous microsphere scaffold in Nile Red working solution, ensuring that the scaffold is completely covered by the solution. Stain for 30 minutes at room temperature in the dark, shaking the scaffold to promote thorough binding of the dye to the scaffold. After staining, wash the porous microsphere scaffold five times with PBS (10 minutes each time) to remove any residual Nile Red working solution.

[0113] Observation: Place the stained porous microsphere scaffold on a confocal dish, and observe the confocal dish under a fluorescence confocal microscope.

[0114] Example 6

[0115] This example performs sterilization of porous microspheres and pre-treatment of cells before inoculation.

[0116] Sterilization: Soak the porous microspheres in 75% ethanol for 12 hours to achieve sterilization.

[0117] Washing and soaking: After sterilization by soaking in 75% ethanol, rinse four times with sterile PBS, letting it sit for 20 minutes each time. After removing the sterile PBS, the microspheres are suspended in sterile complete culture medium and soaked at 4°C for 12 hours to obtain porous microspheres suitable for cell seeding.

[0118] Example 7

[0119] This example involves the extraction and identification of rat adipose-derived mesenchymal stem cells (rADSCs).

[0120] Animal Experimentation and Tissue Procurement: All animal experiments were approved by the Ethics Committee of Huazhong University of Science and Technology. Freshly harvested adipose tissue was rinsed with PBS and allowed to stand for stratification. The rinse solution was discarded and repeated four times. The adipose tissue was then cut into a paste, and fascia and blood clots were removed with forceps. The rinsed adipose tissue was sterilely stored in a 50 mL centrifuge tube.

[0121] Cell digestion and collection: Mix equal volumes of adipose tissue with complete medium containing 0.2% NB4 collagenase and incubate at 37°C in a shaker at 120 rpm for 3 h. After digestion, centrifuge at 1000 rpm for 5 min, collect the white flocculent precipitate at the bottom of the tube, rinse with PBS, and resuspend.

[0122] Cell filtration and plating: Filter the resuspension through a 70 μm cell strainer, collect the filtrate, centrifuge at 1000 rpm for 5 minutes, resuspend in 10 mL of complete culture medium, and then plate the cells in a 10 cm culture dish. Place the dish in a cell culture incubator, changing the medium every two days until the cells reach a confluency of at least 80%. The cells can then be digested and passaged.

[0123] Flow cytometry identification: rADSCs were collected after digestion, washed with PBS three times, filtered with a 200 μm cell strainer, and single cell suspension was collected. 2×10 cells were collected for each sample. 6 Cells were analyzed by flow cytometry. Flow cytometry antibodies were added to the samples and incubated for 45 minutes at room temperature in the dark. Isotype and negative controls were established for each antibody. Unbound antibodies were removed by centrifugation, and the cells were washed twice with PBS. Flow cytometry was performed on the samples, and data were analyzed using CytExpert 2.4 software.

[0124] Example 8

[0125] In this example, rADSCs were differentiated into adipogenic, osteogenic, and chondrogenic types.

[0126] Adipogenic Differentiation: Plate cells into a suitable culture dish. After cells adhere, discard the culture medium and add Saiye Adipogenic Induction Medium. Incubate in a 37°C, 5% CO2 incubator, replacing the medium every two days. After 12 days of induction, discard the culture medium, rinse the cells three times with PBS, fix them with 4% paraformaldehyde for 20 minutes, rinse twice with PBS, stain with Oil Red O working solution for 13 minutes, and finally rinse once with PBS before observing under a microscope.

[0127] Osteogenic Differentiation: Cells were seeded at an appropriate density in culture plates and cultured in medium containing 10% FBS until cell confluence reached 70%-90%. The medium was replaced with Saiye Osteogenic Induction Medium and cultured in a 37°C, 5% CO2 incubator, changing the medium every 3 days. After 2-3 weeks of induction, cells were rinsed twice with PBS, fixed with 4% paraformaldehyde, and stained with Alizarin Red. The appearance of red calcium nodules in the extracellular matrix confirmed successful osteoblastic differentiation.

[0128] Chondrogenic Differentiation: Take an appropriate amount of cells, digest them with trypsin, and prepare a cell suspension. Adjust the cell density. Centrifuge the cell suspension to form cell microclusters, which are then cultured in Saiyecheng Chondrogenic Induction Medium. Incubate at 37°C, 5% CO2, changing the medium every 2-3 days. After 3-4 weeks of induction, perform Alcian Blue staining. Fix the cell microclusters with 4% paraformaldehyde and stain with Alcian Blue. A blue appearance of the extracellular matrix indicates that the cells have differentiated into chondrocytes.

[0129] The results of rADSCs identification were as follows Figure 6 As shown, Figure A is flow cytometry identification, from left to right are rADSCs positive cell markers CD29 + 、CD90 + and negative cell marker CD86 - 、CD34 - 、CD45 - ; Figure B shows the calcium nodules stained with Alizarin red during osteogenic differentiation of rADSCs; Figure C shows the fat droplets stained with Oil Red O during adipogenic differentiation of rADSCs; Figure D shows the chondrogenic differentiation of rADSCs stained with Alcian blue. The results show that the extracted rADSCs have multidirectional differentiation potential.

[0130] Example 9

[0131] In this example, rADSCs were differentiated into SCs.

[0132] SCs differentiation: When rADSCs were passaged to 3-5, cells with good growth status were selected and cultured at a rate of 1×10 5 pieces / cm 2Density seeded in a 10cm culture dish. After the cells adhered, replaced with pre-induction medium (DMEM / F12 medium containing 1mM β-mercaptoethanol) and cultured for 24h. First step of induction: discard the pre-induction medium, wash the cells twice with PBS, add induction medium (DMEM / F12 medium containing 10% fetal bovine serum and 3.5ng / mL all-trans retinoic acid) and culture for 2 days. Second step of induction: After 3 days, discard the induction medium, wash the cells twice with PBS, replace the second step induction medium (DMEM / F12 medium containing 10% fetal bovine serum, 5uM forskolin, 10ng / mL basic fibroblast growth factor, 5ng / mL platelet-derived growth factor, 200ng / mL Heregulin-β1), and continue to culture for 11 days. During the induction of differentiation, replace the induction medium every 2 days.

[0133] The results of rADSCs differentiation into SCs were as follows Figure 7 Figure 1 shows the timeline of rADSC-induced SC differentiation and the process of adding induction factors. Figure 1 shows the schematic diagram of rADSC-induced SC differentiation. Figure 1 shows the cell morphology during the induction phase of rADSC-induced SC differentiation. Figure 1 shows the immunofluorescence staining of Schwann cells. The results show that during the process of rADSC-induced SC differentiation, significant changes in cell morphology were observed, and the gradual appearance of differentiation markers was detected. The cell morphology gradually changed from the typical triangular shape of rADSCs to the characteristic morphology of SCs with elongated morphology and increased processes.

[0134] Example 10

[0135] In this example, P3HB3HV3HHx nanofibers were prepared by electrospinning.

[0136] Schematic diagram of the synthesis of P3HB3HV3HHx nanofibers Figure 8 shown.

[0137] Preparation of P3HB3HV3HHx nanofiber by electrospinning: P3HB3HV3HHx powder was dissolved in chloroform to prepare a 3% (w / v) P3HB3HV3HHx solution, which was sealed and stirred overnight in the dark. The solution was loaded into a glass syringe and installed into the microflow pump of the electrospinning device. The nozzle was adjusted to be perpendicular to the drum of the electrospinning instrument, with a distance of 15 cm from the drum. The parameters of electrospinning were set, with a positive voltage of 17 kV and a negative voltage of -1 kV. The speed of the nozzle moving from left to right was 6 mm / s, and the rotation speed of the roller was 200 rpm (disordered fiber membrane) and 2000 rpm (ordered fiber membrane). The high-voltage power supply was turned on, and the voltage was gradually increased until the solution formed a jet from the nozzle. At the same time, the syringe pump was turned on, and the solution flowed out at a flow rate of 2.5 mL / h. The morphology of the jet and the deposition of the fibers were observed in real time. Electrospinning was carried out at a microflow pump flow rate of 2.5 mL / h for 24 h. After the spinning was completed, the aluminum foil collecting the fibers was removed from the drum and placed in a fume hood to dry, removing the residual solvent.

[0138] Example 11

[0139] In this example, P3HB3HV3HHx fiber membrane characterization and SCs inoculation were performed.

[0140] Fiber membrane characterization: After spraying, SEM was used to observe the morphology of P3HB3HV3HHx nanofibers. For each sample, more than 200 P3HB3HV3HHx fibers were selected in 5 randomly selected fields to take images. Image J software was used to measure and calculate the diameter distribution of the electrospinning membrane.

[0141] Figure 9 Figure for autonomic nerve structure and electrospinning fiber membrane morphology characterization results, wherein, A is the rat sciatic nerve cross-section and longitudinal-section NF200 and S100β immunofluorescence staining (red: S100β; green: NF200; blue: DAPI); B is the rat sciatic nerve axon average diameter distribution; C is the rat sciatic nerve axon directional distribution; D is the SEM images of P3HB3HV3HHx ordered fiber and disordered fiber membrane and contact angle; E is the average diameter distribution of P3HB3HV3HHx ordered fiber and disordered fiber, the results show that the rat sciatic nerve expresses rat neurofilament protein (Neurofilament 200, NF200) and central nervous system specific protein (S100β), the diameter distribution of sciatic nerve axons is between 2 μm and 7 μm, with an average diameter of 4.15 ± 1.21 μm. These nerve fibers have a specific directional distribution to ensure that signals can be accurately and rapidly transmitted to the target tissue.

[0142] P3HB3HV3HHx nanofibers were prepared by electrospinning a 3% solution of P3HB3HV3HHx powder in chloroform. The electrospun fiber membranes exhibited uniform fiber diameters and smooth surfaces without obvious beading. Ordered and disordered electrospun fibers were prepared by varying the speed of the collecting drum. The average diameter of the ordered nanofibers was 3.54±1.19μm, while the average diameter of the disordered nanofibers was 6.43±1.97μm. The contact angles of the nanofibers were 138.52±0.88° (ordered) and 130.88±1.18° (disordered), respectively.

[0143] The characterization results of P3HB3HV3HHx electrospun fibers are shown in Figure 10 Figure 1 shows the tensile stress measurement images of P3HB3HV3HHx electrospun membranes. Figure 1 shows the contact angle quantitative analysis of P3HB3HV3HHx electrospun membranes. Figure 1 shows the maximum tensile strength of P3HB3HV3HHx electrospun membranes. Figure 1 shows the Young's modulus of P3HB3HV3HHx electrospun membranes. Figure 1 shows the Young's modulus of P3HB3HV3HHx electrospun membranes. The results show that the Young's modulus of ordered and disordered fiber membranes is similar, but the tensile stress of the ordered fiber membrane is higher. The elongation at break of both ordered and disordered fiber conduits is less than 30%, indicating poor elasticity. The Young's modulus and tensile stress of the ordered fiber conduits are much greater than those of the disordered fiber membrane. These results demonstrate that the mechanical properties of the P3HB3HV3HHx fiber membrane meet the requirements for neural tissue engineering.

[0144] Schematic diagram of P3HB3HV3HHx nanofiber cell seeding Figure 11 shown.

[0145] Schematic diagram of P3HB3HV3HHx porous microsphere cell seeding Figure 2 shown.

[0146] SCs seeding: Sterilize the microspheres and electrospun membranes by soaking them in 75% ethanol overnight. Wash them three times with sterile PBS and twice with sterile medium without FBS. 6 2 mL of culture medium for each SC was added to a 6-well plate containing microspheres and electrospun membranes and cultured for 12 h at 37°C and 5.0% CO2. After 12 h of incubation, the microspheres were transferred to a new 6-well plate and fresh complete culture medium was added.

[0147] Figure 12The results of the study examined the cytocompatibility of P3HB3HV3HHx porous microspheres. The results showed that SCs expressed the SC markers GFAP and S100β on the microspheres, and neurites of the SCs were clearly observed. After 7 days of culture, the cells continued to proliferate, covering the entire surface of the porous microspheres (OPMs) and partially migrating into the interior of the OPMs. However, no cells were observed within the nonporous microspheres (SMs).

[0148] Nanofiber cytocompatibility Figure 13 Figure 1 shows immunofluorescence images of the SC cytoskeleton (green: βⅢ-tubulin; blue: DAPI) cultured on ordered and disordered fiber membranes for 7 days. Figure 1 shows the coordinate histogram of SC tentacles extending on nanofibers. The results show that the cells not only adhere and spread well on the fiber membranes, but also arrange themselves in an orderly manner under the guidance of the ordered fiber membranes. In contrast, the cells are distributed disorderly on the disordered electrospun membranes. The coordinate histogram shows that compared with the disordered distribution of SCs cultured on the disordered membranes, SCs cultured on the ordered membranes tend to grow along the electrospun fibers. Clearly, the specific surface ordered topology of the conduits plays a guiding role in inducing the directional distribution of cells. Guided by its parallel topology, the cultured cells are induced to grow in a directional manner and tend to extend along the fiber grooves of the conduits.

[0149] Example 12

[0150] In this example, cytoskeleton staining observation was performed.

[0151] Cytoskeleton staining: Samples were fixed with 4% paraformaldehyde for 20 minutes at room temperature and washed three times with PBS. Samples were then treated with 0.25% Triton X-100 for 10 minutes and washed three times with PBS. Samples were blocked with 1% (w / v) bovine serum albumin solution for 40 minutes at room temperature and then incubated with phalloidin labeled with fluorescein isothiocyanate at a working concentration for 40 minutes in the dark at room temperature. Samples were incubated with DAPI at a working concentration for 10 minutes at room temperature in the dark. Samples were observed using a laser confocal microscope and analyzed using Image J software.

[0152] Example 13

[0153] This embodiment performs an integrated long-distance peripheral nerve catheter assembly.

[0154] Catheter Assembly: Wrap the cell-loaded P3HB3HV3HHx ordered or random fiber membrane around a 1mm inner diameter mold, ensuring the fibers align with the catheter axis to maintain the catheter's order. Cut the ordered nanofiber membrane catheter into 12mm ordered nerve conduits. Secure the catheter ends with 7-0 absorbable surgical sutures to prevent subsequent loosening. Remove the catheter from the mold and inspect its size and shape for compliance.

[0155] The characterization results of P3HB3HV3HHx electrospun catheters are as follows Figure 14 As shown, Figure A is the tensile stress detection image of the P3HB3HV3HHx electrospun catheter; Figure B is the elongation of the P3HB3HV3HHx electrospun catheter; Figure C is the maximum tensile strength of the P3HB3HV3HHx electrospun catheter; Figure D is the Young's modulus of the P3HB3HV3HHx electrospun catheter, indicating that the mechanical properties of the catheter meet the requirements of neural tissue engineering.

[0156] Schematic diagram of nerve conduit assembly Figure 15 shown.

[0157] Microspheres containing 20 SCs were suspended in complete culture medium and injected into the hollow, ordered conduits via syringe. After preparation, the conduits were transferred to a 6-well plate and incubated with fresh complete culture medium for 24 hours.

[0158] Example 14

[0159] This example conducts a nerve catheter application test.

[0160] Nerve conduit microstructure and implantation process Figure 16 As shown, Figure A is an electron microscope scan of the nerve catheter showing the catheter microstructure; Figure B is a photo of the nerve catheter and the implantation process.

[0161] The specific experimental process includes:

[0162] (1) Rats were housed in a specific pathogen-free environment. Thirty adult male Sprague Dawley (SD) rats (200-220 g) were randomly divided into five groups: negative control (NC), aligned conduit-Schwann cells (ACS), porous microspheres combined with aligned conduits (not loaded with Schwann cells) (AM), porous microspheres combined with aligned conduits (loaded with Schwann cells) (AMS), and autologous nerve transplantation (Autograft).

[0163] (2) using a 12 mm long, 1 mm inner diameter nerve conduit to repair the nerve defect;

[0164] (3) After anesthetizing the rats, shave the hair on their hind limbs and disinfect the skin of their hind limbs with iodine solution;

[0165] (4) Cut the skin and muscle layer of the leg to expose the sciatic nerve;

[0166] (5) Isolate the sciatic nerve and remove 12 mm of the sciatic nerve;

[0167] (6) For the porous microsphere combined with ordered conduit group, the distal nerve terminal was sutured 1 mm into the nerve conduit with 8-0 monofilament nylon suture. 200 μL of cell culture medium containing 20 porous microspheres (loaded with rADSCs) was injected into the nerve conduit using a 1 mL syringe without a needle. The proximal stump was then sutured 1 mm into the conduit, and the muscle and skin layers were sutured with 6-0 and 3-0 nylon sutures.

[0168] (7) Rats were killed 2, 6, and 12 weeks after catheter implantation and behavioral studies and histological sections were performed;

[0169] (8) For the ordered catheter group, 200 μL of pure cell culture medium without OPMs was injected into the catheter. The other steps were the same as those for the porous microsphere combined with ordered catheter group. For the porous microsphere combined with ordered catheter group without cells, the microsphere catheter was sterilized and used for nerve transplantation repair. A 1 mL syringe without a needle was used to inject 200 μL of cell culture medium containing 20 porous microspheres (without rADSCs) into the nerve catheter. The other steps were the same. For the Autograft group, 12 mm of sciatic nerve was resected, the resected nerve was flipped, and the nerve stump was sutured.

[0170] Figure 17 Figure 1 is the evaluation result of promoting sciatic nerve regeneration in rats by nerve conduit. Figure A is the timeline of AMS repair of long-distance sciatic nerve defects in rats; Figure B is a representative footprint image of rats at 12 weeks; Figure C is the real-time claw area of ​​rat footprints at 12 weeks after repair; Figure D is the quantitative analysis of the claw area of ​​rat footprints at 12 weeks after repair; Figure E is the quantitative analysis of the angle of rat footprints at 12 weeks after repair; Figure F is the sciatic nerve function index (SFI) value of each group 12 weeks after surgery. The results show that when the repair effect of sciatic nerve defects in rats is better, the rat's hind limbs can better bear the force on the treadmill and the claw area is also larger. After 6 weeks, the average claw area of ​​rats in each group was 0.23±0.04cm (0.23±0.04cm) and 0.23±0.04cm (0.23±0.04cm) respectively. 2 ), NC group (0.11±0.04cm 2 ), ACS group (0.15±0.04cm 2), AM group (0.20±0.01cm 2 ), AMS group (0.28±0.04cm 2 After 12 weeks, the average paw area of ​​rats in each group was significantly higher than that in the autologous nerve repair group (0.34±0.04cm 2 ), NC group (0.17±0.02cm 2 ), ACS group (0.22±0.06cm 2 ), AM group (0.23±0.05cm 2 ), AMS group (0.32±0.05cm 2 ). The results of the study showed that the area of ​​the rats' hind limb paws in contact with the treadmill was close to that of the autologous nerve repair group at weeks 6 and 12, suggesting that the nerve conduit in the AMS group can improve the walking ability of the rats. The angle of the rats' footprints was also quantitatively analyzed to evaluate the posture and stability of the rats' paws when walking, so as to further understand the recovery of the sciatic nerve's control over muscles. Sciatic nerve defect will cause the angle of the rats' footprints to increase, reflecting the impairment of motor function. When the repair effect of the rats' sciatic nerve defect was better, the angle of the rats' hind limb footprints when walking on the treadmill was smaller. When the repair effect of the rats' sciatic nerve defect was poor, the angle of the rats' hind limb footprints was larger. After 6 weeks, the average claw angles of the rats in each group were the autologous nerve repair group (13.42±9.32 degrees), NC group (11.82±2.95 degrees), ACS group (7.38±3.79 degrees), AM group (11.66±2.27 degrees), and AMS group (13.42±8.66 degrees).

[0171] After 12 weeks, the average paw angles of rats in each group were as follows: autologous nerve repair group (4.80±1.88 degrees), NC group (10.37±3.96 degrees), ACS group (15.00±3.62 degrees), AM group (9.56±3.70 degrees), and AMS group (4.80±1.94 degrees).

[0172] The sciatic nerve function index (SFI) is a key indicator for measuring motor function recovery, with values ​​ranging from -100 (complete loss of function) to 0 (normal function). Twelve weeks after implantation, the SFI in the AMS group was similar to that in the autologous transplant group and higher than that in the negative control group (-9.08±5.33), the ACS group (-38.57±10.31), and the AM group (-23.41±8.25). These results indicate that the AMS group has a significant advantage in promoting sciatic nerve function recovery and improving gait and stability in rats.

[0173] Figure 18Figure for evaluation results of nerve conduit promoting sciatic nerve repair in rats, wherein, A is a representative composite muscle action potential (CMAP) curve of nerve conduit promoting sciatic nerve regeneration in rats; B is the CMAP latency of sciatic nerve of rats after conduit repair in each group; C is the CMAP amplitude peak of sciatic nerve of rats after conduit repair in each group, and the results show that the composite muscle action potential (CMAP) amplitude potential of A-C-S is smaller, indicating that the nerve conduction ability is poorer. The CMAP potential (5.91±2.59 mV) of A-M-S group is similar to that (7.43±1.47 mV) of autologous transplantation group, higher than that (2.97±1.21 mV) of A-C-S group and that (2.18±0.65 mV) of A-M group. In addition, the CMAP latency of A-M-S group (2.76±0.65 ms) is significantly lower than that (3.59±1.03 ms) of A-C-S group and that (3.99±0.52 ms) of A-M group, and there is no significant difference with that (2.5±0.3 ms) of autologous transplantation group.

[0174] Figure 19 Figure for evaluation results of nerve conduit promoting sciatic nerve repair in rats, and the Masson staining images (blue staining area represents collagen fibers) of gastrocnemius muscle photographs and gastrocnemius muscle cross-section slices.

[0175] The research results show that the A-M-S group promotes nerve function recovery and exhibits similar effect to autologous transplantation, thereby providing a new effective strategy for sciatic nerve injury repair.

[0176] Figure 20 Figure for evaluation results of nerve conduit promoting sciatic nerve repair in rats, wherein, A is a conduit photograph after 12 weeks of repair, B is a nerve myelin sheath transmission electron microscope (TEM) photograph, C is the wet weight of gastrocnemius muscle in each group, D is the average area of gastrocnemius muscle fiber cross-section in each group, and E is the myelin sheath thickness of myelinated axons in each group, and the results show that the myelin sheath thickness of myelin sheath nerve fiber of A-M-S group reaches 0.69±0.22 μm, and the wet weight of gastrocnemius muscle and the average area of gastrocnemius muscle fiber cross-section are significantly higher than those of other groups, and there is no statistical difference with the autologous transplantation group (0.92±0.12 μm).

[0177] In summary, the long-distance tissue engineering nerve conduit based on P3HB3HV3HHx three-dimensional porous microspheres and ordered nerve duct is designed in the application, which can simulate the human autologous nerve conduit and provide a valuable conduit for long-distance peripheral nerve injury. At the same time, the adipose mesenchymal stem cells induced to differentiate into Schwann cells are used as seed cells, which solves the problem of limited nerve cell donors, reduces the immune risk, and has a broad clinical application prospect.

[0178] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A tissue engineering nerve conduit, characterized in that: The tissue engineering nerve conduit comprises polyhydroxyalkanoate porous microspheres and a conduit. The polyhydroxyalkanoate porous microspheres are loaded inside the conduit, and the conduit is made of a polyhydroxyalkanoate fiber membrane.

2. The tissue engineering nerve conduit according to claim 1, characterized in that: The catheter is obtained by winding the polyhydroxyalkanoate fiber membrane on a mold; Preferably, the polyhydroxyalkanoate in the polyhydroxyalkanoate porous microspheres and the polyhydroxyalkanoate fiber membranes independently comprises at least one of P3HB3HV3HHx, P34HB or PHBHHx.

3. The tissue engineering nerve conduit according to claim 1 or 2, characterized in that: The polyhydroxyalkanoate fiber membrane includes a polyhydroxyalkanoate ordered fiber membrane or a polyhydroxyalkanoate disordered fiber membrane; Preferably, the average diameter of the polyhydroxyalkanoate ordered fiber membrane is 3.54±1.19 μm; Preferably, the average diameter of the polyhydroxyalkanoate random fiber membrane is 6.43±1.97 μm.

4. The tissue engineering nerve conduit according to any one of claims 1 to 3, characterized in that: The diameter of the polyhydroxyalkanoate porous microspheres is 362.1±127.4 μm; Preferably, the polyhydroxyalkanoate porous microspheres are also loaded with Schwann cells.

5. The method for preparing a tissue engineering nerve conduit according to any one of claims 1 to 4, characterized in that: The preparation method comprises: The polyhydroxyalkanoate porous microspheres and the conduit are prepared, and the polyhydroxyalkanoate porous microspheres are placed in the conduit to obtain the tissue engineering nerve conduit.

6. The method for preparing a tissue engineering nerve conduit according to claim 5, characterized in that: The preparation method of the polyhydroxyalkanoate porous microspheres comprises: mixing the polyhydroxyalkanoate solution with the gelatin solution to obtain an emulsion; mixing a stabilizer with water to obtain an aqueous phase; injecting the sample using a syringe pump to mix the emulsion and the aqueous phase in a T-shaped microfluidic device, generating polymer droplets in the T-shaped tube, and collecting the polymer droplets; The solvent and gelatin in the polymer droplets are removed to obtain the polyhydroxyalkanoate porous microspheres.

7. The method for preparing a tissue engineering nerve conduit according to claim 6, characterized in that: The solvent of the polyhydroxyalkanoate solution includes any one of dichloromethane, chloroform or acetonitrile or a combination of at least two thereof; Preferably, the mass concentration of polyhydroxyalkanoate in the polyhydroxyalkanoate solution is 1% to 30%; Preferably, the solvent of the gelatin solution includes water; Preferably, the gelatin solution further contains polyvinyl alcohol; Preferably, the mass concentration of gelatin in the gelatin solution is 1% to 10%; Preferably, the mass concentration of polyvinyl alcohol in the gelatin solution is 0.1% to 2%.

8. The method for preparing a tissue engineering nerve conduit according to any one of claims 5 to 7, characterized in that: The stabilizer includes polyvinyl alcohol; Preferably, the mass concentration of polyvinyl alcohol in the aqueous phase is 0.1% to 2%.

9. The method for preparing a tissue engineering nerve conduit according to any one of claims 5 to 8, characterized in that: The preparation method of the catheter comprises: A polyhydroxyalkanoate fiber membrane is prepared by an electrospinning method, and the polyhydroxyalkanoate fiber membrane is wound on a mold to assemble and prepare a catheter.

10. The method for preparing a tissue engineering nerve conduit according to any one of claims 5 to 9, characterized in that: The preparation method further comprises the step of mixing the polyhydroxyalkanoate porous microspheres with Schwann cells.

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