Preparation method for bionic multi-channel nerve conduit, and bionic multi-channel nerve conduit

Abstract

A preparation method for a bionic multi-channel nerve conduit, and a bionic multi-channel nerve conduit. A double-layer shape memory nanofiber membrane is used as a receiving substrate, strip-shaped and spaced gel strips are printed on an oriented side, seed cells are planted on the strip-spaced nanofiber membrane, temperature changes are adjusted to cause the fiber membrane at the bottom layer to have a deforming response and curl up, gel strips wrapped by the self-curling fiber membrane self-shape into a conduit, gaps between the gel strips form a multi-channel structure inside the conduit, an inner wall of the multi-channel nerve conduit becomes oriented natural polymer nanofibers which can provide directional guidance for nerve regeneration, and the multi-channel tube structure promotes the formation of a vascular network in the conduit and provides more space for nerve cell growth. The present invention can be applied to long-distance peripheral thick nerve defects, and has good application prospects.

Description

Preparation method of bionic multi-channel nerve conduit and bionic multi-channel nerve conduit Technical Field

[0001] The present application relates to the field of biomedical material preparation, and in particular to a preparation method of a bionic multi-channel nerve conduit and a bionic multi-channel nerve conduit. Background Art

[0002] The incidence of peripheral nerve injury caused by accidents, trauma, and diseases is on the rise. According to statistics, there are more than 5 million cases of peripheral nerve injury worldwide each year, and the resulting medical expenses exceed 10 billion yuan. Nerve defects often lead to loss of sensory and motor function, muscle paralysis, and even lifelong disability in patients. Due to long-term denervation and irreversible muscle atrophy, the restoration of nerve function is very difficult and remains a bottleneck problem. Autologous nerve transplantation is currently the gold standard for the clinical treatment of long-segment nerve defects, but there are problems such as insufficient donors, impaired nerve function in the donor area, and size mismatch. Allogeneic nerve transplantation usually causes immune rejection reactions. Therefore, finding suitable substitutes to repair long-segment peripheral nerve defects and restore nerve function is of great clinical significance.

[0003] Using tissue engineering techniques to construct nerve conduits based on natural or synthetic polymers to replace autologous or allogeneic nerve transplants and repair peripheral nerve defects has become a key area of ​​research in the field of tissue engineering. Currently used nerve conduits in clinical practice have achieved some degree of defect repair effectiveness, but their effectiveness in nerve regeneration remains insufficient, particularly for long-segment nerve injuries. The axial channels of multi-channel nerve conduits can mimic the perineurium structure. Their larger surface area provides more space for cell adhesion, proliferation, and migration, providing more attachment points and contact guidance for the growth of axonal growth cones. These conduits can also help reduce the dispersion of regenerated fibers, enhance nerve function recovery, and are more beneficial for the repair of large, long-segment nerve defects.

[0004] While currently reported multi-channel nerve conduits have improved nerve defect repair to some extent compared to hollow nerve conduits, they still have numerous shortcomings. First, the conduit preparation process is cumbersome and complex. Second, their mechanical properties are poor, making them prone to collapse. Furthermore, the prepared conduits have a single function, making it impossible to load active factors and seed cells that promote nerve repair during the preparation process. Consequently, for long peripheral nerve defects, current nerve conduits still cannot effectively provide physical guidance for the directional extension of axons.

[0005] Summary of the Invention

[0006] In view of this, it is necessary to provide a method for preparing a bionic multi-channel nerve conduit and a bionic multi-channel nerve conduit to address the technical defect that the current nerve conduits are still unable to effectively provide physical guidance for the directional extension of axons for long peripheral nerve defects, which can provide directional guidance for nerve regeneration.

[0007] To solve the above problems, this application adopts the following technical solutions:

[0008] One of the purposes of this application is to provide a method for preparing a biomimetic multi-channel nerve conduit, comprising the following steps:

[0009] Obtaining a double-layer shape memory nanofiber membrane;

[0010] Using the double-layer shape memory nanofiber membrane as a receiving substrate, printing gel strips with strip intervals on one side of the oriented arrangement;

[0011] Seed cells are planted on the nanofiber membrane with strip spacing, and the temperature is adjusted to make the underlying fiber membrane deform and curl up.

[0012] The gel strip wrapped by the self-curling fiber membrane is self-shaped into a conduit, and the gaps between the gel strips form a multi-channel structure inside the conduit, thereby obtaining the bionic multi-channel nerve conduit.

[0013] In some embodiments, the step of obtaining a double-layer shape memory nanofiber membrane specifically includes the following steps:

[0014] dissolving a polylactic acid-polycarbonate copolymer in an organic solvent and stirring to dissolve the copolymer to obtain a polymer solution;

[0015] The polymer solution is formed into randomly arranged nanofibers by an electrospinning process;

[0016] The polymer solution is received on the surface of the randomly arranged nanofibers by adopting an electrostatic spinning process to obtain a double-layer shape memory nanofiber membrane composed of random fibers and oriented fibers.

[0017] In some embodiments, in the step of dissolving the polylactic acid-polycarbonate copolymer in an organic solvent and stirring to dissolve to obtain a polymer solution, the copolymerization ratio of polylactic acid and polycarbonate in the polylactic acid-polycarbonate copolymer is 60:40 to 80:20, and the viscosity is 1-2 dl / g.

[0018] In some embodiments, in the step of forming randomly arranged nanofibers from the polymer solution using an electrospinning process, the electrospinning parameters include: a voltage of 14-16 kV; a propulsion speed of preferably 1-1.8 mL / h; a receiving distance of preferably 10-20 cm, and the needle used is a 22G stainless steel needle; and a low speed of 100-500 rpm for the receiving roller.

[0019] In some embodiments, in the step of receiving the polymer solution on the surface of the randomly arranged nanofibers using an electrospinning process, the roller rotates at a speed of 2000-4000 rpm.

[0020] In some embodiments, the step of printing gel strips with strip spacing on one side of the oriented arrangement using the double-layer shape memory nanofiber membrane as a receiving substrate specifically includes the following steps:

[0021] The double-layer shape memory nanofiber membrane is cut into strips of a certain size, rolled onto a glass rod of a certain diameter, with the fiber layer in contact with the glass rod, and the axial direction of the glass rod is consistent with the fiber orientation direction, and is subjected to high-temperature treatment to give it an initial tubular shape;

[0022] The fiber membrane after the high temperature treatment is removed from the glass rod, flattened at room temperature to maintain a planar membrane structure, and sterilized for later use;

[0023] A double-layer shape memory nanofiber membrane that maintains a planar structure is used as the receiving substrate, and gel strips are printed on the oriented side.

[0024] In some embodiments, the printing ink includes one or more of methacrylated gelatin, methacrylated chitosan, methacrylated silk fibroin, gelatin, collagen, and sodium alginate. The printing ink is also added with a photoinitiator and a nerve growth factor, the photoinitiator includes LAP, and the nerve growth factor includes at least one of NGF, BDNF, and NT-3NGF.

[0025] In some embodiments, the printing parameters are as follows: printing conditions: pressure: 100-150 kPa; moving speed: 3-5 mm / s; strip thickness: 0.15-0.3 mm; number of printing layers: 2-4 layers; number of strips: 3-5 strips; needle: 24-26G.

[0026] In some embodiments, the step of planting seed cells on the nanofiber membrane with strip spacing and adjusting the temperature change to cause the underlying fiber membrane to deform and curl itself in response specifically includes the following steps:

[0027] The Schwann cells are seeded on the fiber membrane separated by strips and placed at room temperature for 2-3 hours to allow the Schwann cells to adhere to the fiber membrane;

[0028] Then it was transferred to a 37°C cell culture incubator, where the underlying fiber membrane underwent temperature-responsive shape memory and curled up.

[0029] The second purpose of this application is to provide a bionic multi-channel nerve conduit, including one prepared by the preparation method, wherein the bionic multi-channel nerve conduit is self-shaped into a conduit by a gel strip wrapped around a self-curling fiber membrane, and the gaps between the gel strips form a multi-channel structure inside the conduit.

[0030] This application adopts the above technical solution, and its beneficial effects are as follows:

[0031] The preparation method and bionic multi-channel nerve conduit provided in the present application use the double-layer shape memory nanofiber membrane as the receiving substrate, print strip-spaced gel strips on the oriented side, plant seed cells on the strip-spaced nanofiber membrane, adjust the temperature change to make the underlying fiber membrane deform in response and curl itself, and the gel strips wrapped thereon by the self-curling fiber membrane are self-formed into a conduit, and the gaps between the gel strips will form a multi-channel structure inside the conduit. The inner wall of the multi-channel nerve conduit is oriented natural polymer nanofibers, which can provide directional guidance for nerve regeneration. The multi-channel structure and the multi-channel lumen structure promote the formation of a vascular network in the conduit and provide more space for nerve cell growth. It is applied to long-distance defects of peripheral thick nerves and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments of the present application or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0033] FIG1 is a flow chart showing the steps of a method for preparing a bionic multi-channel nerve conduit provided by an embodiment of the present invention.

[0034] FIG2 is a macroscopic photograph and SEM image of the multi-channel catheter provided in Example 2 of the present invention.

[0035] FIG3 is an in vitro release curve of catheter-loaded NGF provided in Example 3 of the present invention.

[0036] FIG4 shows the distribution of living cells loaded on the catheter in the catheter provided by Example 5 of the present invention.

[0037] DETAILED DESCRIPTION

[0038] The following describes in detail embodiments of the present application, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application.

[0039] In the description of this application, it should be understood that the terms "upper", "lower", "horizontal", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on this application.

[0040] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. Throughout the description of this application, "plurality" means two or more, unless otherwise specifically defined.

[0041] In order to make the purpose, technical solutions and advantages of this application more clear, this application is further described in detail below with reference to the accompanying drawings and embodiments.

[0042] Please refer to FIG1 , which is a flowchart of the method for preparing a bionic multi-channel nerve conduit provided in this embodiment, including the following steps S110 to S140 . The specific implementation of each step is described in detail below.

[0043] Step S110: obtaining a double-layer shape memory nanofiber membrane.

[0044] In this embodiment, the step of obtaining a double-layer shape memory nanofiber membrane specifically includes the following steps S111 to S113 , and the implementation method of each step is described in detail below.

[0045] Step S111: dissolving the polylactic acid-polycarbonate copolymer in an organic solvent, and stirring to dissolve the mixture to obtain a polymer solution.

[0046] Specifically, polylactic acid-polycarbonate copolymer (PLATMC) is dissolved in an organic solvent hexafluoroisopropanol, and is placed on a magnetic stirrer and continuously stirred until it is completely dissolved to obtain a uniform polymer solution.

[0047] Furthermore, the copolymerization ratio of polylactic acid to polycarbonate in the polylactic acid-polycarbonate copolymer is 60:40 to 80:20, the viscosity is 1-2 dl / g, and the glass transition temperature is close to 37°C.

[0048] Step S112: using an electrospinning process to form randomly arranged nanofibers from the polymer solution.

[0049] Specifically, the dissolved PLATMC solution is drawn into a syringe and placed on a propulsion pump. The positive electrode of a high-voltage power supply is connected to the needle tip, while the negative electrode is connected to a collection device, a rotating roller with controllable speed. The propulsion speed, load voltage, and receiving distance are set, and the propulsion pump is activated. Under the action of high-voltage static electricity, the polymer solution forms fibers. The receiving roller is controlled to rotate at a low speed (100-500 rpm) to produce randomly arranged nanofibers.

[0050] Furthermore, the electrospinning parameters used in this embodiment include: voltage of 14-16 kV; propulsion speed of preferably 1-1.8 mL / h; receiving distance of preferably 10-20 cm, and the needle used is a 22G stainless steel needle; the speed of the receiving roller is a low speed of 100-500 rpm.

[0051] Step S113: using an electrospinning process to receive the polymer solution on the surface of the randomly arranged nanofibers to obtain a double-layer shape memory nanofiber membrane composed of random fibers and oriented fibers.

[0052] Specifically, the PLATMC solution is sucked into the syringe, and the rotation speed of the receiving roller is increased to above 2500 rpm on the propulsion pump. The oriented nanofibers are further received on the random fibers received in step S112, and finally a double-layer nanofiber membrane composite of random fibers and oriented fibers is obtained.

[0053] Furthermore, the roller speed in the electrospinning process is 2000-4000 rpm, and the thickness of the fiber membrane finally prepared is about 0.1 mm.

[0054] It can be understood that this embodiment uses shape memory polymer to utilize electrospinning technology to prepare a double-layer nanofiber membrane with random and oriented fibers stacked by adjusting the rotation speed of the receiving roller. The fibers in the random layer have temperature-responsive shape memory properties, and the oriented layer can effectively guide the directional growth of cells.

[0055] Step S120: using the double-layer shape memory nanofiber membrane as a receiving substrate, printing gel strips with strip intervals on the oriented side.

[0056] In this embodiment, the step of printing gel strips with strip intervals on one side of the oriented arrangement using the double-layer shape memory nanofiber membrane as a receiving substrate specifically includes the following steps S121 to S123, and the implementation method of each step is described in detail below.

[0057] Step S121: Cut the double-layer shape memory nanofiber membrane into strips of a certain size, roll it onto a glass rod of a certain diameter, with the side in contact with the glass rod being an oriented fiber layer, and the axial direction of the glass rod being consistent with the fiber orientation direction, and give it an initial tubular shape through high-temperature treatment.

[0058] In some embodiments, the high temperature treatment temperature is 80° C., and the high temperature treatment time is 20 min.

[0059] Step S122: The fiber membrane after the high-temperature treatment is removed from the glass rod, flattened at room temperature to maintain a planar membrane structure, and sterilized (sterilized by soaking in 75% alcohol) for later use.

[0060] It can be understood that in this embodiment, the shape memory fiber is curled on a glass rod of a certain size and subjected to high-temperature plasticization at 80°C to obtain a fiber scaffold with an initial shape of a tube. The fiber layer of the orientation layer is located on the inner wall of the tube, and the orientation direction is consistent with the axial direction of the glass rod.

[0061] Step S123: using the double-layer shape memory nanofiber membrane maintaining a planar structure as a receiving substrate, printing a gel strip on the oriented side.

[0062] In this embodiment, the printing ink includes one or more of methacrylated gelatin, methacrylated chitosan, methacrylated silk fibroin, gelatin, collagen, and sodium alginate. The printing ink is also added with a photoinitiator and a nerve growth factor. The photoinitiator includes LAP, and the nerve growth factor includes at least one of NGF, BDNF, and NT-3NGF to achieve the loading of active factors.

[0063] In this embodiment, the printing parameters are as follows: printing conditions: pressure: 100-150 kPa; moving speed: 3-5 mm / s; strip thickness: 0.15-0.3 mm; number of printing layers: 2-4 layers; number of strips: 3-5 strips; needle: 24-26G.

[0064] It can be understood that in this embodiment, the shaped tubular scaffold is unfolded at room temperature to maintain a temporary planar membrane structure, and 3-5 gel strips are printed on the surface of the planar fiber membrane using biological 3D printing technology.

[0065] It is understood that the size of the gel strip can be controlled by adjusting printing parameters, including the size of the printing needle, the number of printing layers, the printing speed, etc.

[0066] Step S130: Planting seed cells on the nanofiber membranes spaced apart by strips, and adjusting the temperature to cause the fiber membranes at the bottom to deform and curl themselves up.

[0067] In this embodiment, the steps of planting seed cells on the nanofiber membrane with strip spacing and adjusting the temperature change to cause the underlying fiber membrane to deform and curl up specifically include the following steps:

[0068] Schwann cells were seeded on the fiber membrane separated by strips and placed at room temperature for 2-3 hours to allow the Schwann cells to adhere to the fiber membrane; then the membrane was transferred to a 37°C cell culture incubator, and the underlying fiber membrane developed temperature-responsive shape memory and self-curled.

[0069] After printing, Schwann cells are seeded onto the fiber membrane between the strips and left at room temperature for 3 hours to allow the cells to fully adhere to the fiber membrane. The tube is then transferred to a 37°C cell culture incubator, where the underlying fiber membrane undergoes temperature-responsive shape memory, restoring its initial conduit shape. The gel strips are then wrapped inside, and the gaps between the gel strips become a multi-channel structure within the conduit, resulting in a bioactive conduit carrying both active factors and seed cells.

[0070] Step S140: The gel strip wrapped by the self-curling fiber membrane is self-shaped into a conduit, and the gaps between the gel strips form a multi-channel structure inside the conduit, thereby obtaining the bionic multi-channel nerve conduit.

[0071] It can be understood that the number of channels inside the nerve conduit can be controlled by controlling the number of gel strips.

[0072] Compared with the existing multi-channel catheter preparation method, the bionic multi-channel nerve catheter preparation method provided by the present invention uses the shape memory of the material itself to achieve rapid self-forming of the catheter, eliminating the step of manually curling it into a catheter. The construction of the multi-channel structure does not require the use of a mold, thus avoiding the damage to the catheter structure caused by removing the mold. By achieving dual loading of active factors and seed cells during the catheter preparation and molding process, the catheter is given more functions. By controlling the number and size of the printed gel strips, the number and size of the channels in the catheter can be controlled, and it can be used for bridging repair of peripheral nerve defects in different locations and diameters.

[0073] The bionic multi-channel nerve conduit provided in this application has an inner wall of oriented natural polymer nanofibers that can provide directional guidance for nerve regeneration. The multi-channel structure and multi-channel lumen structure promote the formation of a vascular network in the conduit and provide more space for nerve cell growth. It is used for long-distance defects of peripheral thick nerves and has good application prospects.

[0074] The technical solution of the present application is described in detail below using this principle diagram in combination with specific embodiments.

[0075] Example 1: Preparation of a double-layer shape memory nanofiber membrane

[0076] 1) Weigh 1.8 g of a 70:30 polylactic acid-polycarbonate copolymer (PLATMC) and dissolve it in 15 mL of hexafluoroisopropanol to make a 12% (w / v) solution. Stir on a magnetic stirrer until the solution is completely dissolved.

[0077] 2) First, take 10 mL of the polylactic acid-polycarbonate copolymer solution and add it to a 10 mL syringe. Place the syringe on a propulsion pump and set the propulsion speed to 1.8 mL / h, the receiving distance to 12 cm, and the load voltage to 16 kV. The receiving roller speed is set to 300 rpm to produce a random PLATMC nanofiber layer. Then, take the remaining 5 mL of PLATMC solution and add it to the syringe to continue electrospinning. Now, set the receiving roller speed to 2600 rpm to receive a layer of aligned fibers on the surface of the random PLATMC nanofiber layer.

[0078] Example 2: Self-forming preparation of NGF-loaded multi-channel conduits

[0079] 1) Weigh 1.8 g of a 70:30 polylactic acid-polycarbonate copolymer (PLATMC) and dissolve it in 15 mL of hexafluoroisopropanol to make a 12% (w / v) solution. Stir on a magnetic stirrer until the solution is completely dissolved.

[0080] 2) First, take 10 mL of the polylactic acid-polycarbonate copolymer solution and add it to a 10 mL syringe. Place the syringe on a propulsion pump and set the propulsion speed to 1.8 mL / h, the receiving distance to 12 cm, and the load voltage to 16 kV. The receiving roller speed is set to 300 rpm to produce a random PLATMC nanofiber layer. Then, take the remaining 5 mL of PLATMC solution and add it to the syringe to continue electrospinning. Now, set the receiving roller speed to 2600 rpm to receive a layer of aligned fibers on the surface of the random PLATMC nanofiber layer.

[0081] 3) The prepared double-layer fiber membrane was cut into rectangular strips of a desired size, with the fibers oriented along the long side of the strip. The strips were 15 mm long and 10 mm wide, respectively. The strips were then rolled onto a 0.6 mm diameter glass rod, with the oriented fiber layer in contact with the rod and the fiber orientation aligned with the rod. The strips were then placed in an 80°C oven for 20 minutes. The membranes were then removed from the rod and flattened at room temperature.

[0082] 4) Prepare a mixed solution of methacrylated gelatin (GelMA) and gelatin at a concentration of 10% (w / v) GelMA and 2% (w / v) gelatin. Add 0.25% photoinitiator LAP and a certain amount of nerve growth factor (NGF) (10 mg / mL) to the ink. Load the prepared printing ink into the cartridge of a 3D bioprinter. Set the printing parameters as follows: pressure: 130 kPa; travel speed: 4 mm / s; strip thickness: 0.2 mm; strip spacing: 1.2 mm; number of print layers: 3; printing tip: 26G (0.21 mm); blue light curing time: 10-20 seconds. Print five gel strips onto the fiber membrane prepared in 3).

[0083] 5) After printing, the tube is transferred to 37°C. The underlying fiber membrane undergoes temperature-responsive shape memory self-curling, restoring its original tube shape and enveloping the gel strips. The spaces between the gel strips form the multi-channel structure within the tube. The tube structure morphology, observed using a scanning electron microscope, is shown in Figure 2.

[0084] Example 3: In vitro release of NGF loaded by gel strips

[0085] 1) Weigh 1.8 g of a 70:30 polylactic acid-polycarbonate copolymer (PLATMC) and dissolve it in 15 mL of hexafluoroisopropanol to make a 12% (w / v) solution. Stir on a magnetic stirrer until the solution is completely dissolved.

[0086] 2) First, take 10 mL of the polylactic acid-polycarbonate copolymer solution and add it to a 10 mL syringe. Place the syringe on a propulsion pump and set the propulsion speed to 1.8 mL / h, the receiving distance to 12 cm, and the load voltage to 16 kV. The receiving roller speed is set to 300 rpm to produce a random PLATMC nanofiber layer. Then, take the remaining 5 mL of PLATMC solution and add it to the syringe to continue electrospinning. Now, set the receiving roller speed to 2600 rpm to receive a layer of aligned fibers on the surface of the random PLATMC nanofiber layer.

[0087] 3) The prepared double-layer fiber membrane was cut into rectangular strips of a desired size, with the fibers oriented along the long side of the strip. The strips were 15 mm long and 10 mm wide, respectively. The strips were then rolled onto a 0.6 mm diameter glass rod, with the oriented fiber layer in contact with the rod and the fiber orientation aligned with the rod. The strips were then placed in an 80°C oven for 20 minutes. The membranes were then removed from the rod and flattened at room temperature.

[0088] 4) Prepare a mixed solution of methacrylated gelatin (GelMA) and gelatin at a concentration of 10% (w / v) GelMA and 2% (w / v) gelatin. Add 0.25% photoinitiator LAP and a certain amount of nerve growth factor (NGF) (10 mg / mL) to the ink. Load the prepared printing ink into the cartridge of a 3D bioprinter. Set the printing parameters as follows: pressure: 130 kPa; travel speed: 4 mm / s; strip thickness: 0.2 mm; strip spacing: 1.2 mm; number of print layers: 3; printing tip: 26G (0.21 mm); blue light curing time: 10-20 seconds. Print five gel strips onto the fiber membrane prepared in 3).

[0089] 5) After printing, the tube is transferred to 37°C. Under the influence of temperature, the underlying fiber membrane undergoes temperature-responsive shape memory self-curling, restoring the initial catheter shape and wrapping the gel strips inside. The gaps between the gel strips become a multi-channel structure inside the catheter, resulting in a multi-channel nerve catheter.

[0090] 6) The prepared catheters were immersed in EP tubes containing 1 mL of PBS and placed in a shaker at 37°C. The PBS solution was removed from the EP tubes at 3 hours, 7 hours, 1 day, 7 days, 14 days, 30 days, and 60 days. The amount of NGF released from the PBS was measured using an ELISA kit (Rat NGF / NGF beta ELISA Kit). The in vitro release curve of NGF was plotted based on the release amount at different time points, as shown in Figure 3.

[0091] Example 4: Preparation of Schwann cell-loaded multichannel catheters

[0092] 1) Weigh 1.8 g of a 70:30 polylactic acid-polycarbonate copolymer (PLATMC) and dissolve it in 15 mL of hexafluoroisopropanol to make a 12% (w / v) solution. Stir on a magnetic stirrer until the solution is completely dissolved.

[0093] 2) First, take 10 mL of the polylactic acid-polycarbonate copolymer solution and add it to a 10 mL syringe. Place the syringe on a propulsion pump and set the propulsion speed to 1.8 mL / h, the receiving distance to 12 cm, and the load voltage to 16 kV. The receiving roller speed is set to 300 rpm to produce a random PLATMC nanofiber layer. Then, take the remaining 5 mL of PLATMC solution and add it to the syringe to continue electrospinning. Now, set the receiving roller speed to 2600 rpm to receive a layer of aligned fibers on the surface of the random PLATMC nanofiber layer.

[0094] 3) Cut the prepared double-layer fiber membrane into rectangular strips of a desired size, with the fibers oriented along the long side of the strip. The length and width of the strips are 15 mm and 10 mm, respectively. Wrap the strips around a 0.6 mm diameter glass rod, with the oriented fiber layer in contact with the rod and the fiber orientation aligned with the rod. Heat-harden the strips in an 80°C oven for 20 minutes. Remove the strips from the rod and flatten them at room temperature. Sterilize the strips by soaking them in 75% alcohol before use.

[0095] 4) Prepare a mixed solution of methacrylated gelatin (GelMA) and gelatin at a concentration of 10% (w / v) GelMA and 2% (w / v) gelatin. Add 0.25% photoinitiator LAP and a certain amount of nerve growth factor (NGF) (10 mg / mL) to the ink. Load the prepared printing ink into the cartridge of a 3D bioprinter. Set the printing parameters as follows: pressure: 130 kPa; travel speed: 4 mm / s; strip thickness: 0.2 mm; strip spacing: 1.2 mm; number of print layers: 3; printing tip: 26G (0.21 mm); blue light curing time: 10-20 seconds. Print five gel strips onto the fiber membrane prepared in 3).

[0096] 5) After printing, Schwann cells were seeded onto the fiber membrane between the strips and allowed to stand at room temperature for 3 hours to allow the cells to fully adhere to the membrane. The membrane was then transferred to a 37°C cell culture incubator. The underlying fiber membrane, influenced by the temperature, self-curled in response to the temperature, regaining its original conduit shape and enveloping the gel strips. The gaps between the gel strips formed the multi-channel structure within the conduit, resulting in a bioactive Schwann cell-loaded neural conduit.

[0097] Example 5: Distribution of Schwann cells in a multi-channel catheter

[0098] 1) Weigh 1.8 g of a 70:30 polylactic acid-polycarbonate copolymer (PLATMC) and dissolve it in 15 mL of hexafluoroisopropanol to make a 12% (w / v) solution. Stir on a magnetic stirrer until the solution is completely dissolved.

[0099] 2) First, take 10 mL of the polylactic acid-polycarbonate copolymer solution and add it to a 10 mL syringe. Place the syringe on a propulsion pump and set the propulsion speed to 1.8 mL / h, the receiving distance to 12 cm, and the load voltage to 16 kV. The receiving roller speed is set to 300 rpm to produce a random PLATMC nanofiber layer. Then, take the remaining 5 mL of PLATMC solution and add it to the syringe to continue electrospinning. Now, set the receiving roller speed to 2600 rpm to receive a layer of aligned fibers on the surface of the random PLATMC nanofiber layer.

[0100] 3) Cut the prepared double-layer fiber membrane into rectangular strips of a desired size, with the fibers oriented along the long side of the strip. The length and width of the strips are 15 mm and 10 mm, respectively. Wrap the strips around a 0.6 mm diameter glass rod, with the oriented fiber layer in contact with the rod and the fiber orientation aligned with the rod. Heat-harden the strips in an 80°C oven for 20 minutes. Remove the strips from the rod and flatten them at room temperature. Sterilize the strips by soaking them in 75% alcohol before use.

[0101] 4) Prepare a mixed solution of methacrylated gelatin (GelMA) and gelatin at a concentration of 10% (w / v) GelMA and 2% (w / v) gelatin. Add 0.25% photoinitiator LAP and a certain amount of nerve growth factor (NGF) (10 mg / mL) to the ink. Load the prepared printing ink into the cartridge of a 3D bioprinter. Set the printing parameters as follows: pressure: 130 kPa; travel speed: 4 mm / s; strip thickness: 0.2 mm; strip spacing: 1.2 mm; number of print layers: 3; printing tip: 26G (0.21 mm); blue light curing time: 10-20 seconds. Print five gel strips onto the fiber membrane prepared in 3).

[0102] 5) After printing, Schwann cells are seeded onto the fiber membrane between the strips and allowed to stand at room temperature for 3 hours to allow the cells to fully adhere to the fiber membrane. The membrane is then transferred to a 37°C cell culture incubator. The temperature influences the underlying fiber membrane, where it undergoes temperature-responsive shape memory self-curling, restoring its original conduit shape and enveloping the gel strips. The gaps between the gel strips form the multi-channel structure within the conduit, resulting in a bioactive Schwann cell-loaded neural conduit.

[0103] 6) After culturing the Schwann cell-loaded ducts in vitro at 37°C in a cell culture incubator for 5 days, the cells were fixed with 4% paraformaldehyde for 2 hours, washed three times with PBS, treated with 500-fold diluted Triton-X100 for 10 minutes, washed three times with PBS, and then treated with rhodamine phalloidin (1:200 dilution) for 40 minutes in the dark to stain the cytoskeleton (protect from light). After staining, the cells were washed three times with PBS.

[0104] 7) The stained, cell-loaded catheters were dehydrated in a 30% (w / v) sucrose solution for 24 hours. The sections were then cryo-embedded in OCT at -80°C for 24 hours. The embedded catheters were then cryo-sectioned (cross-sectioned) using a cryostat, and the distribution of cells within the catheters was observed using a fluorescence microscope. The results demonstrated that the cells were evenly distributed and grown throughout the various channels of the catheters. See Figure 4 for the results.

[0105] The multi-channel nerve conduit involved in the present invention is not limited to the preparation method in the example, and includes a stent having the same structure as the multi-channel nerve conduit involved in the present invention obtained by using similar shape memory polymers and bio-inks and using similar preparation methods.

[0106] It can be understood that the various technical features of the above-described embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the various technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0107] The above are merely preferred embodiments of the present application and only specifically describe the technical principles of the present application. These descriptions are intended only to explain the principles of the present application and should not be construed in any way as limiting the scope of protection of the present application. Based on the explanations herein, any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present application, as well as other specific implementations of the present application that can be conceived by those skilled in the art without inventive effort, shall be included within the scope of protection of the present application.

Claims

1. A method for preparing a bionic multi-channel nerve conduit, characterized in that: The steps include: Obtaining a double-layer shape memory nanofiber membrane; Using the double-layer shape memory nanofiber membrane as a receiving substrate, printing gel strips with strip intervals on one side of the oriented arrangement; Seed cells are planted on the nanofiber membranes separated by strips, and the temperature is adjusted to make the underlying fiber membranes deform and curl themselves up. The gel strip wrapped by the self-curling fiber membrane is self-shaped into a conduit, and the gaps between the gel strips form a multi-channel structure inside the conduit, thereby obtaining the bionic multi-channel nerve conduit.

2. The method for preparing the bionic multi-channel nerve conduit according to claim 1, characterized in that: The step of obtaining a double-layer shape memory nanofiber membrane specifically includes the following steps: Dissolving a polylactic acid-polycarbonate copolymer in an organic solvent and stirring to dissolve the copolymer to obtain a polymer solution; The polymer solution is formed into randomly arranged nanofibers by an electrospinning process; The polymer solution is received on the surface of the randomly arranged nanofibers by an electrostatic spinning process to obtain a double-layer shape memory nanofiber membrane composited with random fibers and oriented fibers.

3. The method for preparing the bionic multi-channel nerve conduit according to claim 2, characterized in that: In the step of dissolving the polylactic acid-polycarbonate copolymer in an organic solvent and stirring to dissolve to obtain a polymer solution, the copolymerization ratio of polylactic acid and polycarbonate in the polylactic acid-polycarbonate copolymer is 60:40 to 80:20, and the viscosity is 1-2dl / g.

4. The method for preparing the bionic multi-channel nerve conduit according to claim 2, characterized in that: In the step of using an electrospinning process to form randomly arranged nanofibers from the polymer solution, the electrospinning parameters include: a voltage of 14-17 kV; a propulsion speed of preferably 1-1.8 mL / h; a receiving distance of preferably 10-20 cm, and a 22G stainless steel needle; and a low speed of 100-500 rpm for the receiving roller.

5. The method for preparing the bionic multi-channel nerve conduit according to claim 2, characterized in that: In the step of receiving the polymer solution on the surface of the randomly arranged nanofibers by adopting an electrospinning process, the roller speed is 2000-4000 rpm.

6. The method for preparing the bionic multi-channel nerve conduit according to claim 1, characterized in that: The step of using the double-layer shape memory nanofiber membrane as a receiving substrate and printing gel strips with strip intervals on one side of the oriented arrangement specifically includes the following steps: The double-layer shape memory nanofiber membrane is cut into strips of a certain size, and is rolled onto a glass rod of a certain diameter, wherein the side in contact with the glass rod is an oriented fiber layer, and the axial direction of the glass rod is consistent with the fiber orientation direction, and is subjected to high temperature treatment to give it an initial tubular shape; The fiber membrane after the high temperature treatment is taken off from the glass rod, flattened at room temperature to keep a planar membrane structure, and sterilized for later use; A double-layer shape memory nanofiber membrane that maintains a planar structure is used as a receiving substrate, and gel strips are printed on the oriented side.

7. The method for preparing the bionic multi-channel nerve conduit according to claim 6, characterized in that: The printing ink comprises one or more of methacrylated gelatin, methacrylated chitosan, methacrylated silk fibroin, gelatin, collagen, and sodium alginate. The printing ink is also added with a photoinitiator and a nerve growth factor. The photoinitiator comprises LAP, and the nerve growth factor comprises at least one of NGF, BDNF, NT-3, and NGF.

8. The method for preparing the bionic multi-channel nerve conduit according to claim 6, characterized in that: The printing parameters are as follows: printing conditions: pressure: 100-150 kpa; moving speed: 3-5 mm / s; strip thickness: 0.15-0.3 mm; number of printing layers: 2-4 layers; number of strips: 3-5 strips; needle: 24-26G.

9. The method for preparing the bionic multi-channel nerve conduit according to claim 1, characterized in that: The step of planting seed cells on the nanofiber membrane with strip spacing and adjusting the temperature change to make the fiber membrane at the bottom deform and curl itself, specifically includes the following steps: The Schwann cells are planted on the fiber membrane separated by strips and placed at room temperature for 2-3 hours to allow the Schwann cells to adhere to the fiber membrane; Then it was transferred to a 37°C cell culture incubator, where the underlying fiber membrane underwent temperature-responsive shape memory and self-curled.

10. A bionic multi-channel nerve conduit, characterized in that: The bionic multi-channel nerve conduit is prepared by the preparation method described in claims 1-9, wherein the bionic multi-channel nerve conduit is self-shaped into a conduit by a gel strip wrapped by a self-curling fiber membrane, and the gaps between the gel strips form a multi-channel structure inside the conduit.

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