Nerve cap and preparation method thereof

Abstract

The invention relates to the field of biomedicine, and discloses a nerve cap and a preparation method thereof. The nerve cap is a tubular body with an open end and a closed end, and the open end is provided with a convergent section with the inner diameter decreasing gradually from the open end to the closed end. The nerve cap is composed of nanofibers which are arranged perpendicular to the axial direction. The nerve cap can gradually limit growth of axons in a three-dimensional space, disordered growth of the axons is reduced to a greater extent, and then formation of neuroma is better prevented.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a neural cap and a preparation method thereof. Background Art

[0002] Neuromas are caused by the proliferation of Schwann cells at the site of injury, which produces axonal growth as cells attempt to restore axonal continuity at the ends of damaged nerves. The regenerated axons are unable to reach their distal targets, but instead form a tangled ball of material, which traps the axons and causes neuropathic pain. For example, a higher incidence of neuromas can occur after nerve surgery or the severance of a body part (limb amputation). The typical characteristics of neuroma pain are spontaneous hyperalgesia and allodynia, which can persist after the wound is completely remodeled, seriously affecting the patient's quality of life.

[0003] Current surgical treatment options include neuroma resection, traction neurectomy (high recurrence rate), or various forms of nerve coaptation or nerve burial (complex process). Although there are many ways to treat neuromas clinically, there is no "gold standard" for neuroma treatment. Surgery is not always feasible, it may expose patients to higher risks, and it cannot fully isolate the damaged nerve from the external environment.

[0004] Studies have shown that capping nerve ends (nerve caps) can isolate axons from external signals, reduce inappropriate and irregular regeneration of axons, and prevent the formation of neuromas. The existing nerve cap structure is mainly a tubular body with a closed end and an open end (such as patent CN107427605B), and the tubular structure at the open end maintains a consistent diameter throughout the body. This spatial characteristic lacks the ability to further limit the formation of neuromas. In addition, no prior art has paid attention to the microscopic morphology of the nerve cap, and there has been no relevant report on changing the arrangement of nanofibers in the nerve cap to affect its effect in preventing the formation of neuromas. Summary of the invention

[0005] In order to solve the above technical problem, that is, the existing nerve cap has limited effect in preventing the formation of neuroma, the present invention provides a nerve cap and a preparation method thereof. The nerve cap of the present invention can gradually restrict the growth of axons in three-dimensional space and reduce the disordered growth of axons to a greater extent, thereby better preventing the formation of neuroma.

[0006] The specific technical scheme of the present invention is: In a first aspect, the present invention provides a neural cap, which is a tubular body with an open end and a closed end, wherein the open end is provided with a converging section whose inner diameter decreases from the open end to the closed end; the neural cap is composed of nanofibers arranged perpendicular to the axial direction.

[0007] In the nerve cap of the present invention, by providing a structure with an inner diameter that changes in a specific manner at the open end, the growth of axons can be gradually restricted in a three-dimensional spatial structure; at the same time, by using nanofibers arranged in a directional manner (perpendicular to the axial direction of the nerve cap), the regeneration state of the transected nerve can be affected through contact guidance, the disordered growth of axons can be inhibited, and the inappropriate and irregular regeneration of nerve fibers can be reduced. Through the above-mentioned comprehensive design of macroscopic and microscopic structures, the structure and morphology of the severed nerve can be improved to a greater extent, and at the same time, an isolation barrier can be provided for the damaged nerve to be protected from nerve-affecting factors and mechanical stimulation in the external environment, thereby better reducing the development of painful nerves and preventing and inhibiting the formation of neuromas.

[0008] Preferably, the converging section is between the open end and the closed end of the nerve cap; or, in the nerve cap, the converging section has a constant diameter section with a constant inner diameter from the end with the smallest inner diameter to the closed end.

[0009] Preferably, in the convergent section, the ratio of the inner diameters at both ends is 2:0.5-1.9, the inner diameter of the end with the smallest inner diameter is 2-5 mm; and the length of the convergent section is 5-20 mm.

[0010] In the contraction section, when the inner diameter of the smallest end is too small, the space at the position of the nerve cap will be too small, and the effective length of the insertable nerve stump will be limited, making it inconvenient for the nerve cap to be anchored in the surrounding tissue, which will make the surgical operation process more complicated.

[0011] Preferably, the average diameter of the nanofibers is 240-410 nm.

[0012] Preferably, the nanofibers are collagen fibers.

[0013] Collagen materials have good cell affinity and can gradually integrate into the patient's own tissues to form a physical barrier to the surrounding soft tissues.

[0014] In a second aspect, the present invention provides a method for preparing the neural cap, comprising the following steps: injecting a nanofiber dispersion into a mold, arranging the nanofibers perpendicular to the axis of the neural cap by directional freeze drying, adding a crosslinking agent for crosslinking treatment, and demolding to obtain a neural cap.

[0015] The present invention aims at a more complex neural cap structure, and can obtain an integrally formed neural cap by filling a mold with a nanofiber dispersion, directional freeze drying and demoulding, which has a reference significance for the design and construction of complex biomaterials.

[0016] Preferably, the directional freeze-drying comprises the following steps: placing the mold horizontally (ie, in the direction in which the neural cap lies horizontally, with the axis of the neural cap parallel to the horizontal plane), placing it on the liquid nitrogen surface for freezing, and then freeze-drying.

[0017] The mechanism of directional freeze drying is Figure 7 As shown ( Figure 7 It is only used to illustrate the mechanism of directional freeze-drying, and the cylindrical container structure and direction do not represent the structure and direction of the nerve cap and mold in the present invention). Specifically: during the directional freeze-drying process, when the mold is placed horizontally on the liquid nitrogen surface, the heat is absorbed by the evaporation of the liquid nitrogen, so that the nanofiber dispersion in the mold begins to freeze from the bottom of the mold, causing ice crystals to grow in a single temperature gradient direction, thereby affecting the arrangement direction of the nanofibers, making them perpendicular to the axial arrangement of the nerve cap.

[0018] Furthermore, the freezing time is 35 to 60 minutes, the depth of the liquid nitrogen is 15 to 24 cm, and the liquid nitrogen is replenished every 25 to 50 minutes during the freezing period.

[0019] Furthermore, the freeze-drying time is 35 to 48 hours.

[0020] Preferably, the material of the mold is one or more of silicone, acrylonitrile-butadiene-styrene copolymer (ABS) and polystyrene (PS).

[0021] Preferably, the cross-linking agent is EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide) and NHS (N-hydroxysuccinimide); the addition of the cross-linking agent for cross-linking treatment comprises the following steps: immersing the mold in a solution containing the cross-linking agent to perform a cross-linking reaction, and then transferring the mold to water for immersion, and then taking it out for freeze-drying.

[0022] Preferably, the concentration of the nanofiber dispersion is 15-20wt%; the preparation steps of the nanofiber dispersion include: spinning the polymer material into a nanofiber membrane through solution spinning; crushing the nanofiber membrane and dispersing it in a dispersion medium to obtain the nanofiber dispersion.

[0023] Furthermore, the thickness of the nanofiber membrane is 0.07-0.18 mm.

[0024] Furthermore, the solution spinning adopts air-spinning method, and the parameters are designed as follows: the inner diameter of the syringe needle is 23-30G, the propulsion speed of the syringe is 4-9mL / h, the air pressure at the syringe needle is 0.24-0.64Mpa, the distance from the syringe to the receiving roller is 24-50cm, and the rotation speed of the receiving roller is 350-1000rpm.

[0025] Furthermore, the process of crushing the nanofiber membrane and dispersing it into a dispersion medium comprises the following steps: after cutting the nanofiber membrane, crushing it with a homogenizer and dispersing it into a dispersion medium, the speed of the homogenizer is 10000-15000 rpm, and the stirring time is 20-45 min.

[0026] Compared with the prior art, the present invention has the following advantages: (1) The nerve cap of the present invention is capable of gradually restricting the growth of axons in three-dimensional space and reducing the disordered growth of axons to a greater extent by providing a converging section with an inner diameter decreasing from the open end to the closed end at the open end and arranging the fibers in the nerve cap perpendicular to the axial direction, thereby better preventing the formation of neuromas.

[0027] (2) The nerve cap of the present invention has good adaptability during surgery and is suitable for anatomical areas with limited or no alternative muscle tissue, such as buried muscles or bones of various diameters. The sealed end in the nerve cap facilitates anchoring the nerve cap to the surrounding tissue, away from surgical incision and mechanical stimulation.

[0028] (3) The present invention uses collagen fibers as the material of the nerve cap, which can make the nerve cap have better cell affinity and can gradually integrate into the patient's own tissues to form a physical barrier to the surrounding soft tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a three-dimensional structural schematic diagram of the neural cap in the present invention.

[0030] Figure 2 yes Figure 1 main view.

[0031] Figure 3 It is a schematic diagram of the external structure of the mold in the present invention.

[0032] Figure 4 It is a structural schematic diagram of the half mold in the present invention.

[0033] Figure 5 This is a scanning electron microscope image of the nanofiber membrane prepared in Example 1.

[0034] Figure 6 This is a statistical diagram of the fiber diameters in the nanofiber membrane prepared in Example 1.

[0035] Figure 7 It is a schematic diagram of the mechanism of directional freeze-drying in the present invention.

[0036] The figures are marked as follows: 1-open end, 2-closed end, 3-constriction section, 4-injection port, 5-drainage groove. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with the embodiments.

[0038] Overall embodiment A neural cap is a tubular body with an open end and a closed end, wherein the open end is provided with a convergence section with an inner diameter decreasing from the open end to the closed end; the neural cap is composed of nanofibers arranged perpendicular to the axial direction.

[0039] In some embodiments, the nerve cap has a constriction section between its open end and its closed end. In other embodiments, the nerve cap has a constant diameter section between the end with the smallest inner diameter of the constriction section and the closed end.

[0040] In some specific embodiments, in the converging section, the ratio of the inner diameters at both ends is 2:0.5-1.9, the inner diameter of the end with the smallest inner diameter is 2-5 mm; and the length of the converging section is 5-20 mm.

[0041] In some embodiments, the nanofibers are collagen fibers with an average diameter of 240-410 nm.

[0042] A method for preparing the neural cap comprises the following steps: injecting a nanofiber dispersion into a mold, arranging the nanofibers perpendicular to the axis of the neural cap by directional freeze drying, adding a crosslinking agent for crosslinking treatment, and demoulding to obtain the neural cap.

[0043] In some specific embodiments, the concentration of the nanofiber dispersion is 15-20wt%; the preparation steps of the nanofiber dispersion include: spinning the polymer material into a nanofiber membrane through solution spinning; crushing the nanofiber membrane and dispersing it into a dispersion medium to obtain a nanofiber dispersion. In this specific embodiment: Optionally or preferably, the thickness of the nanofiber membrane is 0.07 to 0.18 mm; Optionally or preferably, the solution spinning adopts air-spinning method, and the parameters are designed as follows: the inner diameter of the syringe needle is 23-30G, the propulsion speed of the syringe is 4-9mL / h, the air pressure at the syringe needle is 0.24-0.64Mpa, the distance from the syringe to the receiving roller is 24-50cm, and the rotation speed of the receiving roller is 350-1000rpm; Optionally or preferably, the process of crushing the nanofiber membrane and dispersing it into a dispersion medium comprises the following steps: after cutting the nanofiber membrane, crushing it with a homogenizer and dispersing it into a dispersion medium, the speed of the homogenizer is 10000-15000 rpm, and the stirring time is 20-45 min.

[0044] In some specific embodiments, the directional freeze-drying includes the following steps: placing the mold horizontally (i.e., in the direction in which the neural cap lies horizontally, the axis of the neural cap is parallel to the horizontal plane, and the "horizontal placement" in the following embodiments and comparative examples has the same meaning), placing it on the liquid nitrogen surface for freezing, and then freeze-drying; the freezing time is 35 to 60 minutes, the depth of the liquid nitrogen is 15 to 24 cm, and the liquid nitrogen is replenished every 25 to 50 minutes during the freezing period; the freeze-drying time is 35 to 48 hours.

[0045] In some specific embodiments, the material of the mold is one or more of silicone, acrylonitrile-butadiene-styrene copolymer (ABS) and polystyrene (PS).

[0046] In some specific embodiments, the cross-linking agent is EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide) and NHS (N-hydroxysuccinimide). The cross-linking treatment by adding the cross-linking agent comprises the following steps: immersing the mold in a solution containing 10-50 mmol / L EDC and 15-30 mmol / L NHS to perform a cross-linking reaction, and then transferring the mold to water for immersion, taking it out and freeze-drying it. Specific embodiments The present invention is described below by specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims and any equivalents thereof are the protection scope of the present invention.

[0048] Unless otherwise defined, all technical terms and scientific terms used in the present invention have the same meanings as those commonly understood by ordinary technicians in the field to which the present disclosure belongs. Unless otherwise specified, the raw materials and equipment used in the present invention are conventional raw materials and equipment in the field and can be obtained from conventional commercial channels; unless otherwise specified, the methods used in the present invention are conventional methods in the field.

[0049] Example 1 The neural cap structure of this embodiment is as follows Figure 1 and Figure 2 As shown, specifically as follows: the neural cap is a tubular body with an open end 1 and a closed end 2, and the portion between the open end 1 and the closed end 2 in the tubular body is a convergent segment 3; in the convergent segment 3, the inner diameter decreases from the open end 1 to the closed end 2, and the inner diameters at both ends of the convergent segment 3 are 3 mm and 4 mm respectively; the length of the convergent segment 3 is 10 mm; the neural cap is composed of nanofibers, and the nanofibers are arranged perpendicular to the axial direction of the neural cap (i.e., distributed circumferentially along the tubular body).

[0050] The neural cap of this embodiment is prepared by the following steps: (1) Preparation of nanofiber membrane: Prepare a 20% (v / v) acetic acid aqueous solution, dissolve collagen in the acetic acid aqueous solution to make a 12% (w / v) collagen solution, i.e., the spinning precursor solution. Use air-spinning technology to spin the solution, collect the fibers on a winding roller, and obtain a nanofiber membrane with a thickness of 0.07 mm. Its microstructure is as follows Figure 5 As shown in Figure 2, the diameter distribution of the internal nanofibers is Figure 6 As shown, the average diameter of the internal nanofibers is 240nm. In the above air spinning process, the parameters are set as follows: 9mL of spinning precursor solution is sucked by a syringe, the inner diameter of the syringe needle is 30G, the pushing speed of the syringe is 5mL / h, the air pressure at the syringe needle is 0.6MPa, the distance from the syringe to the receiving roller is 30cm, and the speed of the receiving roller is 500rpm.

[0051] (2) Preparation of nanofiber dispersion: The nanofiber membrane was cut into small pieces of 1.5 cm×1.5 cm in size, and then crushed with an IKA T18 homogenizer and dispersed in tert-butyl alcohol to obtain a nanofiber dispersion with a concentration of 15 wt%. During the above homogenization process, the parameters were designed as follows: the rotation speed was 10,000 rpm and the stirring time was 25 min.

[0052] (3) Design and preparation of mold: The structure of the mold is as follows Figure 3 and Figure 4 As shown, it is composed of two symmetrical, complementary and detachable half molds; a drainage groove 5 is provided inside the mold, and an injection port 4 connecting the drainage groove 5 with the outside is provided on the surface of the mold; the entire mold is 11 cm long, 9 cm wide and 4 cm high. After the neural cap physical model is made by 3D printing, the model is used as a template and the mold is obtained by silicone molding process.

[0053] (4) Directional freeze drying: The mold is placed with the injection port facing upwards, and a disposable sterile syringe is used to absorb an appropriate amount of nanofiber dispersion, which is injected into the mold until it is completely immersed. The injection port of the mold is then sealed and placed horizontally on the surface of a metal disc with a diameter of 12 cm. The metal disc is placed on the surface of a 24 cm deep liquid nitrogen pool, and the heat is absorbed by the evaporation of liquid nitrogen, so that the nanofiber dispersion begins to freeze from the bottom of the mold, and the ice crystals grow in a single temperature gradient direction, thereby arranging the nanofibers perpendicular to the axis of the neural cap. The metal disc is placed on top of the liquid nitrogen pool for freezing for 35 minutes, and the liquid nitrogen in the liquid nitrogen pool is replenished every 25 minutes. After freezing, freeze-dry at -40°C for 35 hours to obtain a neural cap precursor (installed in the mold, not yet demolded).

[0054] (5) Cross-linking: Prepare an ethanol solution of EDC and NHS, wherein the concentrations of EDC and NHS are 50mmol / L and 30mmol / L, respectively. Unseal the injection port of the mold containing the neural cap precursor, immerse it in the ethanol solution of EDC and NHS, take it out after soaking for 12 hours, immerse the mold in ultrapure water, take it out after soaking for 40 minutes, and then freeze-dry it. Remove the mold to obtain the neural cap.

[0055] Example 2 The neural cap structure of this embodiment is as follows Figure 1 and Figure 2 As shown, specifically as follows: the neural cap is a tubular body with an open end 1 and a closed end 2, and the portion between the open end 1 and the closed end 2 in the tubular body is a convergent segment 3; in the convergent segment 3, the inner diameter decreases from the open end 1 to the closed end 2, and the inner diameters at both ends of the convergent segment 3 are 4 mm and 6.7 mm respectively; the length of the convergent segment 3 is 15 mm; the neural cap is composed of nanofibers, and the nanofibers are arranged perpendicular to the axial direction of the neural cap (i.e., distributed circumferentially along the tubular body).

[0056] The neural cap of this embodiment is prepared by the following steps: (1) Preparation of nanofiber membrane: Prepare 30% (v / v) acetic acid aqueous solution, dissolve collagen in acetic acid aqueous solution, make 20% (w / v) collagen solution, i.e. spinning precursor solution. Utilize air-spinning technology to carry out solution spinning, the fiber is collected on the winding roller, obtains the nanofiber film with a thickness of 0.09mm, and the average diameter of the internal nanofiber is 300nm. In the above air-spinning process, the parameters are set as follows: use a syringe to draw 8.5mL of spinning precursor solution, the syringe needle inner diameter is 25G, the propulsion speed of the syringe is 4mL / h, the air pressure at the syringe needle is 0.35MPa, the distance from the syringe to the receiving roller is 28cm, and the rotating speed of the receiving roller is 1000rpm.

[0057] (2) Preparation of nanofiber dispersion: The nanofiber membrane was cut into small pieces of 1.5 cm×1.5 cm in size, and then crushed with an IKA T18 homogenizer and dispersed in tert-butyl alcohol to obtain a nanofiber dispersion with a concentration of 15 wt%. During the above homogenization process, the parameters were designed as follows: the rotation speed was 11000 rpm and the stirring time was 20 min.

[0058] (3) Design and preparation of mold: The structure of the mold is as follows Figure 3 and Figure 4 As shown, it is composed of two half molds that are symmetrical, complementary and separable. A drainage groove 5 is provided inside the mold, and an injection port 4 connecting the drainage groove 5 with the outside is provided on the surface of the mold. The entire mold is 8 cm long, 4 cm wide and 3 cm high. ABS material is used to directly prepare two half molds through 3D printing, and the two half molds are combined to form a mold.

[0059] (4) Directional freeze drying: The mold is placed with the injection port facing upwards, and a disposable sterile syringe is used to absorb an appropriate amount of nanofiber dispersion, which is injected into the mold until it is completely immersed. The injection port of the mold is then sealed and placed horizontally on the surface of a metal disc with a diameter of 10 cm. The metal disc is placed on the surface of a 15 cm deep liquid nitrogen pool, and the heat is absorbed by the evaporation of liquid nitrogen, so that the nanofiber dispersion begins to freeze from the bottom of the mold, and the ice crystals grow in a single temperature gradient direction, thereby arranging the nanofibers perpendicular to the axis of the neural cap. The metal disc is placed on top of the liquid nitrogen pool for freezing for 55 minutes, and the liquid nitrogen in the liquid nitrogen pool is replenished every 32 minutes. After freezing, freeze-dry at -40°C for 40 hours to obtain a neural cap precursor (installed in the mold, not yet demolded).

[0060] (5) Cross-linking: Prepare an ethanol solution of EDC and NHS, wherein the concentrations of EDC and NHS are 10mmol / L and 15mmol / L, respectively. Unseal the injection port of the mold containing the neural cap precursor, immerse it in the ethanol solution of EDC and NHS, take it out after soaking for 20 hours, immerse the mold in ultrapure water, take it out after soaking for 40 minutes, and then freeze-dry it. Remove the mold to obtain the neural cap.

[0061] Example 3 The neural cap structure of this embodiment is as follows Figure 1 and Figure 2As shown, specifically as follows: the neural cap is a tubular body with an open end 1 and a closed end 2, and the portion between the open end 1 and the closed end 2 in the tubular body is a convergent segment 3; in the convergent segment 3, the inner diameter decreases from the open end 1 to the closed end 2, and the inner diameters at both ends of the convergent segment 3 are 2 mm and 8 mm respectively; the length of the convergent segment 3 is 5 mm; the neural cap is composed of nanofibers, and the nanofibers are arranged perpendicular to the axial direction of the neural cap (i.e., distributed circumferentially along the tubular body).

[0062] The neural cap of this embodiment is prepared by the following steps: (1) Preparation of nanofiber membrane: Prepare 42% (v / v) acetic acid aqueous solution, dissolve collagen in acetic acid aqueous solution, make 16% (w / v) collagen solution, i.e. spinning precursor solution. Utilize air-spinning technology to carry out solution spinning, fiber is collected on winding roller, obtains nanofiber film with thickness of 0.18mm, and the average diameter of internal nanofiber is 370nm. In the above air-spinning process, parameter setting is as follows: utilize syringe to draw 13mL spinning precursor solution, syringe needle inner diameter is 27G, syringe propulsion speed is 9mL / h, syringe needle place air pressure is 0.64MPa, syringe is 50cm to receiving roller, and the rotating speed of receiving roller is 600rpm.

[0063] (2) Preparation of nanofiber dispersion: The nanofiber membrane was cut into small pieces of 1.5 cm×1.5 cm in size, and then crushed with an IKA T18 homogenizer and dispersed in tert-butyl alcohol to obtain a nanofiber dispersion with a concentration of 20 wt%. During the above homogenization process, the parameters were designed as follows: the rotation speed was 15000 rpm and the stirring time was 45 min.

[0064] (3) Design and preparation of mold: The structure of the mold is as follows Figure 3 and Figure 4 As shown, it is composed of two half molds that are symmetrical, complementary and separable. A drainage groove 5 is provided inside the mold, and an injection port 4 connecting the drainage groove 5 with the outside is provided on the surface of the mold. The entire mold is 5 cm long, 4 cm wide and 4 cm high. PS material is used to directly prepare two half molds through 3D printing, and the two half molds are combined to form a mold.

[0065] (4) Directional freeze drying: The mold is placed with the injection port facing upwards, and a disposable sterile syringe is used to absorb an appropriate amount of nanofiber dispersion, which is injected into the mold until it is completely immersed. The injection port of the mold is then sealed and placed horizontally on the surface of a metal disc with a diameter of 6 cm. The metal disc is placed on the surface of a 17 cm deep liquid nitrogen pool, and the heat is absorbed by the evaporation of liquid nitrogen, so that the nanofiber dispersion begins to freeze from the bottom of the mold, and the ice crystals grow in a single temperature gradient direction, thereby arranging the nanofibers perpendicular to the axis of the neural cap. The metal disc is placed on top of the liquid nitrogen pool for freezing for 60 minutes, and the liquid nitrogen in the liquid nitrogen pool is replenished every 40 minutes. After freezing, freeze-dry at -40°C for 48 hours to obtain a neural cap precursor (installed in the mold, not yet demolded).

[0066] (5) Cross-linking: Prepare an ethanol solution of EDC and NHS, wherein the concentrations of EDC and NHS are 30mmol / L and 30mmol / L, respectively. Unseal the injection port of the mold containing the neural cap precursor, immerse it in the ethanol solution of EDC and NHS, take it out after soaking for 14 hours, immerse the mold in ultrapure water, take it out after soaking for 45 minutes, and then freeze-dry it. Remove the mold to obtain the neural cap.

[0067] Example 4 The neural cap structure of this embodiment is as follows Figure 1 and Figure 2 As shown, specifically as follows: the neural cap is a tubular body with an open end 1 and a closed end 2, and the portion between the open end 1 and the closed end 2 in the tubular body is a convergent segment 3; in the convergent segment 3, the inner diameter decreases from the open end 1 to the closed end 2, and the inner diameters at both ends of the convergent segment 3 are 3 mm and 3.75 mm respectively; the length of the convergent segment 3 is 16 mm; the neural cap is composed of nanofibers, and the nanofibers are arranged perpendicular to the axial direction of the neural cap (i.e., distributed circumferentially along the tubular body).

[0068] The neural cap of this embodiment is prepared by the following steps: (1) Preparation of nanofiber membrane: Prepare 50% (v / v) acetic acid aqueous solution, dissolve collagen in acetic acid aqueous solution, make 35% (w / v) collagen solution, i.e. spinning precursor solution. Utilize air-spinning technology to carry out solution spinning, the fiber is collected on the winding roller, obtains the nanofiber film with a thickness of 0.18mm, and the average diameter of the internal nanofiber is 410nm. In the above air-spinning process, the parameters are set as follows: use a syringe to draw 14mL of spinning precursor solution, the syringe needle inner diameter is 27G, the propulsion speed of the syringe is 6mL / h, the air pressure at the syringe needle is 0.24MPa, the distance from the syringe to the receiving roller is 24cm, and the rotating speed of the receiving roller is 450rpm.

[0069] (2) Preparation of nanofiber dispersion: The nanofiber membrane was cut into small pieces of 1.5 cm×1.5 cm in size, and then crushed with an IKA T18 homogenizer and dispersed in tert-butyl alcohol to obtain a nanofiber dispersion with a concentration of 19 wt%. During the above homogenization process, the parameters were designed as follows: the rotation speed was 12000 rpm and the stirring time was 24 min.

[0070] (3) Design and preparation of mold: The structure of the mold is as follows Figure 3 and Figure 4 As shown, it is composed of two half molds that are symmetrical, complementary and separable. A drainage groove 5 is provided inside the mold, and an injection port 4 connecting the drainage groove 5 with the outside is provided on the surface of the mold. The entire mold is 7 cm long, 7 cm wide and 6 cm high. PS material is used to directly prepare two half molds through 3D printing, and the two half molds are combined to form a mold.

[0071] (4) Directional freeze drying: Place the mold with the injection port facing upward, use a disposable sterile syringe to absorb an appropriate amount of nanofiber dispersion, and inject it into the mold until it is completely immersed. Then seal the injection port of the mold and place it horizontally on the surface of a metal disc with a diameter of 8 cm. Place the metal disc on the surface of a 20 cm deep liquid nitrogen pool, and absorb heat through the evaporation of liquid nitrogen, so that the nanofiber dispersion begins to freeze from the bottom of the mold, and the ice crystals grow in a single temperature gradient direction, so that the nanofibers are arranged perpendicular to the axis of the neural cap. The metal disc is placed on the top of the liquid nitrogen pool for freezing for 50 minutes, and the liquid nitrogen in the liquid nitrogen pool is replenished every 35 minutes. After freezing, freeze-dry at -40°C for 42 hours to obtain the neural cap precursor (installed in the mold, not yet demolded).

[0072] (5) Cross-linking: Prepare an ethanol solution of EDC and NHS, wherein the concentrations of EDC and NHS are 25mmol / L and 20mmol / L, respectively. Unseal the injection port of the mold containing the neural cap precursor, immerse it in the ethanol solution of EDC and NHS, take it out after soaking for 24 hours, immerse the mold in ultrapure water, take it out after soaking for 60 minutes, and then freeze-dry it. Remove the mold to obtain the neural cap.

[0073] Example 5 The neural cap structure of this embodiment is as follows Figure 1 and Figure 2As shown, specifically as follows: the neural cap is a tubular body with an open end 1 and a closed end 2, and the portion between the open end 1 and the closed end 2 in the tubular body is a convergent segment 3; in the convergent segment 3, the inner diameter decreases from the open end 1 to the closed end 2, and the inner diameters at both ends of the convergent segment 3 are 5 mm and 5.6 mm respectively; the length of the convergent segment 3 is 20 mm; the neural cap is composed of nanofibers, and the nanofibers are arranged perpendicular to the axial direction of the neural cap (i.e., distributed circumferentially along the tubular body).

[0074] The neural cap of this embodiment is prepared by the following steps: (1) Preparation of nanofiber membrane: Prepare 40% (v / v) acetic acid aqueous solution, dissolve collagen in acetic acid aqueous solution, make 25% (w / v) collagen solution, i.e. spinning precursor solution. Utilize air-spinning technology to carry out solution spinning, the fiber is collected on the winding roller, obtains the nanofiber film with a thickness of 0.08mm, and the average diameter of the internal nanofiber is 390nm. In the above air-spinning process, the parameters are set as follows: use a syringe to draw 10mL of spinning precursor solution, the syringe needle inner diameter is 23G, the propulsion speed of the syringe is 4mL / h, the air pressure at the syringe needle is 0.6MPa, the distance from the syringe to the receiving roller is 38cm, and the rotating speed of the receiving roller is 350rpm.

[0075] (2) Preparation of nanofiber dispersion: The nanofiber membrane was cut into small pieces of 1.5 cm×1.5 cm in size, and then crushed with an IKA T18 homogenizer and dispersed in tert-butyl alcohol to obtain a nanofiber dispersion with a concentration of 18.5 wt%. During the above homogenization process, the parameters were designed as follows: the rotation speed was 13000 rpm and the stirring time was 28 min.

[0076] (3) Design and preparation of mold: The structure of the mold is as follows Figure 3 and Figure 4 As shown, it is composed of two half molds that are symmetrical, complementary and separable. A drainage groove 5 is provided inside the mold, and an injection port 4 connecting the drainage groove 5 with the outside is provided on the surface of the mold. The entire mold is 8.5 cm long, 5.5 cm wide and 8 cm high. ABS material is used to directly prepare two half molds through 3D printing, and the two half molds are combined to form a mold.

[0077] (4) Directional freeze drying: The mold is placed with the injection port facing upwards, and a disposable sterile syringe is used to absorb an appropriate amount of nanofiber dispersion, which is injected into the mold until it is completely immersed. The injection port of the mold is then sealed and placed horizontally on the surface of a metal disc with a diameter of 9 cm. The metal disc is placed on the surface of a 22 cm deep liquid nitrogen pool, and the heat is absorbed by the evaporation of liquid nitrogen, so that the nanofiber dispersion begins to freeze from the bottom of the mold, and the ice crystals grow in a single temperature gradient direction, thereby arranging the nanofibers perpendicular to the axis of the neural cap. The metal disc is placed on top of the liquid nitrogen pool for freezing for 38 minutes, and the liquid nitrogen in the liquid nitrogen pool is replenished every 25 minutes. After freezing, freeze-dry at -40°C for 42 hours to obtain a neural cap precursor (installed in the mold, not yet demolded).

[0078] (5) Cross-linking: Prepare an ethanol solution of EDC and NHS, wherein the concentrations of EDC and NHS are 35mmol / L and 25mmol / L, respectively. Unseal the injection port of the mold containing the neural cap precursor, immerse it in the ethanol solution of EDC and NHS, take it out after soaking for 15 hours, immerse the mold in ultrapure water, take it out after soaking for 50 minutes, and then freeze-dry it. Remove the mold to obtain the neural cap.

[0079] Example 6 The difference between the nerve cap structure of this embodiment and that of embodiment 3 is that the portion between the open end 1 and the closed end 2 in the tubular body is composed of a convergent section 3 and a constant diameter section with a constant inner diameter; the convergent section 3 is located at the open end 1, and the constant diameter section is located at the closed end 2; the inner diameter of the constant diameter section is 2 mm and the length is 10 mm. The rest of the structure is the same as that of embodiment 3.

[0080] The only difference between the preparation steps of the nerve cap of this embodiment and that of the third embodiment is that the shape of the drainage groove 5 in the mold used is designed according to the shape of the nerve cap of this embodiment. The remaining steps are the same as those of the third embodiment.

[0081] Example 7 The difference between the nerve cap structure of this embodiment and that of the third embodiment is that the inner diameters of the two ends of the convergence section 3 are 1 mm and 8 mm respectively. The rest of the structure is the same as that of the third embodiment.

[0082] The only difference between the preparation steps of the nerve cap of this embodiment and that of the embodiment 3 is that the shape of the drainage groove 5 in the mold used is designed according to the shape of the nerve cap of this comparative example. The remaining steps are the same as those of the embodiment 3.

[0083] Compared with Example 3, in the nerve cap of this embodiment, the space at the smallest end of the inner diameter of the contracting section is too small, and the effective length of the insertable nerve stump is limited, which makes it difficult to anchor the nerve cap to the surrounding tissue, making the surgical operation process more complicated.

[0084] Comparative Example 1 The difference between the nerve cap structure of this comparative example and that of Example 1 is that the nerve cap does not have a constricting section 3, and the inner diameter of the tubular body from the open end 1 to the closed end 2 is the same, which is 6.5 mm. The rest of the structure is the same as that of Example 1.

[0085] The only difference between the preparation steps of the nerve cap of this comparative example and Example 1 is that the shape of the drainage groove 5 in the adopted mold is designed according to the shape of the nerve cap of this comparative example. The remaining steps are the same as those of Example 1.

[0086] Compared with Example 1, the nerve cap of this comparative example cannot gradually limit the growth of axons in a three-dimensional space by utilizing the structure of the convergent segment, has a weak ability to inhibit inappropriate and irregular regenerated axons, and has a poor effect in preventing the formation of neuromas.

[0087] Comparative Example 2 The difference between the nerve cap structure of this comparative example and that of Example 1 is that the nanofibers in the nerve cap are arranged in a disordered manner. The rest of the structure is the same as that of Example 1.

[0088] The only difference between the preparation steps of the nerve cap in this comparative example and Example 1 is that in step (4), the sealed mold is subjected to ordinary freeze drying instead of directional freeze drying. The remaining steps are the same as in Example 1.

[0089] Although this comparative example has a certain effect in preventing the formation of neuromas, compared with Example 1, this comparative example cannot further utilize the contact guidance of the microstructure to hinder the disordered regeneration of nerve fibers based on the effect of the contraction segment, and thus the effect of preventing or inhibiting the formation of neuromas is relatively poor.

[0090] Comparative Example 3 The difference between the nerve cap structure of this comparative example and that of Example 1 is that the nanofibers in the nerve cap are arranged parallel to the axial direction of the nerve cap. The rest of the structure is the same as that of Example 1.

[0091] The only difference between the preparation steps of the nerve cap in this comparative example and Example 1 is that in step (4), the sealed mold is placed vertically on the surface of the metal disc (i.e., the axial direction of the nerve cap is perpendicular to the horizontal plane according to the vertical placement direction of the nerve cap). The remaining steps are the same as Example 1.

[0092] Compared with Example 1, the contact guidance of the nanofibers in this comparative example will promote axon growth, which is not conducive to the synergistic effect with the special three-dimensional spatial structure formed by the contraction segment, and is therefore not conducive to achieving the effect of inhibiting the regeneration of disordered axons to a greater extent, thereby preventing the formation of neuromas.

[0093] Comparative Example 4 The difference between the nerve cap structure of this comparative example and that of Example 4 is that the contraction section 3 is replaced by an expansion section; in the expansion section, the inner diameter increases from the open end 1 to the closed end 2, and the inner diameters at both ends of the expansion section are 3.75 mm and 3 mm respectively. The rest of the structure is the same as that of Example 4.

[0094] The only difference between the preparation steps of the nerve cap of this comparative example and Example 4 is that the shape of the drainage groove 5 in the adopted mold is designed according to the shape of the nerve cap of this comparative example. The remaining steps are the same as those of Example 4.

[0095] Compared with Example 4, in the nerve cap of this comparative example, the structure of the enlarged segment provides more space for axon growth, which is not conducive to inhibiting disordered regenerating axons, and thus the effect of preventing or inhibiting neuroma formation is weaker.

[0096] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0097] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A neural cap, the neural cap being a tubular body having an open end and a closed end, characterized in that: The open end is provided with a convergence section whose inner diameter decreases from the open end to the closed end; the nerve cap is composed of nanofibers arranged perpendicular to the axial direction.

2. The neural cap according to claim 1, characterized in that The constriction section is between the open end and the closed end of the nerve cap; or, in the nerve cap, the constriction section has a constant diameter section with a constant inner diameter from the end with the smallest inner diameter to the closed end.

3. The neural cap according to claim 1 or 2, characterized in that: In the convergence section, the ratio of the inner diameters at both ends is 2:0.5-1.9, and the inner diameter of the end with the smallest inner diameter is 2-5 mm; the length of the convergence section is 5-20 mm.

4. The neural cap according to claim 1, characterized in that: The average diameter of the nanofibers is 240-410 nm.

5. The neural cap according to claim 1 or 4, characterized in that: The nanofibers are collagen fibers.

6. A method for preparing a neural cap according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: injecting nanofiber dispersion into a mold, arranging the nanofibers perpendicular to the axial direction of the nerve cap by directional freeze drying, adding a crosslinking agent for crosslinking treatment, and demoulding to obtain the nerve cap.

7. The preparation method according to claim 6, wherein the directional freeze-drying comprises the following steps: The mold is placed horizontally on the liquid nitrogen surface to freeze, and then freeze-dried.

8. The preparation method according to claim 7, wherein the freezing time is 35 to 60 minutes, the depth of the liquid nitrogen is 15 to 24 cm, and the liquid nitrogen is replenished every 25 to 50 minutes during the freezing period.

9. The preparation method according to claim 6, wherein the crosslinking agent is EDC and NHS; and the adding of the crosslinking agent for crosslinking treatment comprises the following steps: The mold is immersed in a solution containing a cross-linking agent to carry out a cross-linking reaction, and then the mold is transferred to water for immersion, and then taken out for freeze-drying.

10. The preparation method according to claim 6, wherein the concentration of the nanofiber dispersion is 15-20wt%; The preparation steps of the nanofiber dispersion include: The polymer material is made into a nanofiber membrane by solution spinning; The nanofiber membrane is crushed and dispersed in a dispersion medium to obtain a nanofiber dispersion.

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