Artificial outer nerve membrane for sutureless nerve repair as well as preparation method and application of artificial outer nerve membrane

Abstract

The invention provides an artificial outer nerve membrane for sutureless nerve repair and a preparation method and application thereof.The artificial outer nerve membrane comprises a hydrophilic viscous layer, a middle elastic layer and an outer elastic layer which are arranged in a stacked mode, and through the specific structure matching of the three layers, the obtained artificial outer nerve membrane has the excellent bonding performance and mechanical performance, and the service life of the artificial outer nerve membrane is prolonged. Compared with the prior art, stable suture of nervous tissues can be realized without traditional suture and external stimulation, so that the operation time is remarkably shortened, the operation complexity is reduced, postoperative complications are reduced, the recovery effect of neurological functions is greatly improved, and a safe and effective choice is provided for repair of peripheral nerves.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical materials, and in particular relates to an artificial epineurium for sutureless nerve repair, and a preparation method and application thereof. Background Art

[0002] Currently, microsurgery is the standard method for peripheral nerve repair surgery, especially in nerve anastomosis and nerve transplantation. This microsurgical suturing technique has been widely used in clinical practice by providing optimal tissue sealing strength and durability. However, although microsuturing technology has been widely accepted in nerve repair, it still has many limitations. First, microsuturing technology requires surgeons to have superb technical skills and requires the use of expensive and complex surgical equipment. Second, the sutures used during microsuturing surgery may interfere with postoperative axonal regeneration and cause a series of complications such as bleeding, chronic inflammation and fibrosis, ultimately leading to unsatisfactory recovery of nerve function.

[0003] To address the above problems, researchers have developed a variety of alternative methods based on adhesive materials, which aim to reduce dependence on sutures and accelerate the process of nerve connection. For example, in recent years, the use of materials such as fibrin glue or cyanoacrylate derivatives for nerve repair has made certain progress. However, these adhesive materials reported above still face challenges in practical applications, such as insufficient adhesion strength and limited tolerance to mechanical stress. In addition, these adhesive materials usually require external stimulation (such as pressure, light or heating) to achieve effective adhesion, which limits their widespread application in clinical practice, especially in cases where surgical operations are complex or space is limited.

[0004] Therefore, in order to solve the above technical problems, it is of great significance to develop an artificial epineurium with excellent bonding and mechanical properties for application in sutureless nerve repair. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the purpose of the present invention is to provide an artificial epineurium for sutureless nerve repair, and its preparation method and application. The artificial epineurium is composited through a three-layer structure, and has excellent bonding and mechanical properties. It can achieve stable adhesion to nerve tissue and strong mechanical support, thereby effectively shortening the operation time, reducing the complexity of the operation, and helping to reduce postoperative complications. It is suitable for use in sutureless nerve repair and has important research significance.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an artificial epineurium for sutureless nerve repair, the artificial epineurium comprising a hydrophilic adhesive layer, a middle elastic layer, and an outer elastic layer arranged in layers;

[0008] The raw materials of the hydrophilic adhesive layer include hyaluronic acid, catechol, crosslinking agent A and crosslinking agent B;

[0009] The raw materials of the middle elastic layer include polyethylene glycol diamine A and toluene diisocyanate A;

[0010] The raw materials of the outer elastic layer include polyethylene glycol diamine B, toluene diisocyanate B and diphenylmethane-4,4'-diisocyanate.

[0011] The artificial epineurium provided by the present invention includes a three-layer structure, which is a hydrophilic adhesive layer, an intermediate elastic layer and an outer elastic layer in sequence. When used in a seamless nerve repair process, the hydrophilic adhesive layer is in direct contact with the nerve tissue. Its raw materials include hyaluronic acid, catechol, crosslinking agent A and crosslinking agent B. The substance formed by crosslinking the above raw materials can not only quickly adhere to the surface of the nerve tissue without external stimulation, but also form a chemical bond with the nerve tissue to ensure a stable connection without suturing during surgery; the intermediate elastic layer is located in the middle position, and its raw materials include polyethylene glycol diamine A and toluene diisocyanate A. The substance formed by the combination of the two has a relatively low modulus, can effectively disperse the strain caused by external force, effectively buffer the mechanical stress applied externally during surgery, reduce the physical damage to the nerve tissue caused by external stress, and provide necessary mechanical support in nerve repair, thereby reducing the risk of strain damage during repair, and reducing The complexity of the operation is reduced; the outer elastic layer is arranged on the outermost side, and its raw materials include polyethylene glycol diamine B, toluene diisocyanate B and diphenylmethane-4,4'-diisocyanate. The material formed by the combination of the above three raw materials has a relatively high modulus, which can provide higher mechanical stability and reduce the residual stress in the material, thereby reducing the fibrosis and inflammatory reaction that may occur after the operation, improving the biocompatibility of the obtained artificial nerve membrane, further improving the durability and biosafety of the artificial nerve membrane, and helping to reduce postoperative complications, ensuring the long-term effect of nerve repair, and providing a safe and effective option for the repair of peripheral nerves; in summary, the artificial nerve membrane provided by the present invention has excellent bonding properties and mechanical properties through the above-mentioned specific three-layer structure design, has a certain strain gradient, can achieve stable adhesion to nerve tissue and strong mechanical support, is suitable for use in seamless nerve repair, and has important research significance.

[0012] Preferably, the thickness of the hydrophilic adhesive layer is 20-40 μm, for example, 20 μm, 22 μm, 24 μm, 26 μm, 28 μm, 30 μm, 32 μm, 34 μm, 36 μm, 38 μm or 40 μm.

[0013] Preferably, the mass ratio of the hyaluronic acid, catechol, crosslinker A and crosslinker B is 1:(0.2-0.4):(0.6-0.8):(0.05-0.15), for example, 1:0.2:0.6:0.05, 1:0.3:0.6:0.05, 1:0.4:0.6:0.05, 1:0.2:0.7:0.05, 1:0.3:0.7:0.05, 1:0.4:0.8:0.05, 1:0.2:0.7:0.05, 1:0.3:0.7:0.1 or 1:0.4:0.8:015, etc.

[0014] Preferably, the cross-linking agent A comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC).

[0015] Preferably, the cross-linking agent B comprises N-hydroxysuccinimide (NHS).

[0016] Preferably, the thickness of the intermediate elastic layer is 60-80 μm, for example, 60 μm, 62 μm, 64 μm, 66 μm, 68 μm, 70 μm, 72 μm, 74 μm, 76 μm, 78 μm or 80 μm.

[0017] Preferably, the molar ratio of the polyethylene glycol diamine A to toluene diisocyanate A is 100:(2-3), for example, 100:2, 100:2.2, 100:2.4, 100:2.6, 100:2.8 or 100:3.

[0018] Preferably, the outer elastic layer has a thickness of 90 to 110 μm, for example, 90 μm, 92 μm, 94 μm, 96 μm, 98 μm, 100 μm, 102 μm, 104 μm, 106 μm, 108 μm or 110 μm.

[0019] Preferably, the mass ratio of the polyethylene glycol diamine B, toluene diisocyanate B and diphenylmethane-4,4'-diisocyanate is 100:(2-3):(3-4.5), for example, 100:2:3, 100:2.2:3.3, 100:2.4:3.6, 100:2.6:3.9, 100:2.8:4.2 or 100:3:4.5, etc.

[0020] Preferably, the number average molecular weight of the polyethylene glycol diamine A and the polyethylene glycol diamine B is independently 5000 to 7000, for example, 5000, 5200, 5400, 5600, 5800, 6000, 6200, 6400, 6600, 6800 or 7000.

[0021] In a second aspect, the present invention provides a method for preparing the artificial epineurium for sutureless nerve repair as described in the first aspect, the preparation method comprising the following steps:

[0022] (1) reacting hyaluronic acid and catechol in a MES buffer solution containing a crosslinking agent A and a crosslinking agent B, dialyzing and drying to obtain a hyaluronic acid / catechol crosslinked product, and then dissolving the obtained hyaluronic acid / catechol crosslinked product in water to obtain a hyaluronic acid / catechol composite solution;

[0023] Polyethylene glycol diamine A and toluene diisocyanate A react in the presence of catalyst A, precipitate and dry to obtain an intermediate layer elastomer, then dissolve the obtained intermediate layer elastomer in solvent A, and place in container A for drying to form a film to obtain an intermediate elastic layer;

[0024] Polyethylene glycol diamine B, toluene diisocyanate B, and diphenylmethane-4,4'-diisocyanate are reacted in the presence of a catalyst B, and precipitated and dried to obtain an outer elastomer. The obtained outer elastomer is then dissolved in a solvent B and placed in a container B for drying to form a film, thereby obtaining an outer elastic layer.

[0025] (2) The middle elastic layer and the outer elastic layer obtained in step (1) are stacked to form a double-layer structure, and the hyaluronic acid / catechol composite solution obtained in step (1) is dripped onto one surface of the middle elastic layer of the double-layer structure, and dried to form a film to form a hydrophilic adhesive layer, thereby obtaining the artificial epineurium.

[0026] Preferably, the reaction temperature of reacting hyaluronic acid and catechol in the MES buffer solution containing crosslinker A and crosslinker B in step (1) is 20-40°C, for example, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C or 40°C.

[0027] Preferably, the reaction time of reacting hyaluronic acid and catechol in the MES buffer solution containing crosslinker A and crosslinker B in step (1) is 12 to 48 hours, for example, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 35 hours, 37 hours, 39 hours, 40 hours or 45 hours.

[0028] Preferably, the mass percentage of the hyaluronic acid / catechol cross-linked product in the hyaluronic acid / catechol composite solution in step (1) is 0.003-0.007%, for example, 0.003%, 0.004%, 0.005%, 0.006% or 0.007%.

[0029] Preferably, the catalyst A and the catalyst B in step (1) both comprise triethylamine.

[0030] Preferably, based on 100 g of the polyethylene glycol diamine A in step (1), the amount of the catalyst A is 5 to 15 mL, for example, 5 mL, 7 mL, 9 mL, 11 mL, 13 mL or 15 mL.

[0031] Preferably, based on 100 g of the polyethylene glycol diamine B in step (1), the amount of the catalyst B is 5 to 15 mL, for example, 5 mL, 7 mL, 9 mL, 11 mL, 13 mL or 15 mL.

[0032] Preferably, the reaction of polyethylene glycol diamine A and toluene diisocyanate A in the presence of catalyst A and the reaction of polyethylene glycol diamine B, toluene diisocyanate B and diphenylmethane-4,4'-diisocyanate in the presence of catalyst B in step (1) are both carried out under an inert gas environment.

[0033] Preferably, the inert gas comprises argon.

[0034] Preferably, in step (1), the reaction temperatures for reacting polyethylene glycol diamine A and toluene diisocyanate A in the presence of catalyst A and for reacting polyethylene glycol diamine B, toluene diisocyanate B and diphenylmethane-4,4'-diisocyanate in the presence of catalyst B are each independently 0 to 40°C, for example, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or 40°C.

[0035] Preferably, the reaction time of reacting polyethylene glycol diamine A and toluene diisocyanate A in the presence of catalyst A and reacting polyethylene glycol diamine B, toluene diisocyanate B and diphenylmethane-4,4'-diisocyanate in the presence of catalyst B in step (1) is independently 12 to 48 hours, for example, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours or 48 hours.

[0036] Preferably, the solvent A and the solvent B in step (1) both comprise anhydrous chloroform.

[0037] Preferably, the container A and the container B in step (1) both comprise Teflon culture dishes.

[0038] As a preferred technical solution of the present invention, the preparation method comprises the following steps:

[0039] (1) Dissolve hyaluronic acid (HA) in MES buffer, stir until completely dissolved, add NaOH to adjust the pH of the system to obtain a hyaluronic acid solution; then dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in deionized water, and add them to the obtained hyaluronic acid solution to react to form a HA / EDC / NHS solution; then dissolve catechol in distilled deionized water, and add it dropwise to the obtained HA / EDC / NHS solution, stir and react at room temperature overnight, and after completion, dialyze the obtained mixture in distilled deionized water containing sodium chloride (molecular weight cutoff value is 3.5 kDa), and then dialyze in distilled deionized water, and finally freeze-dry and store in a refrigerator to obtain a hyaluronic acid / catechol cross-linked product, and dissolve the obtained hyaluronic acid / catechol cross-linked product in distilled deionized water to obtain a hyaluronic acid / catechol composite solution;

[0040] Dissolving polyethylene glycol diamine in anhydrous chloroform, first adding triethylamine, then adding toluene diisocyanate, reacting in an ice-water bath, and then stirring at room temperature, precipitating the resulting solution with methanol, and then adding chloroform to dissolve the product after a white precipitate appears, repeating this process three times, and finally drying by vacuum evaporation to obtain an intermediate layer elastomer; then dissolving the obtained intermediate layer elastomer in anhydrous chloroform, and thoroughly mixing under stirring to obtain a uniform solution, and then pouring the obtained uniform solution into a Teflon petri dish and drying at room temperature to obtain an intermediate elastic layer;

[0041] Dissolve polyethylene glycol diamine in anhydrous chloroform, add triethylamine, then add diphenylmethane-4,4'-diisocyanate and toluene diisocyanate, first react in an ice-water bath, then stir and react at room temperature, after the reaction is completed, precipitate the resulting solution with methanol, and after a white precipitate appears, add chloroform to dissolve the product, repeat this process three times, and finally dry by vacuum evaporation to obtain an outer elastomer; then dissolve the obtained outer elastomer in anhydrous chloroform, and fully mix under stirring to obtain a uniform solution, then pour the obtained uniform solution into a Teflon culture dish and dry at room temperature to obtain an outer elastic layer;

[0042] (2) The dried middle elastic layer and the outer elastic layer are stacked and allowed to stand at room temperature to allow the two layers of elastomer to adhere to form a double-layer structure, and then the obtained double-layer structure is placed in an oxygen plasma device for treatment to enhance surface activity; finally, the HA / Catechol composite solution is added dropwise to the surface of the middle elastic layer of the treated double-layer structure, and the solution is allowed to stand at room temperature to allow the solvent to evaporate naturally to form a hydrophilic adhesive layer, thereby obtaining the artificial epineurium.

[0043] In a third aspect, the present invention provides a use of the artificial epineurium for sutureless nerve repair as described in the first aspect as a medical material.

[0044] The numerical range described in the present invention includes not only the point values listed above, but also any point values between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The artificial epineurium for seamless nerve repair provided by the present invention includes a hydrophilic adhesive layer, a middle elastic layer and an outer elastic layer arranged in a stacked manner. Through the combination of the above-mentioned specific three-layer structure, the obtained artificial epineurium has both excellent bonding properties and mechanical properties, so that it can quickly achieve a stable connection to the nerve tissue without the need for traditional suturing and without external stimulation, ensuring stability and safety during the repair process. It can also effectively and significantly shorten the operation time, reduce postoperative complications, and greatly improve the recovery effect of nerve function, providing a safe and effective option for peripheral nerve repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 1 is a schematic diagram of the cross-sectional structure of the artificial epineurium provided by the present invention;

[0048] Figure 2 This is a physical picture of the artificial epineurium provided in Example 1. DETAILED DESCRIPTION

[0049] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0050] Unless otherwise specified, the raw materials involved in the following specific embodiments are all conventional materials in the art and can be purchased from commercial products.

[0051] Example 1

[0052] An artificial epineurium for sutureless nerve repair, the cross-sectional structure of which is shown in FIG. Figure 1 As shown, it includes a hydrophilic adhesive layer 1, a middle elastic layer 2 and an outer elastic layer 3 which are stacked;

[0053] The thickness of the hydrophilic adhesive layer 1 is 30 μm, the thickness of the middle elastic layer 2 is 70 μm, and the thickness of the outer elastic layer 3 is 100 μm.

[0054] The method for preparing the artificial epineurium provided in this embodiment comprises the following steps:

[0055] (1) Under normal temperature and pressure, 1 g of hyaluronic acid (HA) was dissolved in 250 mL of MES buffer and stirred until completely dissolved. 0.1 M NaOH was added to adjust the pH of the system to 4.5 to obtain a hyaluronic acid solution. Subsequently, 0.7 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.1 g of N-hydroxysuccinimide (NHS) was dissolved in 10 mL of distilled deionized water and then added to the resulting hyaluronic acid solution for 10 minutes to form a HA / EDC / NHS solution. 0.3 g of catechol was then dissolved in 10 mL of distilled deionized water and added dropwise to the resulting HA / EDC / NHS solution. The mixture was stirred and reacted overnight at room temperature. After completion, the resulting mixture was dialyzed against 5 L of distilled deionized water containing 45 g of sodium chloride for 3 days (molecular weight cutoff value was 3.5 kDa), then dialyzed against 5 L of distilled deionized water for 4 hours, and finally freeze-dried for 4 days and stored in a refrigerator to obtain a hyaluronic acid / catechol cross-linked product, which was dissolved in distilled deionized water to obtain a hyaluronic acid / catechol composite solution with a mass percentage of 0.005%.

[0056] 100 g of polyethylene glycol diamine (Mn=6000) was dissolved in 400 mL of anhydrous chloroform, 10 mL of triethylamine was added, and the mixture was stirred at 0° C. under argon for 1 hour. Then, 2.7 g of toluene diisocyanate was added, and the mixture was reacted in an ice-water bath for 1 hour. The mixture was stirred at room temperature for 4 days. After the reaction, the resulting solution was precipitated with methanol. After a white precipitate appeared, the mixture solution was precipitated for 30 minutes, and chloroform was added to dissolve the product. This process was repeated three times, and finally dried by vacuum evaporation to obtain an intermediate layer elastomer. Subsequently, 1 g of the obtained intermediate layer elastomer was dissolved in 20 mL of anhydrous chloroform and thoroughly mixed under stirring to obtain a uniform solution. The obtained uniform solution was then poured into a Teflon culture dish with a diameter of 100 mm and dried at room temperature to obtain an intermediate elastic layer.

[0057] 100 g of polyethylene glycol diamine (Mn=6000) was dissolved in 400 mL of anhydrous chloroform, 10 mL of triethylamine was added, and the mixture was stirred at 0°C under argon for 1 hour. Then, 2.0 g of diphenylmethane-4,4'-diisocyanate and 2.7 g of toluene diisocyanate were added, and the mixture was reacted in an ice-water bath for 1 hour, and then stirred at room temperature for 4 days. After the reaction, the resulting solution was first precipitated with methanol. After a white precipitate appeared, the mixture solution was precipitated for 30 minutes, and then chloroform was added to dissolve the product. This process was repeated three times, and finally dried by vacuum evaporation to obtain an outer elastomer; then, 1 g of the obtained outer elastomer was dissolved in 20 mL of anhydrous chloroform and thoroughly mixed under stirring to obtain a uniform solution. The obtained uniform solution was then poured into a Teflon culture dish with a diameter of 100 mm and dried at room temperature to obtain an outer elastic layer;

[0058] (2) The dried middle elastic layer and the outer elastic layer are stacked and allowed to stand at room temperature for 12 hours to allow the two layers of elastomer to adhere to form a double-layer structure. The resulting double-layer structure is then placed in an oxygen plasma device with a power of 100 W for 3 minutes to enhance surface activity. Finally, 350 μL of HA / Catechol composite solution is added to the surface of the middle elastic layer of the treated double-layer structure, and the solution is allowed to stand at room temperature for 12 hours to allow the solvent to evaporate naturally, thereby forming a hydrophilic adhesive layer to obtain the artificial epineurium.

[0059] Examples 2 to 5

[0060] An artificial epineurium for sutureless nerve repair, which differs from Example 1 only in that the thickness of the middle elastic layer is 60 μm (Example 2), 40 μm (Example 3), 80 μm (Example 4), and 100 μm (Example 5), respectively. The other structures, parameters, and preparation methods are the same as those in Example 1.

[0061] Examples 6 to 9

[0062] An artificial epineurium for sutureless nerve repair is disclosed. The difference between the embodiment 1 and the embodiment 1 is that the thickness of the outer elastic layer is 90 μm (embodiment 6), 70 μm (embodiment 7), 110 μm (embodiment 8), and 150 μm (embodiment 9), respectively. The other structures, parameters, and preparation methods are the same as those in embodiment 1.

[0063] Comparative Example 1

[0064] An artificial epineurium for sutureless nerve repair, comprising a hydrophilic adhesive layer and an elastic layer arranged in layers;

[0065] The thickness of the hydrophilic adhesive layer is 30 μm, and the thickness of the elastic layer is 170 μm.

[0066] The preparation method of the artificial epineurium provided in this comparative example comprises the following steps:

[0067] (1) Under normal temperature and pressure, 1 g of hyaluronic acid (HA) was dissolved in 250 mL of MES buffer and stirred until completely dissolved. 0.1 M NaOH was added to adjust the pH to 4.5 to obtain a hyaluronic acid solution. 0.7 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.1 g of N-hydroxysuccinimide (NHS) was dissolved in 10 mL of distilled deionized water and then added to the resulting hyaluronic acid solution for 10 minutes to form a HA / EDC / NHS solution. 0.3 g of catechol was then dissolved in 10 mL of distilled deionized water and added dropwise to the resulting HA / EDC / NHS solution. The mixture was stirred and reacted overnight at room temperature. After completion, the resulting mixture was dialyzed against 5 L of distilled deionized water containing 45 g of sodium chloride for 3 days (molecular weight cutoff value was 3.5 kDa), then dialyzed against 5 L of distilled deionized water for 4 hours, and finally freeze-dried for 4 days and stored in a refrigerator to obtain a hyaluronic acid / catechol cross-linked product, which was dissolved in distilled deionized water to obtain a hyaluronic acid / catechol composite solution with a mass percentage of 0.005%.

[0068] 200 g of polyethylene glycol diamine (Mn=6000) was dissolved in 800 mL of anhydrous chloroform, 20 mL of triethylamine was added, and the mixture was stirred at 0° C. under argon for 1 hour. Then, 5.4 g of toluene diisocyanate was added, and the mixture was reacted in an ice-water bath for 1 hour. The mixture was stirred at room temperature for 4 days. After the reaction, the resulting solution was precipitated with methanol. After a white precipitate appeared, the mixture solution was precipitated for 30 minutes, and chloroform was added to dissolve the product. This process was repeated three times, and the mixture was finally dried by vacuum evaporation to obtain an elastomer. Subsequently, 2 g of the obtained intermediate layer elastomer was dissolved in 40 mL of anhydrous chloroform and thoroughly mixed under stirring to obtain a uniform solution. The obtained uniform solution was then poured into a Teflon culture dish with a diameter of 100 mm and dried at room temperature to obtain an elastic layer.

[0069] (2) The dried elastic layer was placed in an oxygen plasma device with a power of 100 W for 3 minutes to enhance the surface activity; finally, 350 μL of HA / Catechol composite solution was added to the surface of the treated elastic layer, and the solution was allowed to stand at room temperature for 12 hours to allow the solvent to evaporate naturally, thereby forming a hydrophilic adhesive layer to obtain the artificial epineurium.

[0070] Comparative Example 2

[0071] An artificial epineurium for sutureless nerve repair, comprising a hydrophilic adhesive layer and an elastic layer arranged in layers;

[0072] The thickness of the hydrophilic adhesive layer is 30 μm, and the thickness of the elastic layer is 170 μm.

[0073] The preparation method of the artificial epineurium provided in this comparative example comprises the following steps:

[0074] (1) Under normal temperature and pressure, 1 g of hyaluronic acid (HA) was dissolved in 250 mL of MES buffer and stirred until completely dissolved. 0.1 M NaOH was added to adjust the pH to 4.5 to obtain a hyaluronic acid solution. 0.7 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.1 g of N-hydroxysuccinimide (NHS) was dissolved in 10 mL of distilled deionized water and then added to the resulting hyaluronic acid solution for 10 minutes to form a HA / EDC / NHS solution. 0.3 g of catechol was then dissolved in 10 mL of distilled deionized water and added dropwise to the resulting HA / EDC / NHS solution. The mixture was stirred and reacted overnight at room temperature. After completion, the resulting mixture was dialyzed against 5 L of distilled deionized water containing 45 g of sodium chloride for 3 days (molecular weight cutoff value was 3.5 kDa), then dialyzed against 5 L of distilled deionized water for 4 hours, and finally freeze-dried for 4 days and stored in a refrigerator to obtain a hyaluronic acid / catechol cross-linked product, which was dissolved in distilled deionized water to obtain a hyaluronic acid / catechol composite solution with a mass percentage of 0.005%.

[0075] 200 g of polyethylene glycol diamine (Mn=6000) was dissolved in 800 mL of anhydrous chloroform, 10 mL of triethylamine was added, and the mixture was stirred at 0°C under argon for 1 hour. Then, 4.0 g of diphenylmethane-4,4'-diisocyanate and 5.4 g of toluene diisocyanate were added, and the mixture was reacted in an ice-water bath for 1 hour, and then stirred at room temperature for 4 days. After the reaction, the resulting solution was first precipitated with methanol. After a white precipitate appeared, the mixture solution was precipitated for 30 minutes, and then chloroform was added to dissolve the product. This process was repeated three times, and finally dried by vacuum evaporation to obtain an elastomer. Subsequently, 2 g of the obtained elastomer was dissolved in 40 mL of anhydrous chloroform and thoroughly mixed under stirring to obtain a uniform solution. The obtained uniform solution was then poured into a Teflon culture dish with a diameter of 100 mm and dried at room temperature to obtain an elastic layer.

[0076] (2) The dried elastic layer was placed in an oxygen plasma device with a power of 100 W for 3 minutes to enhance the surface activity; finally, 350 μL of HA / Catechol composite solution was added to the surface of the treated elastic layer, and the solution was allowed to stand at room temperature for 12 hours to allow the solvent to evaporate naturally, thereby forming a hydrophilic adhesive layer to obtain the artificial epineurium.

[0077] Comparative Example 3

[0078] An artificial epineurium for sutureless nerve repair, comprising a middle elastic layer and an outer elastic layer arranged in layers;

[0079] The thickness of the middle elastic layer 2 is 70 μm, and the thickness of the outer elastic layer 3 is 100 μm.

[0080] The preparation method of the artificial epineurium provided in this comparative example comprises the following steps:

[0081] (1) 100 g of polyethylene glycol diamine (Mn = 6000) was dissolved in 400 mL of anhydrous chloroform, 10 mL of triethylamine was added, and the mixture was stirred at 0°C under argon for 1 h. 2.7 g of toluene diisocyanate was then added, and the mixture was reacted in an ice-water bath for 1 h. The mixture was stirred at room temperature for 4 days. After the reaction was completed, the resulting solution was precipitated with methanol. After a white precipitate appeared, the mixture solution was precipitated for 30 min, and chloroform was added to dissolve the product. This process was repeated three times, and the mixture was finally dried by vacuum evaporation to obtain an intermediate layer elastomer. Subsequently, 1 g of the obtained intermediate layer elastomer was dissolved in 20 mL of anhydrous chloroform and thoroughly mixed under stirring to obtain a uniform solution. The obtained uniform solution was then poured into a Teflon culture dish with a diameter of 100 mm and dried at room temperature to obtain an intermediate elastic layer.

[0082] 100 g of polyethylene glycol diamine (Mn=6000) was dissolved in 400 mL of anhydrous chloroform, 10 mL of triethylamine was added, and the mixture was stirred at 0°C under argon for 1 hour. Then, 2.0 g of diphenylmethane-4,4'-diisocyanate and 2.7 g of toluene diisocyanate were added, and the mixture was reacted in an ice-water bath for 1 hour, and then stirred at room temperature for 4 days. After the reaction, the resulting solution was first precipitated with methanol. After a white precipitate appeared, the mixture solution was precipitated for 30 minutes, and then chloroform was added to dissolve the product. This process was repeated three times, and finally dried by vacuum evaporation to obtain an outer elastomer; then, 1 g of the obtained outer elastomer was dissolved in 20 mL of anhydrous chloroform and thoroughly mixed under stirring to obtain a uniform solution. The obtained uniform solution was then poured into a Teflon culture dish with a diameter of 100 mm and dried at room temperature to obtain an outer elastic layer;

[0083] (2) The dried middle elastic layer and the outer elastic layer are stacked and allowed to stand at room temperature for 12 hours to allow the two layers of elastomer to adhere to form a double-layer structure. The resulting double-layer structure is then placed in an oxygen plasma device with a power of 100 W for 3 minutes to enhance surface activity, thereby obtaining the artificial neural epineurium.

[0084] Experimental characterization:

[0085] (1) Appearance: Visually observe the artificial epineurium prepared in Example 1, and obtain the physical picture of the artificial epineurium provided in Example 1 as shown below: Figure 2 As shown;

[0086] from Figure 2 It can be seen that the artificial epineurium was successfully prepared in Example 1, and the appearance of the prepared artificial epineurium was white.

[0087] (2) Mechanical properties:

[0088] ① Cut the artificial epineurium into rectangular samples with a size of 0.5 × 1.0 cm and install them on the fixture of the universal testing machine for testing;

[0089] ② Initial tensile strength and initial strain: Each sample (initial length 10 mm, width 5 mm, thickness 0.3 mm) was tensile tested at a tensile rate of 20 mm / min. A 50 N load cell was used to record the force (N) and elongation (mm) generated during the stretching process. The initial tensile strength and initial strain of the sample were calculated.

[0090] ② Tensile strength and strain after 10 cycles: The sample was first stretched to a strain of 500% and then restored to its initial length at a rate of 50 mm / min. After the stretching and recovery were completed, the sample was maintained at its initial length for 10 minutes, and the above cycle was repeated 10 times. During the test, the changes in load (N) and extension (mm) were monitored and recorded in real time using Instron Bluehill software. The tensile strength and strain of the sample after 10 cycles were calculated.

[0091] In addition, all experiments were repeated three times to ensure the reliability of the data.

[0092] The artificial epineurium provided in Examples 1 to 9 and Comparative Examples 1 to 3 was tested according to the above test method. The test results are shown in Table 1:

[0093] Table 1

[0094]

[0095]

[0096] (3) Adhesion performance:

[0097] The adhesion of the artificial epineurium was evaluated using a universal testing machine (Instron 34SC-1, USA). First, the artificial epineurium or the commercially available medical adhesive Kwik-Cast TM (World Precision Instruments, Inc., USA) was fixed between two skin tissues on a PET matrix (application area of 1 × 1 cm 2 ) and measure the adhesion strength (kPa);

[0098] The test method is as follows: freshly cut skin tissue and sciatic nerve are immersed in 0.1M MES buffer (pH = 4.5) and 1×PBS buffer (pH = 7.4), respectively, and incubated at 25°C for 1 hour. The adhesion strength of the test sample is measured by the maximum load when the sample is separated from the substrate interface. The test rate is set to 20 mm / min. The obtained adhesion strength is expressed in kilopascals (kPa). The adhesion force is calculated by dividing the maximum load (kN) by the contact area of the sample (m 2 ) were calculated, and each sample was tested three times.

[0099] The artificial epineurium provided in Examples 1 to 9 and Comparative Examples 1 to 3 and commercially available medical adhesive were tested according to the above test method. The test results are shown in Table 2:

[0100] Table 2

[0101]

[0102] From the data in Table 1 and Table 2, we can see that:

[0103] The artificial epineurium for sutureless nerve repair provided in Examples 1 to 9 has both excellent adhesive properties and mechanical properties, and has significantly higher adhesion strength than commercially available medical adhesives.

[0104] By comparing the data of Examples 1 to 9 and Comparative Examples 1 to 3, it can be seen that the artificial epineurium provided in Comparative Example 1 does not contain an outer elastic layer, resulting in weak rigidity and strong flexibility, and the mechanical support performance of the overall structure is insufficient; the artificial epineurium provided in Comparative Example 2 does not contain an intermediate elastic layer, resulting in strong rigidity and weak flexibility, which is not conducive to dynamic adaptability during nerve regeneration; the artificial epineurium provided in Comparative Example 3 does not contain an outer elastic layer hydrophilic adhesive layer, and the overall adhesion strength is poor; none of them can have both excellent bonding properties and mechanical properties.

[0105] Further comparison of the data from Examples 1 to 5 shows that the thickness of the intermediate elastic layer also affects the mechanical properties of the resulting artificial neural epineurium. If the thickness of the intermediate elastic layer is too low (Example 3), the resulting artificial neural epineurium will have a relatively high rigidity and a relatively low flexibility, which is not conducive to the dynamic adaptability during nerve regeneration. If the thickness of the intermediate elastic layer is too low (Example 5), the resulting artificial neural epineurium will have a relatively low rigidity and a relatively high flexibility, resulting in insufficient mechanical support performance of the overall structure.

[0106] Finally, a comparison of the data from Example 1 and Examples 6 to 9 shows that the thickness of the outer elastic layer also affects the mechanical properties of the resulting artificial epineurium. If the thickness of the outer elastic layer is too low (Example 7), the resulting artificial repair epineurium will have weak rigidity and strong flexibility, resulting in insufficient mechanical support for the overall structure. If the thickness of the outer elastic layer is too low (Example 9), the resulting artificial repair epineurium will have strong rigidity and weak flexibility, which is detrimental to dynamic adaptability during nerve regeneration.

[0107] (4) Nerve anastomosis experiment

[0108] The animals used in this experiment were male Lewis rats (weighing 250-300 g). In the experiment, 20 rats were randomly divided into four groups: a group in which surgeons used traditional microsuturing nerve anastomosis (suture group, n=10), a group in which surgeons used the artificial epineurium provided by the present invention for nerve anastomosis (nerve anastomosis group, n=10); a group in which non-experts used traditional microsuturing nerve anastomosis (suture group, n=10), and a group in which surgeons did not use the artificial epineurium provided by the present invention for nerve anastomosis (nerve anastomosis group, n=10);

[0109] ① Conventional microsurgery for nerve anastomosis: All rats were initially anesthetized by inhalation of 2.5% isoflurane and maintained in an anesthetized state of 1.5% isoflurane during the operation. The sciatic nerve on the right side of each animal was then exposed under an operating microscope. The exposure range was from the inferior edge of the piriformis muscle to approximately 5 mm below the bifurcation of the sciatic nerve. The sciatic nerve was cut at the mid-thigh using microsurgery scissors. In the conventional microsurgery nerve anastomosis group, six 9-0 epineurial interrupted sutures were used to repair the broken ends under a microscope.

[0110] ② The nerve anastomosis group of the artificial epineurium provided by the present invention: In order to test the application effect of the artificial epineurium for sutureless nerve repair provided by the present invention in in vivo nerve anastomosis and compare it with traditional microsurgery, a rat sciatic nerve injury model (SD rats, 300-400 g, 10 weeks old, male) was used; the following are the detailed steps of the experiment: the experiment used 10-week-old male SD rats (300-400 g); before the operation, the rats were initially anesthetized by inhalation of 2.5% isoflurane, and maintained in an anesthesia state of inhalation of 1.5% isoflurane during the operation; then the right sciatic nerve of the rat was exposed under a surgical microscope and the nerve was cut using microscissors; after cutting, the nerve was rinsed once with 1×PBS; for nerve anastomosis, the traditional microsurgery group (n=4) used 6 9-0 epineurium interrupted sutures for nerve repair; and in the artificial epineurium group for sutureless nerve repair provided by the present invention (n=4), a 1.0×1.0 cm 2The artificial epineurium for seamless nerve repair provided by the present invention is wrapped around the stump nerve for docking and anastomosis, and the excess epineurium is cut off.

[0111] The artificial epineurium provided in Example 1 was tested according to the above test method. The test results are shown in Table 3:

[0112] Table 3

[0113]

[0114] According to the data in Table 3, we can see that:

[0115] Compared with 600 seconds required by surgeons using traditional microsurgery suturing technology and 3000 seconds required by non-experts using traditional microsurgery suturing technology, the artificial epineurium for seamless nerve repair provided in Example 1 of the present invention has a shorter suturing time of only 40 seconds when operated by surgeons, and only 60 seconds when operated by non-experts, proving whether efficient surgical operations can be achieved without the need for advanced microsurgery technology.

[0116] 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. An artificial epineurium for sutureless nerve repair, characterized in that: The artificial epineurium comprises a hydrophilic adhesive layer, a middle elastic layer and an outer elastic layer which are stacked; The raw materials of the hydrophilic adhesive layer include hyaluronic acid, catechol, crosslinking agent A and crosslinking agent B; The raw materials of the middle elastic layer include polyethylene glycol diamine A and toluene diisocyanate A; The raw materials of the outer elastic layer include polyethylene glycol diamine B, toluene diisocyanate B and diphenylmethane-4,4'-diisocyanate.

2. The artificial epineurium according to claim 1, wherein The thickness of the hydrophilic adhesive layer is 20 to 40 μm; Preferably, the mass ratio of the hyaluronic acid, catechol, crosslinking agent A and crosslinking agent B is 1:(0.2-0.4):(0.6-0.8):(0.05-0.15); Preferably, the cross-linking agent A comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; Preferably, the cross-linking agent B comprises N-hydroxysuccinimide.

3. The artificial epineurium according to claim 1 or 2, characterized in that The thickness of the intermediate elastic layer is 60 to 80 μm; Preferably, the mass ratio of the polyethylene glycol diamine A to toluene diisocyanate A is 100:(2-3).

4. The artificial epineurium according to any one of claims 1 to 3, characterized in that: The thickness of the outer elastic layer is 90 to 110 μm; Preferably, the mass ratio of the polyethylene glycol diamine B, toluene diisocyanate B and diphenylmethane-4,4'-diisocyanate is 100:(2-3):(3-4.5); Preferably, the number average molecular weight of the polyethylene glycol diamine A and the polyethylene glycol diamine B is independently 5000 to 7000.

5. A method for preparing an artificial epineurium for sutureless nerve repair according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: (1) reacting hyaluronic acid and catechol in a MES buffer solution containing a crosslinking agent A and a crosslinking agent B, dialyzing and drying to obtain a hyaluronic acid / catechol crosslinked product, and then dissolving the obtained hyaluronic acid / catechol crosslinked product in water to obtain a hyaluronic acid / catechol composite solution; Polyethylene glycol diamine A and toluene diisocyanate A react in the presence of catalyst A, precipitate and dry to obtain an intermediate layer elastomer, and then dissolve the obtained intermediate layer elastomer in solvent A, and place in container A to dry and form a film to obtain an intermediate elastic layer; Polyethylene glycol diamine B, toluene diisocyanate B, and diphenylmethane-4,4'-diisocyanate are reacted in the presence of catalyst B, precipitated, and dried to obtain an outer elastomer, which is then dissolved in solvent B and placed in container B for drying to form a film, thereby obtaining an outer elastic layer; (2) The middle elastic layer and the outer elastic layer obtained in step (1) are superimposed to form a double-layer structure, and the hyaluronic acid / catechol composite solution obtained in step (1) is dripped onto one surface of the middle elastic layer of the double-layer structure, and dried to form a film to form a hydrophilic adhesive layer, thereby obtaining the artificial epineurium.

6. The preparation method according to claim 5, characterized in that In step (1), the reaction temperature of reacting hyaluronic acid and catechol in a MES buffer solution containing crosslinking agent A and crosslinking agent B is 20-40° C., and the reaction time is 12-48 hours; Preferably, the mass percentage of the hyaluronic acid / catechol cross-linked product in the hyaluronic acid / catechol composite solution in step (1) is 0.003-0.007%.

7. The preparation method according to claim 5 or 6, characterized in that: The catalyst A and catalyst B in step (1) both comprise triethylamine; Preferably, based on the amount of polyethylene glycol diamine A used in step (1) being 100 g, the amount of catalyst A used is 5 to 15 mL; Preferably, based on 100 g of the polyethylene glycol diamine B in step (1), the amount of the catalyst A is 5 to 15 mL.

8. The preparation method according to any one of claims 5 to 7, characterized in that The step (1) of reacting polyethylene glycol diamine A and toluene diisocyanate A in the presence of catalyst A and reacting polyethylene glycol diamine B, toluene diisocyanate B and diphenylmethane-4,4'-diisocyanate in the presence of catalyst B are both carried out under an inert gas environment; Preferably, the inert gas comprises argon; Preferably, in step (1), the reaction temperatures of reacting polyethylene glycol diamine A and toluene diisocyanate A in the presence of catalyst A and the reaction of polyethylene glycol diamine B, toluene diisocyanate B and diphenylmethane-4,4'-diisocyanate in the presence of catalyst B are independently 0 to 40° C., and the reaction times are independently 12 to 48 h. Preferably, the solvent A and solvent B in step (1) both comprise anhydrous chloroform; Preferably, the container A and the container B in step (1) both comprise Teflon culture dishes.

9. The preparation method according to any one of claims 5 to 8, characterized in that: Step (2) further includes placing the double-layer structure in an oxygen plasma device for plasma treatment before dripping the hyaluronic acid / catechol composite solution obtained in step (1) onto the surface of one side of the middle elastic layer of the double-layer structure.

10. Use of the artificial epineurium for sutureless nerve repair according to any one of claims 1 to 4 as a medical material.

Images