A directional nerve conduit with dynamic microstructure and its preparation method

Abstract

This invention relates to a method for preparing a directional nerve conduit with a dynamic microstructure, comprising the following steps: S1, preparation of a memory film material precursor solution; S2, preparation of a directional microstructure template; S3, preparation of a directional microstructure memory film; S4, programming and shaping treatment; S5, nerve conduit preparation: the directional microstructure memory film treated in step S4 is rolled and bonded to form a conduit. This invention also claims protection for the nerve conduit prepared by this method. Through the preparation method of this invention, the obtained nerve conduit can undergo specific changes in its directional microstructure by adjusting temperature, thus enabling more precise control of peripheral nerve cells, better acceleration of the physiological nerve regeneration process, and promotion of nerve defect healing and the creation of effector organ functionality; furthermore, the preparation method is simple, environmentally friendly, and easy for industrial production applications.

Description

Technical Field

[0001] This invention relates to the field of medical devices, specifically to a directional nerve conduit with a dynamic microstructure, and a method for preparing the nerve conduit. Background Technology

[0002] Peripheral nerve injury is a common condition caused by various factors, including metabolic disorders, poisoning, nutritional deficiencies, and iatrogenic injuries (such as chemotherapy and radiotherapy). Patients experience varying degrees of sensory and motor dysfunction, including paresthesia at the site of injury, intractable neuralgia, or denervation paralysis of the limbs, severely impacting their daily lives and placing a significant burden on patients and society. According to literature, there are over 20 million existing cases of peripheral nerve injury in my country, with approximately 2 million new cases added annually, making it a significant public health threat. Currently, the gold standard for treating nerve injuries longer than 2mm is autologous nerve transplantation. However, this method has drawbacks such as donor nerve shortages, size mismatch between donor and recipient sites, donor site damage, and time-consuming secondary surgeries. Artificial nerve conduits, on the other hand, can provide a suitable local microenvironment for peripheral nerve repair, accelerating peripheral nerve regeneration. They also offer advantages such as not being limited by donor nerve availability, adjustable size, short surgical time, and rapid recovery. Therefore, artificial nerve conduits have become a promising new trend for treating peripheral nerve injuries, potentially replacing autologous nerve transplantation. Currently, many hollow nerve conduits have been approved for clinical use.

[0003] However, while conventional hollow conduits can guide autologous stump neuronal axons through the injury area, they fail to effectively promote the directional alignment of peripheral nerve repair-related cells. Furthermore, their guidance of peripheral nerve regeneration is often slow and the regeneration direction is not precise enough, ultimately leading to poor neurological function recovery in patients after peripheral nerve repair surgery. Therefore, we need to improve the neural conduit with certain cues. These cues can be mainly divided into physical cues and chemical cues. In recent years, scholars have noticed that materials and physical cues can also efficiently regulate cell behavior with fewer side effects compared to chemical methods such as drugs and growth factors. Among them, the effects of geometric cues on the material surface, such as parallel microgrooves, on the differentiation of neuronal precursor cells, axonal elongation, and Schwann cell proliferation have been widely confirmed. Currently, many scholars have attempted to add topological structures to the surface of neural conduits to further improve their ability to guide nerve regeneration, but these added topological structures are mainly static micropatterns. However, peripheral nerve repair is a finely regulated sequential process, and a single static micropattern cannot accurately match the sequential dynamic process of nerve repair. Recent reports have demonstrated that different geometric cues lead to significantly different cellular behaviors. For example, smooth material surfaces can promote stem cell proliferation, while micropillar-structured surfaces promote cell differentiation. This suggests that we can regulate cell behavior by controlling the microstructure of the material's topological interface to reproduce the different temporal cellular behaviors during neural regeneration. Specifically, we could first use smooth planes to promote Schwann cell adhesion and proliferation, and then at an appropriate time, change the interface microstructure to initiate the differentiation of Schwann cells and neuronal precursor cells. Therefore, creating a novel neural conduit with a controllable, dynamically changing surface microstructure that can precisely match the temporal requirements of the neural repair process could provide a more favorable local microenvironment and biomechanical properties for damaged peripheral nerves.

[0004] Shape memory polymers (SMPs) are a class of intelligent polymer materials that can recover their permanent shape in response to external stimuli (such as temperature changes, electricity, magnetism, or light). Polycaprolactone (PCL), as a mature biomaterial, has long been used in neural tissue engineering due to its non-neurotoxicity, good tissue compatibility, good biodegradability, and non-toxic and pollution-free degradation products. Research has shown that by attaching acrylate double bonds to both ends of the PCL chain and functionalizing the end groups of PCL, it can be endowed with shape memory function, thus becoming a polycaprolactone shape memory polymer (PCL-SMP). Furthermore, the deformation temperature can be adjusted by changing the ratio of the mixed components and the molecular weight of the PCL incorporated, and the mechanical and biological properties of the composite scaffold can be adjusted by incorporating various additives.

[0005] Therefore, a novel 4D directional nerve conduit with dynamic fine microstructure was developed based on PCL-SMP. By remotely controlling the dynamic changes in the shape and surface microstructure of the conduit, multiple temporal processes of nerve repair can be precisely matched and promoted, providing a more personalized and efficient treatment plan for peripheral nerve injury. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a directional nerve conduit with a dynamic and intricate microstructure, and the directional nerve conduit obtained by this method. The directional nerve conduit of this invention has a 3D spatial structure, and its shape and microstructure can be dynamically changed over time by appropriate temperature variations. This allows for precise control of the cell fate related to peripheral nerve repair and accurate matching of the peripheral nerve repair sequence, thereby better accelerating the physiological nerve regeneration process and promoting the healing of nerve defects and the reconstruction of effector organ function. This solves the problem that while conventional nerve conduits can guide nerve regeneration, they cannot precisely coordinate with the long and complex dynamic physiological process of nerve repair.

[0007] To address the aforementioned problems, the technical solution adopted by this invention is as follows:

[0008] A method for preparing a directional nerve conduit with a dynamic microstructure, comprising the following steps:

[0009] S1. Preparation of precursor solution for memory film material: Take a mixture of two PCLDA molecules with different molecular weights, place it in a container and add DMF solvent, then heat to dissolve all PCLDA, then add catalyst BPO, and continue heating to dissolve BPO completely to obtain precursor solution for memory film material.

[0010] S2. Preparation of directional microstructure templates: PDMS is mixed with a curing agent to obtain PDMS printing ink, and then a directional microstructure template with a preset structure is obtained by 3D printing.

[0011] S3. Preparation of oriented microstructure memory film: Construct a mold using the oriented microstructure template obtained in step S2, then pour the memory film material precursor liquid obtained in step S1 into the mold, heat it to polymerize the material to obtain a film sheet, then peel the film sheet out of the mold, remove excess solvent and clean the film sheet to obtain the oriented microstructure memory film.

[0012] S4. Programming and Shaping Process: The oriented microstructure memory film obtained in step S3 is programmed and adjusted by external force, and then it is fixed by quick-freezing.

[0013] S5. Preparation of nerve conduit: The directional microstructure memory film processed in step S4 is rolled and bonded to form a conduit.

[0014] In this invention, a further preferred embodiment is that, in step 1, a mixture is composed of PCLDA with a molecular weight of 2000 and PCLDA with a molecular weight of 10000, wherein the weight ratio of PCLDA with a molecular weight of 2000 to PCLDA with a molecular weight of 10000 in the mixture is 1-4:1.

[0015] In this invention, a further preferred embodiment is that in step S1, the heating temperature is 80°C, the heating time for PCLDA is 3 minutes, and the heating time after adding BPO is 1 minute.

[0016] In this invention, a further preferred embodiment is that the preset structure in step S2 is a series of parallel grooves with a width of 24-36 μm and a depth of 21 μm, and the spacing between adjacent grooves is equal to the width of the grooves.

[0017] In this invention, a further preferred embodiment is that in step S3, the heating temperature is 80°C and the heating time is 2 hours.

[0018] In a further preferred embodiment of the present invention, in step S3, excess solvent is removed by drying in an oven at 60°C; and the film sheet is placed in 75% alcohol for ultrasonic cleaning.

[0019] In a further preferred embodiment of the present invention, the thickness of the thin film in step S3 is 1-2 mm.

[0020] In this invention, a further preferred embodiment is that, in step S4, the programming adjustment process specifically involves: stretching the film sheet at 50°C using external force until the groove width is 85-105μm; the quick-freezing and shaping process specifically involves: quick-freezing the programmed film sheet at -20°C for at least 2 minutes.

[0021] In this invention, a further preferred embodiment is to use the adhesiveness of the memory film material itself to bond the rolled-up film sheet into a conduit.

[0022] Another object of the present invention is to protect the directional nerve conduit with dynamic microstructure obtained by the above-described methods / technical solutions.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (1) This invention utilizes PCL shape memory material, which exhibits excellent biocompatibility and biodegradability, to fabricate a 4D directional nerve conduit with a dynamic and fine microstructure. Compared to traditional nerve conduits, which, while capable of guiding peripheral nerve regeneration, cannot precisely match the lengthy and complex dynamic physiological process of nerve repair, the conduit provided by this invention not only possesses a three-dimensional spatial directional microstructure but also allows for remote control of the dynamic changes of this microstructure over time. This precisely regulates the fate of cells related to peripheral nerve repair and accurately matches the sequential process of peripheral nerve repair, thereby accelerating the physiological nerve regeneration process and promoting the healing of nerve defects and the reconstruction of effector organ function. Furthermore, this invention regulates the dynamic changes of the microstructure by applying appropriate temperature variations, avoiding repeated trauma and reducing the risk of infection. Moreover, this invention uses grooves as micropatterns on the surface of the conduit, forming a more stable mechanical structure with better mechanical properties, thus solving to some extent the problems of easy collapse and breakage of existing conduits.

[0025] (2) The manufacturing method provided by this invention involves first preparing a PCL shape memory film with a programmed directional microstructure on its surface, and then rolling the film to obtain a nerve conduit. This allows us to arbitrarily adjust the inner diameter and length of the manufactured conduit while rolling the film, thereby making it easier to obtain nerve conduits of personalized length and size. In addition, this film is very easy to store. This means that shape memory films with programmed micropatterns on their surface can be mass-produced and stored in various medical institutions as semi-finished products. Each medical institution can use this semi-finished product to manufacture the required personalized conduits at any time, which is conducive to the large-scale promotion and application of this conduit;

[0026] (3) The PCL shape memory material used in this invention can have its deformation temperature adjusted by adjusting the proportion of PCLDA mixing components and by mixing various additives to adjust the mechanical and biological properties of the composite scaffold. In addition, the method provided by this invention can adjust the width and depth of the grooves on the printed PDMS template and adjust the direction and magnitude of the external force applied during programming, thereby preparing personalized neural conduits with grooves of different widths and depths and different dynamic change processes.

[0027] (4) The method in this invention is simple and environmentally friendly, highly operable, non-toxic in preparation process, and has low economic cost, making it easy to mass-produce.

[0028] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0029] Figure 1 This is a physical image of the directional nerve conduit with dynamic and fine microstructure of the present invention;

[0030] Figure 2This is a flowchart of the preliminary preparation process of the present invention;

[0031] Figure 3 These are confocal microscope images showing the surface microstructure before and after dynamic changes in the present invention.

[0032] Figure 4 The results of flow cytometry analysis of PC12 cells after 48 hours of culture;

[0033] Figure 5 Scanning electron micrograph of PC12 cells after 48 hours of culture;

[0034] Figure 6 Immunofluorescence staining image of PC12 cells after 48 hours of culture;

[0035] Figure 7 Image showing nerve conduit implantation in a rat;

[0036] Figure 8 HE staining image of regenerated nerve tissue 16 weeks post-surgery;

[0037] Figure 9 Image of toluidine blue staining of regenerated nerve tissue 16 weeks post-surgery;

[0038] Figure 10 Masson trichrome staining image of muscle tissue distal to the regenerated nerve 16 weeks postoperatively;

[0039] Figure 11 This is a diagram showing the gait analysis results of rats 16 weeks after surgery. Detailed Implementation

[0040] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples in the specific embodiments are all obtained from commercial sources. The specific embodiments are exemplary and are only used to explain this application, and should not be construed as limiting the scope of protection of this application.

[0041] A method for preparing a directional nerve conduit with a dynamic microstructure, comprising the following steps:

[0042] S1. Preparation of precursor solution for memory film material: Take a mixture of two different molecular weight PCLDA [synthetic poly(ε-caprolactone) diacrylate], place it in a container and add DMF (dimethylformamide) solvent, then heat to dissolve all PCLDA, then add catalyst BPO (benzoyl peroxide), continue heating to dissolve BPO completely, and obtain the precursor solution for memory film material;

[0043] S2. Preparation of directional microstructure templates: PDMS (polydimethylsiloxane) is mixed with a curing agent to obtain PDMS printing ink, and then a directional microstructure template with a preset structure is obtained by 3D printing.

[0044] S3. Preparation of oriented microstructure memory film: Construct a mold using the oriented microstructure template obtained in step S2, then pour the memory film material precursor liquid obtained in step S1 into the mold, heat it to polymerize the material to obtain a film sheet, then peel the film sheet out of the mold, remove excess solvent and clean the film sheet to obtain the oriented microstructure memory film.

[0045] S4. Programming and Shaping Process: The oriented microstructure memory film obtained in step S3 is programmed and adjusted by external force, and then it is fixed by quick-freezing.

[0046] S5. Preparation of nerve conduit: The directional microstructure memory film processed in step S4 is rolled and bonded to form a conduit.

[0047] In the preparation method of the directional nerve conduit with dynamic microstructure of the present invention, in step S1, a mixture of two different molecular weight PCLDA [synthetic poly(ε-caprolactone) diacrylate] is added to DMF and BPO to obtain a memory film material precursor solution; the memory film material precursor solution is added to a mold constructed with a directional microstructure template, and then the material is polymerized by heating to obtain a film sheet complementary to the directional microstructure template in step S2. Such a film sheet has a directional microstructure (i.e., grooves with corresponding width, depth and spacing). The nerve conduit made from this film sheet has a more stable mechanical structure and mechanical properties, which can solve the problem of existing conduits being prone to collapse and breakage to a certain extent.

[0048] Through the programming and shaping process in step S4, the directional microstructure on the thin film can be further adjusted. That is, the width, spacing, and depth of the grooves can be adjusted by the magnitude of the external force. To facilitate the programming process, it can be carried out at a certain temperature, such as 60°C. At this temperature, the thin film will soften appropriately, which will make the programming process (i.e., adjusting the depth, width, and spacing of the grooves on the thin film) easier. In order to reduce the deformation of the thin film after the programming process, it can be subjected to a quick-freeze shaping (shaping) process. The preferred conditions for quick-freeze shaping are: quick-freeze temperature of -20°C and quick-freeze time of not less than 2 minutes (not less than 2 minutes in this invention includes 2 minutes). This allows for more complete crystallization inside the thin film material and a more stable structure.

[0049] After the directional microstructure memory film is made through steps S3 and S4, it can be rolled and bonded to obtain a nerve conduit. This method allows us to roll and bond the film according to the actual inner diameter and length of the nerve conduit, which can meet personalized customization needs. In addition, compared with the pre-formed nerve conduit, the film takes up less space and is easier to store and transport. In practice, the film can be used as a semi-finished product, and the corresponding medical structure can be made into a nerve conduit according to the film as needed, which is more convenient for promotion and application.

[0050] The nerve conduit prepared through the above steps not only possesses a directional microstructure, but also exhibits corresponding changes in its microstructure over time due to its main body being composed of two different molecular weight PCLDA molecules. These changes can be induced by adjusting temperature variations, allowing for more precise regulation of peripheral nerve cells, accelerating the physiological nerve regeneration process, promoting the healing of nerve defects, and facilitating the creation of functional effector organs. Compared to traditional nerve conduits, it better complements the long and complex dynamic physiological process of nerve repair. The dynamic changes in the microstructure of the nerve conduit, regulated by temperature variations, avoid repeated trauma and reduce the risk of infection.

[0051] The preparation method of this invention is simple, environmentally friendly, highly operable, non-toxic, low-cost, and easy to implement for large-scale industrial production.

[0052] In step S1, for a mixture of PCLDA with different molecular weights, the deformation temperature of the corresponding neural conduit can be adjusted by changing the specific molecular weights and mixing ratio of the two PCLDA molecules in the mixture. Preferably, a mixture of PCLDA with a molecular weight of 2000 and PCLDA with a molecular weight of 10000 is used. A more preferred PCLDA mixture is one where the ratio of PCLDA with a molecular weight of 2000 to PCLDA with a molecular weight of 10000 is between 1 and 4:1. At this temperature ratio, short-term temperature regulation will not damage cells or cause deformation at body temperature. The inventors found that a deformation temperature of 39°C corresponds to a ratio of 8:2 (i.e., 4:1), and a deformation temperature of 46°C corresponds to a ratio of 5:5 (i.e., 1:1). An even more preferred ratio is 6:4 (i.e., 1.5:1). The neural conduit corresponding to this ratio exhibits a fixation recovery rate exceeding 99% after deformation, allowing for better dynamic adjustment.

[0053] In step S1, a more preferred approach for heating temperature and time is to heat at 80°C, heat PCLDA for 3 minutes, and heat BPO for 1 minute.

[0054] For the grooves in step S2, which are preset to be arranged in parallel, the groove width is 24-36μm, the depth is 21μm, and the spacing between adjacent grooves is equal to the groove width.

[0055] In step S3, the heating temperature is 80℃ and the heating time is 2 hours.

[0056] In step S3, excess solvent is removed by drying in an oven at 60°C; the film is then placed in 75% alcohol for ultrasonic cleaning; this oven temperature promotes ethanol evaporation more effectively.

[0057] In step S3, the thickness of the thin film is 1-2 mm; a more preferred thickness is 1 mm.

[0058] In step S4, the programming adjustment process specifically involves stretching the film sheet at 50°C using external force until the groove width is 85-105μm; the quick-freeze setting process specifically involves quick-freezing the programmed film sheet at -20°C for at least 2 minutes; the use of the above quick-freeze setting conditions allows for more complete internal crystallization of the film sheet material and greater overall stability.

[0059] In step S5, for bonding, it is preferable to use the adhesiveness of the memory film material itself to bond the rolled film into a conduit. This can avoid the addition of excess material and make subsequent use safer.

[0060] The mold construction in step S is specifically as follows: The prepared oriented microstructure template (i.e., PDMS template) is placed into a sealed space mold constructed from a silicone rubber sheet. The cavity formed between the oriented microstructure template and the silicone rubber sheet is the mold cavity. Then, a precursor solution is poured in to form a thin film complementary to the oriented microstructure template. (See reference...) Figure 2 To understand this, in step S3, before heating, the inside of the mold is sealed to isolate it from the outside air and excess oxygen is expelled. This can prevent side reactions from occurring and affecting the quality of subsequent products.

[0061] Furthermore, the nerve conduit of the present invention can also be used in later stages by adding substances to adjust the mechanical and biological properties of the applied structure (such as a scaffold).

[0062] The directional nerve conduits with dynamic microstructures prepared by the above methods / technical solutions have stable mechanical structures and good mechanical properties, which can better accelerate the physiological nerve regeneration process, promote the healing of nerve defects and the creation of effector organ functions.

[0063] The nerve conduit described in this invention can be used in medical devices and other products.

[0064] Example 1

[0065] A method for preparing a directional nerve conduit with a dynamic microstructure, comprising the following steps:

[0066] S1. Preparation of precursor solution for memory film material: Take a mixture of two different molecular weight PCLDA [synthetic poly(ε-caprolactone) diacrylate], place it in a container and add DMF (dimethylformamide) solvent, then heat to dissolve all PCLDA, then add catalyst BPO (benzoyl peroxide), continue heating to dissolve BPO completely, and obtain the precursor solution for memory film material;

[0067] S2. Preparation of directional microstructure templates: PDMS (polydimethylsiloxane, commercially available) is mixed with a curing agent to obtain PDMS printing ink, and then directional microstructure templates with a preset structure are obtained by 3D printing.

[0068] S3. Preparation of oriented microstructure memory film: A mold is constructed using the oriented microstructure template obtained in step S2. Then, the memory film material precursor liquid obtained in step S1 is poured into the mold, and the material is heated to polymerize and obtain a film sheet. The film sheet is then peeled out of the mold, excess solvent is removed, and the film sheet is cleaned to obtain the oriented microstructure memory film. The mold construction is as follows: The prepared oriented microstructure template (i.e., PDMS template) is placed into a sealed space mold constructed from a silicone rubber sheet to form a mold. Before heating, the inside of the mold is sealed to isolate it from the outside air and excess oxygen is discharged.

[0069] S4. Programming and Shaping Process: The oriented microstructure memory film obtained in step S3 is programmed and adjusted by external force, and then it is fixed by quick-freezing.

[0070] S5. Preparation of nerve conduit: The directional microstructure memory film processed in step S4 is rolled and bonded to form a conduit;

[0071] In step 1, a mixture is made of PCLDA with a molecular weight of 2000 and PCLDA with a molecular weight of 10000, and the weight ratio of PCLDA with a molecular weight of 2000 and PCLDA with a molecular weight of 10000 in the mixture is 6:4 (1.5:1).

[0072] In step S1, the heating temperature is 80°C, the heating time for PCLDA is 3 minutes, and the heating time after adding BPO is 1 minute.

[0073] In step S2, the preset structure consists of parallel grooves with a width of 35 μm and a depth of 21 μm. The spacing between adjacent grooves is equal to the groove width (35 μm). This preset structure's directional microstructure template is printed using melt electrospinning direct writing technology and an EFL-MDW5800 printer developed by the Suzhou Institute of Intelligent Manufacturing. The design parameters in the computer are adjusted, and the printing is performed under automatic computer control. The printing parameters are: voltage of 4.7 kV, air pressure of 10 kPa, temperature of 95 °C, and distance between the nozzle and the collector of 5 mm.

[0074] In step S3, the heating temperature is 80℃ and the heating time is 2 hours.

[0075] In step S3, excess solvent is removed by drying in an oven at 60°C; the film sheet is then placed in 75% alcohol for ultrasonic cleaning.

[0076] In a further preferred embodiment of the present invention, the thickness of the thin film in step S3 is 1 mm.

[0077] In this invention, a further preferred embodiment is that, in step S4, the programming adjustment process specifically involves: stretching the film sheet at 50°C using external force until the groove width is 100μm; the quick-freezing and shaping process specifically involves: quick-freezing the programmed film sheet at -20°C for 2 minutes.

[0078] In step S5, the curled film sheet is bonded into a conduit using the adhesive properties of the memory film material itself.

[0079] The nerve conduit is 17 mm long, 2 mm inner diameter, and 1 mm wall thickness. Before programming, the groove width is 35 μm, the depth is 21 μm, the distance between adjacent grooves is 35 μm, and after programming, the groove width is 100 μm.

[0080] Under the same conditions, five neural conduits were prepared for testing. The microstructural deformation time of the conduits at 44°C in vitro was 9 seconds, and at 44°C in vivo it was 150 seconds. The fixation recovery rate after deformation was 99.0%, the radial stiffness was 0.4724±0.05%, the axial stiffness was 156.9071±50.692 kPa, the maximum stress was 815.9971±13.049 MPa, and the maximum load was 3.1194±0.830 N, which meets the application requirements in neural tissue engineering.

[0081] Example 2

[0082] The specific preparation steps are the same as in Example 1, except that the ratio of PCLDA with a molecular weight of 2000 and PCLDA with a molecular weight of 10000 in step S1 is changed to 7:3, and a nerve conduit with a length of 17 mm, an inner diameter of 2 mm, a wall thickness of 1 mm, a groove width of 35 μm and a depth of 21 μm before programming, a spacing of 35 μm between adjacent grooves, and a groove width of 100 μm after programming is finally obtained.

[0083] Under identical conditions, five neural conduits were fabricated for testing. The microstructural deformation time of the conduits at 43°C in vitro was 10 seconds, and at 43°C in vivo, it was 150 seconds. The fixation recovery rate after deformation was 97.3%. The radial stiffness was 0.4382±0.09%, the axial stiffness was 133.8265±39.977 kPa, the maximum stress was 760.9477±7.950 MPa, and the maximum load was 2.5816±0.669 N, meeting the application requirements in neural tissue engineering.

[0084] Example 3

[0085] The specific preparation steps are the same as in Example 1, except that: in step S2, the design parameters in the computer are adjusted so that the groove width on the printed template surface is 25μm, the depth is 21μm, the width of adjacent grooves is 25μm, and the final result is a nerve conduit with a length of 17mm, an inner diameter of 2mm, a wall thickness of 1mm, a groove width of 25μm, a depth of 21μm, a spacing of 25μm between adjacent grooves, and a groove width of 100μm after programming.

[0086] Under identical conditions, five neural conduits were fabricated for testing. The microstructural deformation time of the conduits at 44°C in vitro was 9 seconds, and at 44°C in vivo, it was 150 seconds. The fixation recovery rate after deformation was 98.7%. The radial stiffness was 0.4802±0.08%, the axial stiffness was 150.3890±37.594 kPa, the maximum stress was 806.6195±15.581 MPa, and the maximum load was 3.2806±0.706 N, meeting the application requirements in neural tissue engineering.

[0087] Example 4

[0088] The specific preparation steps are the same as in Example 1, except that in step (5), when the membrane is rolled up to form a conduit, the length of the membrane is controlled to be 13 mm by trimming, and a nerve conduit with a length of 13 mm, an inner diameter of 2 mm, a wall thickness of 1 mm, a groove width of 35 μm and a depth of 21 μm before programming, a distance between adjacent grooves of 35 μm, and a groove width of 100 μm after programming is finally obtained.

[0089] Under identical conditions, five neural conduits were fabricated for testing. The microstructural deformation time of the conduits at 44°C in vitro was 9 seconds, and at 44°C in vivo, it was 150 seconds. The fixation recovery rate after deformation was 99.1%. The radial stiffness was 0.4773±0.05%, the axial stiffness was 148.9628±42.805 kPa, the maximum stress was 810.4807±11.355 MPa, and the maximum load was 3.1696±0.895 N, meeting the application requirements in neural tissue engineering.

[0090] Experiment Example 1 (In Vitro Cell Experiment)

[0091] Take the oriented microstructure memory film processed in step S4 of Example 1, cut a material sheet of appropriate size from the film and place it in a 24-well cell culture plate. Seed immature neuronal cells PC12 onto the surface of the material sheet with micropatterns in the 24-well cell culture plate. After culturing for 24 hours, place the culture plate in a 44°C environment for 9 seconds, then remove it and continue culturing for another 24 hours.

[0092] The viability of cultured PC12 cells was detected by flow cytometry at 12h, 24h, 36h and 48h.

[0093] The proliferation of PC12 cells was detected by immunofluorescence at 24h and 48h of culture, and the differentiation of PC12 cells was observed by scanning electron microscopy.

[0094] Flow cytometry results are shown in Figure 4 As shown in the figure, after 48 hours of culture on the membrane, the vast majority of PC12 cells were viable, with only a very small number of cells showing damage or death, indicating that the membrane has good biocompatibility.

[0095] Scanning electron microscope image (see) Figure 5 As can be seen from the figure, after 48 hours of culture, PC12 cells exhibited good orientation on the thin film surface.

[0096] Immunofluorescence staining results are shown in Figure 6 As can be seen from the figure, after 48 hours of culture, PC12 cells showed good differentiation on the membrane surface and exhibited good directional arrangement.

[0097] Experiment Example 2 (In vivo animal experiment)

[0098] (1) Experimental animals: 6 healthy wild-type male SD rats, 3-4 weeks old

[0099] (2) Animal surgical procedure: Sevoflurane inhalation anesthesia was performed. A midline incision was made in the posterior thigh to expose the sciatic nerve in the middle segment of the right hind limb. 15 mm of the sciatic nerve was removed, and the nerve conduit manufactured in Example 1 was transplanted at the sciatic nerve defect site. The proximal and distal nerve stumps were inserted 1 mm into the 17 mm nerve conduit, and the anastomosis was sutured with surgical sutures. The muscles and skin were sutured. The animals were fed routinely after surgery. Two weeks after surgery, the limb was wrapped with a 44°C heating machine for 150 seconds and then removed. Gait analysis was performed 16 weeks after surgery. The mice were then euthanized, and the regenerated nerve and distal muscle tissue were removed, fixed, dehydrated, cleared, paraffin-impregnated, and embedded. Three regenerated nerve tissue samples were stained with hematoxylin and eosin (HE), and the other three were stained with toluidine blue. The distal muscle tissue was stained with Masson's trichrome stain.

[0100] HE staining results are shown in [the original text]. Figure 8 The toluidine blue staining results are shown in the figure. Figure 9 ,from Figure 7 and Figure 8 As can be seen, 16 weeks post-surgery, the implanted conduit completely degraded, the regenerated nerve structure remained continuous, the myelin sheath thickness was uniform and regeneration was good, there were no signs of neuroma formation, minimal inflammatory cell infiltration, no obvious adhesion to surrounding tissues, and no scar formation, forming an anatomical structure similar to normal nerve tissue. This demonstrates that the 4D directional nerve conduit fabricated in this invention can effectively promote regeneration of peripheral nerves after injury.

[0101] Masson staining results are shown in […]. Figure 10 As can be seen from the figure, the majority of the tissue consists of muscle fibers with only a small amount of collagen, indicating that the muscle tissue distal to the regenerated nerve is growing well 16 weeks after surgery.

[0102] Gait analysis results are shown in Figure 11 As shown in the figure, the experimental rats recovered their motor function well 16 weeks after the operation.

[0103] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a directional nerve conduit with a dynamic microstructure, characterized in that... Includes the following steps: S1. Preparation of precursor solution for memory film material: Take a mixture of two PCLDA molecules with different molecular weights, place it in a container and add DMF solvent, then heat to dissolve all PCLDA, then add catalyst BPO, and continue heating to dissolve BPO completely to obtain precursor solution for memory film material. S2, Preparation of directional microstructure template: PDMS is mixed with a curing agent to obtain PDMS printing ink, and then a directional microstructure template with a preset structure is obtained by 3D printing; the preset structure in step S2 is a parallel groove with a groove width of 24-36μm and a depth of 21μm, and the spacing between adjacent grooves is equal to the groove width. S3. Preparation of oriented microstructure memory film: Construct a mold using the oriented microstructure template obtained in step S2, then pour the memory film material precursor liquid obtained in step S1 into the mold, heat it to polymerize the material to obtain a film sheet, then peel the film sheet out of the mold, remove excess solvent and clean the film sheet to obtain the oriented microstructure memory film. S4. Programming and Shaping Process: The oriented microstructure memory film obtained in step S3 is programmed and adjusted by external force, and then fixed by quick-freezing; In step S4, the programming and adjustment process specifically involves stretching the film sheet at 50°C using external force until the groove width is 85-105μm. S5. Preparation of nerve conduit: The directional microstructure memory film processed in step S4 is rolled and bonded to form a conduit.

2. The preparation method according to claim 1, characterized in that: In step 1, a mixture is made of PCLDA with a molecular weight of 2000 and PCLDA with a molecular weight of 10000, and the weight ratio of PCLDA with a molecular weight of 2000 to PCLDA with a molecular weight of 10000 in the mixture is 1-4:

1.

3. The preparation method according to claim 1, characterized in that: In step S1, the heating temperature is 80°C, the heating time for PCLDA is 3 minutes, and the heating time after adding BPO is 1 minute.

4. The preparation method according to claim 1, characterized in that: In step S3, the heating temperature is 80℃ and the heating time is 2 hours.

5. The preparation method according to claim 1, characterized in that: In step S3, excess solvent is removed by drying in an oven at 60°C; the film is then placed in 75% alcohol for ultrasonic cleaning.

6. The preparation method according to claim 1, characterized in that: In step S3, the thickness of the thin film is 1-2 mm.

7. The preparation method according to claim 1, characterized in that: In step S4, the quick-freezing and shaping process specifically involves quick-freezing the programmed film sheet at -20°C for at least 2 minutes.

8. The preparation method according to claim 1, characterized in that: In step S5, the adhesive properties of the memory film material itself are used to bond the rolled-up film sheet into a conduit.

9. A directional nerve conduit with a dynamic microstructure, characterized in that, It is prepared by the method according to any one of claims 1-8.

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