A customized differentiated interface neural conduit

Abstract

The present invention discloses a differentiated interface nerve conduit that can be customized, including a hollow outer sheath and an inner core filler arranged inside the hollow outer sheath; the surface of the hollow outer sheath is distributed with nano-scale micropores, and the inner core filler is a micron-scale lamellar loose filler arranged in an axial orientation. The nerve conduit structure meets the two different requirements of the internal and external repair microenvironments, respectively, and realizes the important functions of "inner affinity and outer sparseness, inner promotion and outer resistance". The conduit can not only promote the directional myelination of nerves and the directional growth of axons, but also avoid the formation of neuromas. The present invention also uses digital modeling combined with a template leaching method to customize nerve conduits. The preparation process is convenient, and the product parameters are adjustable and controllable. It provides a reliable personalized treatment plan for nerve defects with complex structures such as multiple branches, filling the gap of the current lack of precise repair products for complex nerve defects in clinical practice.

Description

Technical Field

[0001] The present invention relates to the technical field of nerve conduits, and in particular to a differentiated interface nerve conduit that can be customized in an individualized manner and is used for repairing and regenerating defective nerves. Background Art

[0002] Peripheral nerve injury is a common emergency and orthopedic disease, caused by high-energy trauma, post-tumor resection, and chronic systemic diseases. According to the Seddon classification criteria (according to the severity of the injury), it can be divided into neuropraxia, axonal truncation, and neurotruncation. For smaller injuries, "end-to-end anastomosis" is currently used clinically without high tension; for severe nerve defects (defects of more than 5 mm in adults), autologous nerve transplantation is still the gold standard, but this treatment measure has important defects such as donor limitation, size mismatch, and donor site damage. In addition, long-term follow-up shows that the functional recovery of patients after treatment is very limited, which greatly limits its wide clinical application. With the theoretical expansion of the field of life sciences and the advancement of tissue engineering technology, the role of nerve conduit bridging in clinical treatment has been continuously improved. Various functionalized nerve conduits are expected to replace autologous nerve transplantation and become the first choice for clinical treatment by effectively mimicking the structure and function of peripheral nerve tissue.

[0003] At present, there are many improved designs on the nerve catheter market at home and abroad, which can not only bridge the nerve ends and provide certain mechanical support for peripheral nerve regeneration, but also guide the axons to extend to the distal end through auxiliary material exchange or the addition of inducible bioactive factors, so as to achieve directional regeneration of peripheral nerves. However, these inventions and designs are mainly considered from the basic aspects of nerve adaptability, biocompatibility and peripheral nerve regeneration physiology, and the clinical products formed have deficiencies such as uncertain therapeutic effects and rapid degradation in vivo. In addition, the addition of bioactive factors has the risk of inducing gene mutations, tumorigenicity and teratogenicity. In comparison, the regulation effect of physical stimulation is accurate and stable, the effect is continuous and the application in vivo is safe. While exerting its function, it can avoid the deficiencies of biochemical preparations and has a broader application prospect. Among the many physical intervention factors, the topological microstructure (i.e., bionic concept) of nerve grafts by imitating the directional structure of peripheral nerves has attracted widespread attention. Among them, the axially oriented micro-nanostructure has been proven to have the effect of chemotactic axonal directional growth, which is of great significance for long-distance defects of peripheral nerves.

[0004] There are records of nerve catheters in the prior art (CN201810660151.8 A multi-channel peripheral nerve catheter and its preparation method; CN201511003594.2 An artificial nerve scaffold and its preparation method and application). Previous nerve catheter products or experimental studies mostly used electrospinning technology or additive manufacturing to construct hollow nerve catheters. The fibers oriented in the tube wall can induce axial migration of cells, and topological structures such as micro-nanoscale grooves on the fiber surface can increase the adhesion area of ​​cells on the inner surface of the catheter, aiming to establish and maintain a regenerative microenvironment early. However, this structure means that there will be more fibrous tissue adhesion and wrapping on the outer surface of the regenerated nerve, forming an obstacle in the process of nerve repair, and even causing neuromas, making it difficult for nerves to regenerate with high quality. Therefore, the lack of nerve catheters with differentiated internal and external interfaces makes it difficult to reconstruct nerves on demand, which is a huge problem in the current nerve repair market. Summary of the invention

[0005] In view of the deficiencies of the prior art, an object of the present invention is to provide a differentiated interface nerve conduit that can be customized.

[0006] The present invention is a customized differentiated interface nerve conduit. It uses 3D printing technology combined with phase separation molding to customize special-shaped bionic nerve conduits for multi-branch nerve defect repair. Based on fluorescent labeling three-dimensional digital modeling, sucrose is used as the printing raw material, and the printing ink is obtained by in-situ heating and caramelization. After 3D printing and customizing the required shape of the sugar mold, a mixed solution such as polycaprolactone (PCL) with good mechanical strength and relatively slow degradation is cast on the sugar mold, and the bionic nerve graft "outer sheath" is prepared by phase separation and template leaching method; using the gradient freezing method, solutions such as silk fibroin with good biocompatibility and relatively fast degradation are formed in situ inside the "outer sheath" with axially oriented lamellar "inner core filler" to guide the growth of nerve axons.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] The present invention provides a customized differentiated interface nerve conduit, comprising a hollow outer sheath and an inner core filler arranged inside the hollow outer sheath;

[0009] The surface of the hollow outer sheath is distributed with nanometer-scale micropores, and the inner core filler is a micrometer-scale lamellar loose filler arranged in an axial direction.

[0010] The degradation rate of the hollow outer sheath is lower than the degradation rate of the inner core filling.

[0011] Preferably, the pore size of the nanoscale micropores is 400-800 nm.

[0012] The present invention also provides a method for preparing a differentiated interface nerve conduit that can be customized, comprising the following steps:

[0013] S1. Printing sucrose template: setting the nerve defect parameters and performing 3D printing with caramelized ink to obtain a sucrose template;

[0014] S2. Preparing a hollow outer sheath of a nerve conduit: dissolving the raw materials for preparing the outer sheath in a solvent to form a mixed solution a, then immersing the sucrose template in the mixed solution a, taking it out after being fully soaked, and after the solvent is completely evaporated, obtaining the sucrose template on the surface of the outer sheath, then dissolving the sucrose template in the outer sheath, and then freeze-drying to obtain the hollow outer sheath;

[0015] S3. In situ formation of the inner core filling of the nerve conduit: dissolving the raw materials for preparing the inner core filling in water to form a mixed solution b, and then vertically placing the hollow outer sheath in the mixed solution b to fully immerse it, performing gradient freeze-drying or electric field action to form a directional frozen structure, and then freeze-drying to remove ice crystals to obtain the differentiated interface nerve conduit.

[0016] As a preferred solution, the specific steps of step S1 are:

[0017] S11. Open the GeSim software, wait for the machine to complete self-check, load sucrose into the screw barrel, select the needle specification, install the printing parts, and click the "Lock" button to lock;

[0018] S12, after measuring the height of the print head, setting the heating temperature for heating to obtain printable caramelized ink;

[0019] S13. After setting the printed nerve defect parameters and printing conditions, perform 3D printing to obtain a sucrose template.

[0020] As a preferred solution, in step S12, the heating temperature is 150-170° C., and the heating time is 0.5-2 h;

[0021] In step S13, the printing conditions include: a printing temperature of 140-150°C and a printing speed of 1-5 mm / s.

[0022] As a preferred embodiment, in step S2, the raw materials for preparing the outer sheath are raw materials with good casting mechanical strength and relatively slow degradation, specifically including PCL, PLGA, collagen, and decellularized neural matrix;

[0023] The solvent is selected from at least one of water and an organic solvent.

[0024] As a further preferred embodiment, the raw material for preparing the outer sheath is PCL or a mixture of PCL and other bioactive substances, and the other bioactive substances are selected from at least one of decellularized neural matrix, collagen, and PLGA; for example, the mixture of PCL and decellularized neural matrix has a mixing mass ratio of 4:0.5-2.

[0025] As a preferred embodiment, the organic solvent includes at least one of hexafluoroisopropanol, toluene, methanol, acetone, carbon tetrachloride, and dimethyl sulfoxide (DMSO).

[0026] As a preferred embodiment, in the mixed solution a, the concentration of the raw material for preparing the outer sheath is 3-8% (w / v), and more preferably 5% (w / v).

[0027] As a preferred embodiment, in step S2, the step of dissolving the sucrose template is specifically: immersing the sucrose template with the outer sheath on the surface in deionized water to fully dissolve the sucrose template.

[0028] As a preferred embodiment, in step S3, the raw materials for preparing the inner core filling material are raw materials with good biocompatibility and relatively fast degradation, specifically including silk fibroin, acellular neural matrix, hyaluronic acid, gelatin, and sodium alginate.

[0029] As a further preferred embodiment, the raw material for preparing the inner core filling material is a mixture of silk fibroin and at least one of hyaluronic acid, gelatin and acellular neural matrix. For example, the raw material for preparing the inner core filling material is a mixture of silk fibroin, hyaluronic acid and acellular neural matrix, and the mass ratio is 2:2-6:1, and the mass percentage of hyaluronic acid in the obtained mixed solution b is 2-6%.

[0030] As a preferred solution, in step S3, the soaking temperature is 3-5°C and the soaking time is 2-4h.

[0031] As a preferred embodiment, in step S3, the specific steps of the gradient freeze-drying are: precooling the hollow outer sheath containing the mixed solution b in the inner core to 0°C, and then placing it on a -190±10°C cold table for 20-40 minutes to form a directional frozen structure.

[0032] As a preferred embodiment, in the specific step of gradient freeze-drying, the mixture is placed on a -190°C cold table for 30 minutes.

[0033] The elastic modulus of the differentiated interface "inner core and outer sheath" nerve conduit prepared by the present invention ranges from 50 to 80 MPa.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The present invention provides a nerve conduit with a differentiated interface, which uses a substrate approved by the FDA to construct a differentiated functional interface of "inner core and outer sheath". The outer surface has extremely strong anti-adhesion characteristics to prevent the invasion of fibrous tissue, and evenly distributed nanoscale micropores are presented under an electron microscope for molecular exchange; the inner surface is a micron-scale lamellar loose filler arranged in an axial orientation. This structure meets the two different requirements of the repair microenvironment inside and outside, and realizes the important function of "inner affinity and outer sparseness, inner promotion and outer resistance". This conduit can not only promote the directional myelination of nerves and the directional growth of axons, but also avoid the formation of neuromas.

[0036] (2) Based on previous research, the present invention provides a nerve conduit with optimal topological structural parameters, which forms an axially arranged structure suitable for the extension and migration of vascular endothelial cells and neurons under the action of gradient freeze-drying or electric field. This structure has been proven to be conducive to guiding the rapid and efficient reconstruction of micro-vessels and remyelination after injury, and plays the role of a "paving stone" for the extension of axonal stumps.

[0037] (3) The present invention utilizes digital modeling combined with a template leaching method to provide a customizable nerve conduit with a convenient preparation process and adjustable and controllable product parameters. It provides a reliable personalized treatment plan for nerve defects with complex structures such as multiple branches, filling the gap in the current clinical lack of precise repair products for complex nerve defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0039] Figure 1 The surface microscopic topological structure and scanning electron microscope image of the outer "sheath" of the differentiated interface nerve conduit prepared in Example 1; wherein, Figure 1 A is a schematic diagram of the microscopic topological structure; Figure 1 B is a scanning electron microscope image;

[0040] Figure 2 The cross-sectional super-depth-of-field physical image, scanning electron microscope image and bottom cross-sectional scanning electron microscope image of the "core filler" of the differentiated interface nerve conduit prepared in Example 1; wherein, Figure 2 A is a cross-sectional super-depth-of-field physical image; Figure 2 B is a cross-sectional SEM image; Figure 2 C is a scanning electron microscope image of the bottom cross section;

[0041] Figure 3 Schematic diagram of the cell-core interface during the repair of the "core-inner-sheath" nerve conduit;

[0042] Figure 4 The actual image and in vivo implantation image of the differentiated interface nerve conduit prepared in Example 1; wherein, Figure 4 A is the actual picture; Figure 4 B is a diagram of the in vivo implantation of the nerve guide;

[0043] Figure 5 The gait analysis results of rats 12 weeks after the nerve conduits and autologous nerves prepared in Example 1, Comparative Example 1 and Comparative Example 2 were implanted into rats; wherein, Figure 5 A is the footprint shape; Figure 5 B is the shape diagram of rat foot; Figure 5 C is sciatic function index (SFI) analysis;

[0044] Figure 6 Transmission electron microscopic images of regenerated nerves at different magnifications after 12 weeks of implantation of nerve conduits prepared in Example 1, Comparative Example 1 and Comparative Example 2 and 12 weeks of autologous nerve transplantation;

[0045] Figure 7 The Masson staining and HE staining results of the gastrocnemius muscle of rats after 12 weeks of implantation of the nerve conduits prepared in Example 1, Comparative Example 1 and Comparative Example 2 and 12 weeks of autologous nerve transplantation;

[0046] Figure 8 The in vitro experimental results of the inner core fillings prepared in Example 1 and Comparative Example 2 on the migration of Schwann cells;

[0047] Fig. 9 This is a schematic diagram of the structure of a "core-inner-sheath" multi-branch nerve catheter. DETAILED DESCRIPTION

[0048] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0049] The words "preferred", "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that can provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0050] It should be understood that, except in any operating examples, or otherwise indicated, all numbers representing the amount of ingredients used in the specification and claims should be understood to be modified by the term "about" in all cases. Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are approximate values ​​that vary according to the desired performance to be obtained by the present invention. At least, it is not intended to limit the application of the doctrine of equivalents to the scope of the claims, and each numerical parameter should at least be interpreted according to the number of reported significant figures and by applying ordinary rounding techniques.

[0051] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0052] In the previous research of the applicant team, it was found that the currently prepared nerve conduits still have the following problems:

[0053] 1) Lack of internal and external differentiated interfaces

[0054] At present, most studies adopt the hollow structure design inside the catheter, the wall is not permeable, and the internal interface is disordered, which cannot achieve accurate repair of nerves and blood vessels. In response to this problem, the oriented structure topology design has accelerated the speed of nerve repair, but it has brought about the problem of increased adhesion on the outer surface of the catheter. Therefore, there is currently a lack of nerve catheters with differentiated internal and external functional interfaces in clinical practice. The main reason is that the microenvironment required inside and outside the catheter is quite different, and a single substrate or a single preparation process is difficult to achieve the optimal internal and external functional requirements at the same time.

[0055] In addition, according to the pathophysiological characteristics of nerve regeneration, the degradation rates required for the internal and external interfaces are different: the external interface, as a mechanical support and anti-adhesion physical barrier, needs to be degraded slowly; the internal interface needs to be degraded synchronously on the basis of guiding nerve regeneration, otherwise it will form a physical obstacle.

[0056] 2) Unclear bionic parameters

[0057] In the past, the research and development ideas of bionic products were mainly to analyze the natural anatomical structure or physiological characteristics of tissues, and then simulate them at the physical, chemical or biological levels. However, the application team recently discovered that the nerve regeneration microenvironment is significantly different from the natural physiological characteristics, and the repair effect of simulating the regeneration microenvironment after injury is better than natural structure bionics. Therefore, there is currently a lack of unified standards and consensus on the bionic concept of neural structure, and the optimal parameters of bionic nerve catheter products are still unclear, making it difficult to prepare nerve grafts with optimal parameters.

[0058] 3) It is difficult to achieve personalized neural repair

[0059] Currently, the nerve catheters available for clinical use are all single straight tubular products, which cannot meet the needs of multi-branch nerve segment defects (such as the sciatic nerve at the bifurcation of the tibial nerve and the common peroneal nerve). In clinical practice, the above limitations often lead to target organ atrophy and a decrease in the quality of life of patients. The shapes and sizes of post-traumatic nerve defects vary, which brings difficulties to personalized repair, and it is difficult for templated preparation processes or products to form an advantage. Therefore, it is crucial to develop personalized product preparation methods.

[0060] Based on this, the applicant team first proposed the theory of "constructing differentiated interface structures on demand". The inner and outer surfaces of the catheter need to conform to different environmental requirements and have the important functions of "inside closeness and outside sparing, inside promotion and outside resistance". The present invention aims to construct a bionic nerve catheter with a differentiated functional interface that can be customized, and to explain its preparation method, which is used to guide the efficient regeneration of blood vessels and nerves. According to the individual needs of nerve repair, the catheter can obtain the defect parameters, quickly 3D print the sugar mold and calibrate it, and then use the template leaching method to obtain an adaptive outer sheath with anti-adhesion function; the electric field directional guidance combined with freeze-drying method is used to form an inner core with an axial topological structure in situ inside the outer sheath. The shape, size, branching angle and other parameters of the nerve catheter can be customized, and the differentiated interface of "inner core and outer sheath" formed in situ is conducive to improving the repair efficiency of nerve grafts and accelerating peripheral nerve regeneration.

[0061] There are two reasons why sucrose is used as printing ink in the following embodiments: first, sucrose has good fluidity, processability and formability in a molten state; second, sucrose can be quickly dissolved in water and is a good sacrificial template.

[0062] The following example specifically extracts a method for preparing a differentiated interface nerve conduit that can be customized, which specifically includes the following steps:

[0063] S1. Printing sucrose template: setting the nerve defect parameters and performing 3D printing with caramelized ink to obtain a sucrose template;

[0064] S2. Preparing a hollow outer sheath of a nerve conduit: dissolving the raw materials for preparing the outer sheath in a solvent to form a mixed solution a, then immersing the sucrose template in the mixed solution a, taking it out after being fully soaked, and after the solvent is completely evaporated, obtaining the sucrose template on the surface of the outer sheath, then dissolving the sucrose template in the outer sheath, and then freeze-drying to obtain the hollow outer sheath;

[0065] S3. In situ formation of the inner core filling of the nerve conduit: dissolving the raw materials for preparing the inner core filling in water to form a mixed solution b, and then vertically placing the hollow outer sheath in the mixed solution b to fully immerse it, performing gradient freeze-drying or electric field action to form a directional frozen structure, and then freeze-drying to remove ice crystals to obtain the differentiated interface nerve conduit.

[0066] In the following examples, the polycaprolactone (PCL) used was produced by Aldrich Corporation of the United States, and its Mn = 80000 g / mol; the hyaluronic acid used was produced by Shanghai McLean Biochemical Technology Co., Ltd., and its purity was 97%; the decellularized matrix (dnECM) used was homemade in the laboratory, and its specific formula and preparation method refer to "Fangsong Zhang, et al. Decellularized nerve extracellular matrix / chitosan crosslinked by genipinto prepare a moldable nerve repair material. Cell Tissue Bank (2021) 22: 419–430".

[0067] Example 1

[0068] This embodiment provides a differentiated interface "inner core and outer sheath" nerve conduit for use in a 15 mm rat sciatic nerve defect model, and the preparation steps are as follows:

[0069] (1) Determine the area to be implanted as a nerve defect with a diameter of 2.5 mm and a length of 15 mm.

[0070] (2) Printing the sucrose template: First open the GeSim software, wait for the machine to complete the self-test, fill 3 / 4 volume of sucrose into the screw barrel, select a needle with a specification of 1.2mm, install the printing parts, and click the "Lock" button to lock. After measuring the height of the print head, set the heating temperature to 160℃ and the heating time to 1h to obtain printable caramelized ink. Set the temperature to 145℃, wait for the temperature to drop and remain stable, and set the 3D printing parameters in the "Scaffold" software, setting "Height" to 2.0mm, "Angle Change" to +90°, "Infill Distance" to 1.4mm, "Speed" to 2.4mm / s, and "Feed" to 4m / s. And set the corresponding parameters according to the diameter of 2.5mm and the length of 15mm. Then start printing to obtain the sucrose template.

[0071] (3) Preparation of the "outer sheath" of the nerve conduit: 0.4 g of polycaprolactone (PCL) and 0.1 g of decellularized matrix (dnECM) were dissolved in 10 mL of hexafluoroisopropanol (HFIP), and magnetically stirred at room temperature for 8 h to form a colorless clear solution, thereby obtaining a PCL / dnECM mixed solution with a concentration of 5% (w / v). The sucrose template prepared in step (2) was immersed in the above-prepared PCL / dnECM mixed solution for 10-15 seconds to ensure that the solution was fully infiltrated on the surface of the template. The sucrose template was then removed with tweezers, and the solvent (hexafluoroisopropanol) was allowed to evaporate for 10 minutes, and the immersion was repeated 6 times. The sucrose template attached to the PCL / dnECM mixed solution after infiltration was taken out, and after the solvent (hexafluoroisopropanol) evaporated (evaporation for 2 h), it was immersed in deionized water for 2 h. During this period, the deionized water was replaced every 30 minutes to allow the sucrose template to be fully dissolved. After being taken out, it was freeze-dried to obtain a hollow "outer sheath", as shown in FIG. Figure 1 As shown, Figure 1 A is a schematic diagram of the microscopic topological structure of its outer surface. Figure 1 B is a scanning electron microscope image. Figure 1 It can be seen that the surface of the "outer sheath" has uniformly distributed nanoscale micropores (pore size is about 612.5±107nm).

[0072] (4) In situ formation of the "inner core" of the nerve conduit: 0.02 g of decellularized matrix (dnECM), 0.04 g of silk fibroin (SF) and 0.04 g of hyaluronic acid (HA) were dissolved in 2 mL of deionized water to form a colorless clear solution, and a HA / dnECM / SF mixed aqueous solution with a HA concentration of 2% (w / v) was obtained. The hollow "outer sheath" obtained in step (3) was placed vertically in a sample bottle filled with the above-mentioned "inner core filler" mixed aqueous solution, and after being fully soaked at 4°C for 4 hours, the sample bottle was precooled to 0°C and then placed on a -190°C cold table for 30 minutes to form a directional frozen structure, and then the ice crystals were removed by freeze drying (24-36 hours) to obtain a differentiated interface "inner core outer sheath" nerve conduit with a diameter of 2.5 mm and a length of 15 mm (such as Figure 2 , Figure 3 , Figure 4 The cross-sectional super-depth-of-field real-object image of the inner core filling of the obtained nerve conduit is shown in FIG. Figure 2 A, cross-sectional SEM image Figure 2 B shows the SEM image of the bottom cross section. Figure 2 As shown in C, it can be seen from the figure that the inner core filler is a loose micron-level lamellar filler arranged in an axial direction. Figure 3 The interface between cells and the inner core filling in the nerve conduit is shown. As can be seen from the figure, cells (including vascular endothelial cells, Schwann cells and neuronal cells, etc.) extend and migrate in a directional manner on the inner core structure with an axial orientation to achieve the purpose of rapid repair. Figure 4A is a physical picture of the prepared differentiated interface "inner core outer sheath" nerve conduit. After testing, its elastic modulus is 68.5±7.3Mpa.

[0073] Example 2

[0074] This embodiment provides a method for preparing a differentiated interface "inner core and outer sheath" nerve conduit. The specific preparation steps are basically the same as those in Example 1, except that: in step (4), the amount of hyaluronic acid added is 0.08 g, and a HA / dnECM / SF mixed aqueous solution with a HA concentration of 4% (w / v) is obtained.

[0075] The final differentiated interface "inner core and outer sheath" nerve conduit structure is substantially the same as that of Example 1. After testing, its elastic modulus is 66.4±8.1 Mpa.

[0076] Example 3

[0077] This embodiment provides a method for preparing a differentiated interface "inner core and outer sheath" nerve conduit. The specific preparation steps are basically the same as those in Example 1, except that: in step (4), the amount of hyaluronic acid added is 0.12 g, and a HA / dnECM / SF mixed aqueous solution with a HA concentration of 6% (w / v) is obtained.

[0078] The final differentiated interface "inner core outer sheath" nerve conduit structure is basically the same as that of Example 1. After testing, its elastic modulus is 65.1±6.7 Mpa.

[0079] Comparative Example 1

[0080] This comparative example provides a hollow nerve conduit, namely, the "outer sheath" of a nerve conduit that does not contain the "inner core filler" described in Example 1, and its preparation method is the same as steps (1) to (3) of Example 1.

[0081] Comparative Example 2

[0082] This comparative example provides a nerve conduit, and its preparation method is basically the same as that of Example 1, except that: the step of in situ forming the "inner core" of the nerve conduit in step (4) is: 0.02g of decellularized matrix (dnECM), 0.04g of silk fibroin (SF) and 0.04g of hyaluronic acid (HA) are dissolved in 2mL of deionized water to form a colorless clear solution, and a HA / dnECM / SF mixed aqueous solution with a HA concentration of 2% (w / v) is obtained. The hollow "outer sheath" obtained in step (3) is placed vertically in a sample bottle filled with the above-mentioned "inner core filler" mixed aqueous solution, and after being fully soaked at 4°C for 4h, the sample bottle is precooled to 0°C, and then the ice crystals are removed by freeze drying (24-36h) to obtain a "inner core outer sheath" nerve conduit with a diameter of 2.5mm and a length of 15mm. The inner core filler of the nerve conduit does not have an axial orientation arrangement, but is randomly arranged.

[0083] Effect verification:

[0084] 1. In vivo experiments

[0085] The nerve conduits prepared by the methods of Example 1, Comparative Example 1 and Comparative Example 2 were implanted into the nerve defect of rats (such as Figure 4 B), and autologous nerves were transplanted into the nerve defects of rats as a comparison.

[0086] After 12 weeks, the rats' gait was analyzed. Figure 5 As shown, Figure 5 A is the footprint morphology of rats in different experimental groups. Figure 5 B is the shape of rat feet. Figure 5 C is the analysis result of the sciatic nerve function index (SFI) of the rats in each experimental group. According to the footprint morphology of the rats in different experimental groups, the footprint of the group implanted with the nerve conduit of Example 1 is similar to that of the autologous nerve transplantation group, while the footprints of the group implanted with the nerve conduit of Comparative Example 1 and the group implanted with the nerve conduit of Comparative Example 2 are obviously narrow and long, with curled toes. The footprints were analyzed and counted, and the sciatic nerve function index (SFI) was calculated based on the IT, TS and PL distances, ranging from -100 to 0, and the closer to 0, the better the nerve function (SFI calculation method: SFI = (-38.3×(EPL-NPL) / NPL)+(109.5×(ETS-NTS) / NTS)+(13.3×(EIT-NIT) / NIT)-8.8). Figure 5 The variance analysis of the C results showed that the differences were statistically significant, and the neurological function of the Example 1 group was significantly better than that of the Comparative Example 1 group and the Comparative Example 2 group.

[0087] After 12 weeks, the nerve regeneration quality was observed. Figure 6 shown). Figure 6The transmission electron micrographs at different magnifications are respectively the images of the regenerated nerves after 12 weeks of implantation of the nerve conduits prepared in Example 1, Comparative Example 1 and Comparative Example 2 and 12 weeks of autologous nerve transplantation. It can be seen from the results in the images that only a small amount of formed myelin sheath is formed in the nerve conduit prepared in Comparative Example 1, the myelin sheath thickness of the nerve conduit prepared in Comparative Example 2 is increased, and the myelin sheath thickness and axon diameter of the nerve conduit prepared in Example 1 and the autologous nerve transplantation are comparable and are significantly better than the nerve conduits in Comparative Examples 1 and 2.

[0088] After 12 weeks, the gastrocnemius muscles were harvested to investigate the nutritional effects of nerve recovery on the innervated muscles. The results of Masson staining and HE staining of rats in each experimental group are shown in Figure 2. Figure 7 As shown, the results show that the muscle fibers of the nerve conduit group implanted with comparative example 1 and the nerve conduit group implanted with comparative example 2 atrophy and have a large amount of collagen deposition (blue part), while the collagen deposition of the nerve conduit group implanted with example 1 is significantly less than that of the nerve conduit groups of comparative example 1 and comparative example 2. In addition, the muscle fiber density and muscle fiber diameter of the nerve conduit group and the autologous nerve transplantation group of example 1 are significantly better than those of the nerve conduit groups of comparative example 1 and comparative example 2.

[0089] 2. In vitro experiments

[0090] Endothelial cells were seeded on different interfaces and then co-cultured with Schwann cells to observe the migration of Schwann cells. The specific experimental method is as follows: a transwell chamber with a pore size of 8.0 μm was placed in a 24-well plate, and the bottom surface of the lower chamber was covered with oriented inner cores (Example 1), randomly arranged inner cores (Comparative Example 1) or blank control (tissue culture plate control), and then vascular endothelial cells (HUVECs) were spread on the above different interfaces, and 1.5×10 4 Schwann cells (mSCs or RSC96) were fixed 24 hours later, the residual cells on the upper surface of the upper chamber were removed, and the cells that passed through the chamber were marked with crystal violet staining, photographed, counted and analyzed.

[0091] The results are as follows Figure 8 As shown, after the endothelial cells implanted in the inner core filler with an oriented arrangement structure prepared in Example 1 were co-cultured with the Schwann cells surrounding the neurons, the migration of the Schwann cells was significantly promoted (the number of migrating Schwann cells increased in the figure).

[0092] It should be noted that, based on the specific preparation method shown in the embodiment of the present invention, the raw material for preparing the outer sheath can also be pure PCL or a mixed solution of the outer sheath prepared by other mass ratios of PCL and decellularized neural matrix; the raw material for preparing the inner core can also be a mixed solution of the inner core filler prepared by the following different combinations: (1) silk fibroin, hyaluronic acid (such as a content of 1%, 2%, 4%); (2) silk fibroin, gelatin (such as a content of 1%, 2%, 4%); (3) silk fibroin, decellularized neural matrix, hyaluronic acid; (4) silk fibroin, decellularized neural matrix, gelatin. In this way, a differentiated interface nerve conduit with "a hollow outer sheath with nano-scale micropores distributed on the surface and a micron-scale lamellar loose filler with axial orientation as the inner core filler" can be prepared.

[0093] It should also be noted that by setting the parameters in 3D printing, the following can be obtained: Fig. 9 The sucrose template of the multi-branched nerve conduit shown is used to prepare the corresponding multi-branched, differentiated interface nerve conduit.

[0094] The present invention has many specific application paths, and the above is only a preferred embodiment of the present invention. It should be pointed out that the above embodiments are only used to illustrate the present invention, and are not used to limit the protection scope of the present invention. For those of ordinary skill in the art, without departing from the principle of the present invention, several improvements can also be made, and these improvements should also be regarded as the protection scope of the present invention.

Claims

1. A customized differentiated interface neural conduit, It is characterized in that It includes a hollow outer sheath and an inner core filler arranged inside the hollow outer sheath; The surface of the hollow outer sheath is distributed with nano-scale micropores, and the inner core filler is a micron-scale lamellar loose filler arranged in an axial orientation; The method for preparing the personalized and customized differentiated interface nerve conduit comprises the following steps: S1. Printing sucrose template: setting the nerve defect parameters and performing 3D printing with caramelized ink to obtain a sucrose template; S2. Preparing a hollow outer sheath of a nerve conduit: dissolving the raw materials for preparing the outer sheath in a solvent to form a mixed solution a, then immersing the sucrose template in the mixed solution a, taking it out after being fully soaked, and after the solvent is completely evaporated, obtaining the sucrose template on the surface of the outer sheath, then dissolving the sucrose template in the outer sheath, and then freeze-drying to obtain the hollow outer sheath; S3, forming the inner core filling material of the nerve conduit in situ: dissolving the raw materials for preparing the inner core filling material in water to form a mixed solution b, and then vertically placing the hollow outer sheath in the mixed solution b to fully immerse it, performing gradient freeze-drying or electric field action to form a directional frozen structure, and then freeze-drying to remove ice crystals, thereby obtaining the differentiated interface nerve conduit; In step S2, the raw material for preparing the outer sheath is PCL or a mixture of PCL and other bioactive substances, and the other bioactive substances are selected from at least one of acellular neural matrix, collagen, and PLGA; In step S3, the raw material for preparing the inner core filling material is a mixture of silk fibroin and at least one of hyaluronic acid, gelatin and acellular neural matrix; In step S3, the specific steps of the gradient freeze-drying are: precooling the hollow outer sheath with the inner core containing the mixed solution b to 0°C, and then placing it on a -190±10°C cold table for 20-40 minutes to form a directional frozen structure.

2. The customizable differentiated interface nerve conduit according to claim 1, It is characterized in that The specific steps of step S1 are: S11. Open the GeSim software, wait for the machine to complete self-check, load sucrose into the screw barrel, select the needle specification, install the printing parts, and click the "Lock" button to lock; S12, after measuring the height of the print head, setting the heating temperature for heating to obtain printable caramelized ink; S13. After setting the printed nerve defect parameters and printing conditions, perform 3D printing to obtain a sucrose template.

3. The customizable differentiated interface nerve conduit according to claim 2, It is characterized in that In step S12, the heating temperature is 150-170° C. and the heating time is 0.5-2 h; In step S13, the printing conditions include: a printing temperature of 140-150°C and a printing speed of 1-5 mm / s.

4. The customizable differentiated interface nerve conduit according to claim 1, It is characterized in that The solvent in step S2 is selected from at least one of water and an organic solvent.

5. The customizable differentiated interface nerve conduit according to claim 4, It is characterized in that The organic solvent includes at least one of hexafluoroisopropanol, toluene, methanol, acetone, carbon tetrachloride and dimethyl sulfoxide.

6. The customizable differentiated interface nerve conduit according to claim 1, It is characterized in that In step S2, the step of dissolving the sucrose template is specifically as follows: immersing the sucrose template with the outer sheath on the surface in deionized water to fully dissolve the sucrose template.

7. The customizable differentiated interface nerve conduit according to claim 1, It is characterized in that In step S3, the soaking temperature is 3-5°C and the soaking time is 2-4h.

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