A scaffold mimicking spinal cord white matter structure and a preparation method thereof

Abstract

The application provides a grey / white matter partitioned biomimetic spinal cord scaffold which can be customized grey / white matter partitioned and can match the grey / white matter morphology of any segment; the grey / white matter partitioned biomimetic spinal cord scaffold is composed of an internal 'butterfly' shaped grey matter area and an external white matter area, and the whole appearance is in a cylindrical shape; the grey matter area mainly comprises a hydrogel material; the white matter area mainly comprises a hydrogel and a directional fiber material; the direction of the directional fiber material is consistent with the longitudinal direction of the scaffold; the grey matter area and the white matter area are fused into a whole through the hydrogel, and there is no obvious barrier structure between the grey matter area and the white matter area.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of spinal cord nerve repair, in particular to a bionic structure spinal cord injury repair scaffold and a method for preparing the scaffold, and the application of the scaffold in the field of spinal cord injury. BACKGROUND

[0002] After severe spinal cord injury, the disordered structure of regenerated nerve tissue is one of the main factors restricting the recovery of spinal cord function. From the anatomical structure, the normal spinal cord is in the middle of the "butterfly" or "H" shaped gray matter area, surrounded by white matter area. The gray matter area is mainly composed of neuron cell bodies and network-like nerve fibers, responsible for neural signal integration, transformation and participation in neural reflex; the white matter area is mainly composed of longitudinally parallel nerve bundles, responsible for the uplink and downlink transmission of signals between the brain and the peripheral nerves. The gray matter and white matter are not completely isolated but have extensive neural interpenetration and intercommunication, linking their functions together to achieve fine and complex spinal cord neural functions. For patients with spinal cord injury, the reconstruction of gray / white matter structure of the spinal cord is crucial to their functional recovery, but the existing technology has very limited effect on the reconstruction of gray / white matter structure after spinal cord injury.

[0003] At present, the strategy of promoting spinal cord regeneration based on transplantation materials mainly involves hydrogel, directional topological scaffold, 3D printed scaffold, etc., which have their own unique advantages and disadvantages. For example, hydrogel material can optimize the microenvironment of spinal cord regeneration to the greatest extent and promote rapid and large-scale regeneration of nerve fibers, but the isotropy of hydrogel easily leads to scattered nerve regeneration, which is not conducive to the reconstruction of white matter form and function; directional topological scaffold can induce directional regeneration of spinal cord white matter, but it cannot simultaneously consider the network-like nerve regeneration of gray matter; 3D printing can theoretically construct any morphological partition structure, but in the case of millimeter-level size, the current 3D printed bionic spinal cord soft tissue scaffold often has poor precision and is difficult to match the fine structure of the in vivo spinal cord.

[0004] Patent CN114366383B discloses a bionic spinal cord scaffold for promoting directional extension of axons after spinal cord injury, which is printed by three-dimensional printing technology and is composed of an upper and lower two-layer base layer containing spinal cord mesenchymal stem cells and a support layer carrying Schwann cells. It focuses on solving the directional extension of axons after spinal cord injury, which meets the directional regeneration demand of white matter nerve bundles, but does not consider the network-like nerve regeneration of spinal cord gray matter.

[0005] Patent CN109010915B discloses an ordered collagen scaffold for spinal cord injury repair, which has an ordered three-dimensional structure composed of multiple channel structures, and essentially belongs to a multi-channel catheter topological structure. By loading nerve regeneration promoting drugs wrapped by liposomes in the scaffold, the ordered collagen scaffold disclosed in the invention also simplifies the structure of the spinal cord and performs bionic simulation.

[0006] Patent CN103007350A discloses a method for preparing a biomimetic spinal cord scaffold using rat spinal cord acellular preparation, which uses animal-derived spinal cord tissue, relies on the optimization of acellular technology to retain the 3D structure of rat spinal cord natural scaffold, and is not directed to the biomimic of gray / white matter morphogenesis microenvironment. In addition, this invention does not mention the partition and structural difference between gray matter and white matter. SUMMARY

[0007] The present application provides a gray / white matter partitioned biomimetic spinal cord scaffold, which can customize the gray / white matter partition to match the gray / white matter morphology of any segment. The gray matter region can induce neural network structure regeneration, and the white matter region can induce directional nerve bundle structure regeneration. At the same time, there is extensive neural intercommunication between the regenerated gray matter and white matter, solving the problem of difficult biomimetic reconstruction of the current gray / white matter partition structure. The present application also provides a method for preparing a biomimetic spinal cord scaffold, which involves a special mold and its preparation process, as well as the selection and matching of materials in the gray matter and white matter regions.

[0008] The present application achieves the above-mentioned application purposes through the following technical solutions:

[0009] A biomimetic spinal cord scaffold is a cylindrical structure composed of an internal "H"-shaped gray matter region and an external white matter region. The gray matter region and the white matter region are tightly connected as a whole. The gray matter region is filled with injectable hydrogel material. The white matter region is composed of directional fibers. The direction of the directional fiber material is consistent with the longitudinal direction of the scaffold.

[0010] Further, the biomimetic spinal cord scaffold includes a small-diameter biomimetic spinal cord scaffold.

[0011] Further, the preparation process of the biomimetic spinal cord scaffold includes threading, wire arrangement, glue filling, and demolding processes.

[0012] Further, the preparation process of the small-diameter biomimetic spinal cord scaffold includes threading, wire arrangement, heat shrinking, glue filling, and demolding processes.

[0013] Further, the threading process is to longitudinally thread the directional fiber material through a hollow cylindrical pipeline, which is a plastic tube or a heat shrink tube.

[0014] Further, the wire arrangement process needs to use a 3D printing customized wire arrangement mold, which comprises a first body and a second body, the first body and the second body correspond to each other in position and are separated by at least one support part, the plastic tube or the heat shrink tube is arranged between the first body and the second body, the first body and the second body have an 'H' shaped pattern and divide the area outside the 'H' area of the first body and the second body into six areas, and the center of the 'H' shaped pattern has a through hole.

[0015] Further, the directional fiber material is fixed outside the wire arrangement mold after passing through the corresponding partitions of the first body and the second body respectively, and the injectable hydrogel is injected into the center of the plastic tube or the heat shrink tube through the through hole on the first body or the second body.

[0016] Further, the heat shrink tube is a PVC heat shrink tube, the diameter before heat shrinkage is 4 mm, and the diameter after heat shrinkage is 2 mm.

[0017] Further, the injectable hydrogel is selected from hyaluronic acid hydrogel and / or chitosan hydrogel, or modified hyaluronic acid hydrogel, or modified chitosan hydrogel, or hyaluronic acid hydrogel loaded with biological macromolecules, or chitosan hydrogel loaded with biological macromolecules.

[0018] Further, the directional fiber material is tussah silk.

[0019] In summary, the present application has the following beneficial effects: 1) the bionic spinal cord scaffold prepared by the present application has gray / white matter partitions, and the gray / white matter morphology can be adjusted by cooperating with the 3D printed mold, so that it is more suitable for the spinal cord structure of the part to be repaired; 2) the gray matter area and the white matter area are selected according to their bionic characteristics, the homogeneous hydrogel material of the gray matter area can optimize the microenvironment of spinal cord regeneration and promote the formation of neural network structure, and the directional fiber material of the white matter area can provide additional guidance information to promote the formation of white matter nerve bundles; 3) the directional fiber of the white matter area is infiltrated by the hydrogel of the gray matter area, and after the hydrogel is solidified, the white matter area and the gray matter area are tightly combined to form a whole, without the need for any external shaping catheter during use, and the operation is more convenient. 4) The bionic spinal cord scaffold prepared by the present application has no barrier between the gray matter and the white matter, and the nerve fibers can transmit signals between the gray matter and the white matter, which is more consistent with the normal physiological structure of the spinal cord. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a structure schematic diagram of the bionic spinal cord scaffold of embodiment 1 of the present application;

[0021] Figure 2 is a top view schematic diagram of the bionic spinal cord scaffold of embodiment 1 of the present application.

[0022] Figure 3 is a cross-sectional schematic view of the bionic spinal cord scaffold of embodiment 1 of the present application;

[0023] Figure 4 is a flow chart of the preparation process of the bionic spinal cord scaffold of embodiment 1 of the present application;

[0024] Figure 5 is a flow chart of the preparation process of the bionic spinal cord scaffold of embodiment 2 of the present application;

[0025] Figure 6 is a structural view of the wire arrangement mold of embodiment 1 of the present application;

[0026] Figure 7 is a schematic view of each partition in the top view of the wire arrangement mold of embodiment 1 of the present application;

[0027] Figure 8 is a structural view of the support part of the wire arrangement mold of embodiment 5 of the present application;

[0028] Figure 9 is a structural view of the groove of the wire arrangement mold of embodiment 5 of the present application;

[0029] Figure 10 is a photo of the bionic spinal cord scaffold of embodiment 1 of the present application;

[0030] Figure 11 is a scanning electron microscope view of the bionic spinal cord scaffold of embodiment 1 of the present application;

[0031] Figure 12 is a staining picture of the in-vitro effect verification of embodiment 3 of the present application;

[0032] Figure 13 is an in-vivo repair effect picture of the bionic spinal cord scaffold of embodiment 4 of the present application.

[0033] Explanation of reference signs: 1-imitating gray matter area, 2-imitating white matter area, 3-gray-white matter junction, 4-directional fiber, 5-first main body, 6-second main body, 7-connecting column, 8-through hole, 9-coverage area, 10-edge ring, 11-support, 12-groove. DETAILED DESCRIPTION

[0034] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings and technical solutions required to be used in the embodiment or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0035] Example 1:

[0036] As shown in Figure 1 , Figure 2 , Figure 3 , the bionic spinal cord scaffold with gray-white matter partition, the whole is a cylindrical structure, the inside is an "H" shaped simulated gray matter area 2, the area outside the simulated gray matter area 2 to the inside of the cylindrical side surface is a simulated white matter area 1, and the connection part of the simulated white matter area 1 and the simulated gray matter area 2 is a gray-white matter junction 3; the simulated gray matter area 2 is filled with an injectable hydrogel material, the simulated white matter area 1 is composed of directional fiber 4 material, and the direction of the directional fiber 4 is consistent with the longitudinal direction of the cylinder; the simulated gray matter area 2 and the simulated white matter area 1 are fixed into a complete whole by extrusion and filling; the hydrogel in the simulated gray matter area 2 provides a good microenvironment for spinal cord nerve regeneration, and the directional fiber 4 material in the simulated white matter area 1 provides a guidance signal for nerve regeneration.

[0037] The material of the directional fiber 4 selected in this embodiment is boiled degummed tussah silk, and the diameter of a single fiber is between 10-20 microns

[0038] The injectable hydrogel selected in this embodiment is hyaluronic acid hydrogel, which is prepared by crushing the 1,4-butanediol diglycidyl ether cross-linked hyaluronic acid gel with a homogenizer.

[0039] As shown in Figure 4 , the preparation process of the bionic scaffold includes the processes of threading, wire arrangement, glue filling, and film removal; the directional fiber 4 material is threaded into the plastic tube in the form of a fiber bundle in the longitudinal direction, there are six fiber bundles, and the sum of the diameters of all the fiber bundles is less than the inner diameter of the plastic tube, which determines the diameter of the finally prepared scaffold; the plastic tube is a hollow cylindrical shape or a hollow front and back narrow cylindrical shape, and a transparent plastic tube is usually used to facilitate observation of the wire bundle and the filling of the hydrogel in the tube.

[0040] Because the content of gray matter and white matter is different in different segments of the spinal cord, for example, the cervical enlargement and lumbosacral enlargement have more gray matter content, and the cervical spinal cord has more white matter; the wire arrangement mold is printed by a 3D printing method, which can be customized according to the spinal cord segments, and the bionic spinal cord scaffold prepared by the customized mold has a higher matching degree with the patient's spinal cord, as shown in Figure 6 and Figure 7As shown, the wire arrangement mold is composed of a first body 5 and a second body 6 and connecting columns 7, the first body 5 and the second body 6 are completely identical in structure, shape and size, and are fixedly connected together through at least three connecting columns 7, and the first body 5 and the second body 6 completely coincide in the top view after being connected; the first body 5 and the second body 6 are composed of a through hole 8, a covering area 9 and an edge ring 10, the edge ring 10 is a hollow ring, the covering area 9 is in the shape of "H" or butterfly shape, and its size and shape are determined by the segment where the spinal cord is located, the through hole 8 is located in the covering area and at the center of the covering area, the upper part of the covering area 9 is connected to the edge ring 10, and if the lower part of the covering area 9 cannot be connected to the edge ring 10, an elongated connecting shaft is extended outward along the shape of the lower part of the covering area 9, and the lower part of the covering area is also connected to the edge ring 10 through the connecting shaft, in addition, a connecting shaft is fixed on the left side and the right side of the longitudinal middle position of the covering area 9, and the other side of the connecting shaft is fixed on the edge ring 10; the edge ring 10, the connecting shaft and the covering area 9 divide the space within the edge ring 10 and outside the covering area 9 into six small spaces.

[0041] The wire arrangement process is to longitudinally place the fiber bundle and the plastic tube as a whole into the longitudinally placed wire arrangement mold, and six fiber bundles are fixed after passing through the six small spaces of the first body 5 and the second body 6, and the upper end and the lower end of the same fiber bundle pass through the small space at the same position in the top view.

[0042] The syringe passes through the through hole 8 in the covering area from the top to fill the hydrogel into the pores between the fiber bundles, and through the outward force generated by the continuous filling of the hydrogel and the inward force given by the plastic tube, the gray matter area and the white matter area are completely extruded and fixed into a whole, and the transition between the hydrogel and the fiber bundle is natural, which also conforms to the anatomical structure of the spinal cord.

[0043] The fiber bundle fixed outside is cut open, the plastic tube after filling is removed, and the plastic tube is completely cut off at the position close to the lower end port and the lower end port, and then the plastic tube is taken out from the outside of the biomimetic spinal cord scaffold as a whole, and the biomimetic spinal cord scaffold is obtained.

[0044] The photo of the biomimetic spinal cord scaffold is shown in the attached Figure 10 The gray-white matter area with clear boundary can be clearly seen; the scanning electron microscope picture is shown in the attached Figure 11 There are multiple longitudinal fibers in the simulated white matter area, the simulated gray matter area presents a porous structure, and the simulated gray matter area and the simulated white matter area are tightly connected together.

[0045] Example 2:

[0046] On the basis of embodiment 1, different situations of the spinal cord are fully considered in this embodiment. For the small-diameter spinal cord scaffold, the plastic tube used in embodiment 1 is replaced by a heat-shrinkable tube during preparation. After the wire arrangement, the heat-shrinking step is added before the glue filling.

[0047] As shown in Figure 5 , after the wire arrangement is completed, the wire arrangement mold, heat-shrinkable tube and fiber bundle are heated as a whole to make the heat-shrinkable tube shrink, and then the glue filling process is performed. In this way, a small-diameter biomimetic spinal cord scaffold can be prepared.

[0048] As one of the options, the PVC heat-shrinkable tube with a shrinkage ratio of 2:1 is selected in this embodiment. The diameter of the heat-shrinkable tube before shrinking is 2 mm, and the diameter of the biomimetic spinal cord scaffold prepared after shrinking is 1 mm.

[0049] Example 3:

[0050] The biomimetic spinal cord scaffold is prepared by the method of embodiment 2. The dorsal root ganglion (DRG) dissociated cells are cultured in the biomimetic spinal cord scaffold for 5 days for in-vitro effect verification.

[0051] By observing the junction between the gray matter and the white matter after staining, it is found that the axons in the white matter grow along the direction of the oriented fibers, and the nerves in the gray matter present a network structure. The staining method is to mark the myelin structure with green, mark the NF200 nerve axon with red, and mark the cell nucleus with blue DAPI dye. The results are shown in Figure 12 . It can be clearly seen that the axons in the white matter grow along the silk fibers, and the nerves in the gray matter present a network structure.

[0052] Example 4:

[0053] On the basis of embodiment 2, in order to further verify the performance of the scaffold, this embodiment is an experiment of using the scaffold to repair the full transection of the T10 spinal cord of a rat.

[0054] Twenty adult SD rats are divided into four groups, with five animals in each group. The normal group is not treated. The spinal cord injury group is subjected to a full transection operation at the T10 segment of the spinal cord to model a defect distance of 2 mm, and no treatment is performed after the injury. The biomimetic spinal cord scaffold group is implanted with the biomimetic spinal cord scaffold prepared in embodiment 2 at the spinal cord defect.

[0055] As shown in Figure 13 , the differences in spinal cord regeneration morphology after in-vivo transplantation of the biomimetic spinal cord scaffold for one month are shown, Figure 13 , where the white dashed line in the C chart indicates the injury / scaffold transplantation segment, the box indicates the region of interest (ROI-1, 2, 3), and the scale is 1 mm. Figure 13C1 is a zoomed-in view of ROI-1, showing that nerves grow into the scaffold from the spinal cord stumps and maintain the gray / white matter partition structure characteristics; Figure 13 C2 is a zoomed-in view of ROI-2, showing the nerve regeneration morphology in the central gray matter region; Figure 13 C3 is a zoomed-in view of ROI-3, showing the nerve regeneration morphology in the middle white matter region of the scaffold material.

[0056] Example 5:

[0057] The wire arrangement mold used in this embodiment and the use method are different from those in Embodiment 1 and Embodiment 2, as shown in Figure 8 and Figure 9 The first body 1 and the second body 2 are connected through the support part 11, the upper part of the support part 11 is fixedly connected with the outer ring of the first body 1, the lower part of the support part 11 is fixedly connected with the outer ring of the second body 2, and the support part 11 is a hollow column structure, the upper side and the lower side of the support part 11 are respectively connected with the first body 1 and the second body 2 in a plug-in type clamping groove connection, there are downward and upward protruding grooves 12 at the corresponding parts of the first body 1 and the second body 2, and the upper and lower profiles and thicknesses of the support part are just inserted into the grooves 12 for fixation; the support part 7 is printed with transparent material, which facilitates observation of the condition of the internal placed object.

[0058] Due to the change of the wire arrangement mold, the preparation method of the biomimetic spinal cord scaffold is also different, but the prepared biomimetic spinal cord scaffold has no obvious difference, and the specific preparation method is as follows: the upper side or the lower side of the support part 11 is plugged and fixed, the plastic tube or the heat shrink tube and the silk bundle are placed in the support part, and the silk bundle is divided into six partitions, after division, the silk bundle is placed in the support part 11 around the outer contour of the first body 1 or the second body 2, and then the support part 11 is clamped into the first body or the second body for fixation.

[0059] Under the inspiration of this embodiment, any intermediate part that can accommodate the biomimetic spinal cord scaffold, the first body 1 and the second body 2 can be separated by a distance and placed above and below the biomimetic spinal cord scaffold respectively, and the wire arrangement can have a fixed structure or method that can fix the directional fibers on the outside, which can be used as the design structure of the wire arrangement mold of the present application; of course, the data of the "H" area is derived from the modeling of the spinal cord part of the patient's defect.

[0060] Example 6:

[0061] On the basis of Embodiment 1, chitosan hydrogel can also be used as an injection hydrogel to fill the gray matter region, and chitosan has antibacterial effect, and in this embodiment, a composite hydrogel is prepared by using hyaluronic acid and chitosan hydrogel.

[0062] The 0.2 mol / L chitosan acetic acid solution is prepared by using chitosan and acetic acid solution, 5 mmoL of chitosan acetic acid solution is added with 1% pentanediol aqueous solution 5 mL in an ice bath, and is rapidly stirred for 30 s, and is poured into a glass container for solidification, so that the chitosan hydrogel is obtained.

[0063] The 3% chitosan solution, 8% hyaluronic acid solution and 8% glycerol phosphate sodium solution are prepared into a composite hydrogel according to a volume ratio of 2:6:2.

[0064] Example 7:

[0065] On the basis of example 1 and example 6, the injectable chitosan hydrogel has the problems of insufficient strength and too fast degradation speed, the injectable hydrogel used in the embodiment is a modified chitosan hydrogel, and the indexes of strength and degradation speed are improved.

[0066] The chitosan solution prepared in example 6 is taken as 5 mmoL, 1 ml, 3 ml and 5 ml of 1% glutaraldehyde aqueous solution are sequentially added in an ice bath, stirring is performed for 30 s, and the chitosan gel is prepared by pouring into a glass container.

[0067] Example 8:

[0068] On the basis of example 1, the hyaluronic acid hydrogel can also be used as a carrier to load biomolecules, such as mesenchymal stem cells; the hyaluronic acid hydrogel loaded with bone marrow mesenchymal stem cells promotes the paracrine effect of mesenchymal stem cells, releases extracellular vesicles, reduces cell death at the damage site, reduces scar formation, and has a certain promoting effect on the recovery of motor function.

[0069] Bone marrow stem cells are isolated from rabbit bone marrow, and are routinely isolated, cultured and expanded, and are subcultured to the second or third generation, and are collected and adjusted to a final concentration of 1x10 5 / mL, and are wrapped in 8% w / v hyaluronic acid hydrogel precursor solution to prepare hyaluronic acid hydrogel at 37 DEG C.

[0070] The in vitro experimental results of example 6, example 7 and example 8 are similar to the animal experiment results of example 3 and example 4, so it can be known that the hyaluronic acid hydrogel, the chitosan hydrogel and the modified or biomolecule-loaded injectable hydrogel can all be prepared into a biomimetic spinal cord scaffold.

[0071] The above merely illustrates the specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any change or replacement without creative labor should be covered in the protection scope of the present application.

Claims

1. A gray matter partitioned biomimetic spinal cord scaffold, consisting of an inner "butterfly" shaped gray matter region and an outer white matter region, overall appearance in a cylindrical shape, characterized in that, The gray matter region mainly comprises a hydrogel material, the white matter region comprises the hydrogel and a directional fiber material, the directional fiber material is in the same direction as the longitudinal direction of the scaffold, the gray matter region and the white matter region are fused into a whole through the hydrogel, there is no obvious barrier structure between the gray matter region and the white matter region, and there is also no obvious barrier structure between the directional fiber and the surrounding hydrogel. The preparation process of the biomimetic spinal cord scaffold comprises the processes of threading, wire arranging, glue filling and demolding. The process of threading is to longitudinally thread the directional fiber material through a hollow cylindrical pipeline, and the cylindrical pipeline is a plastic tube or a heat-shrinkable tube. The wire arranging process needs to use a 3D-printed wire arranging mold, which comprises a first body and a second body. The first body and the second body are in one-to-one correspondence in the up-down position, and at least one support part is arranged between the first body and the second body to separate them by a distance. The plastic tube or the heat-shrinkable tube is arranged between the first body and the second body. The first body and the second body have a "butterfly" pattern and divide the area outside the "butterfly" area of the first body and the second body into six areas. There is a through hole at the center position of the "butterfly" pattern. The directional fiber material is fixed outside the wire arranging mold after being threaded upwards and downwards through the corresponding partitions of the first body and the second body, respectively. An injectable hydrogel syringe is used to inject the hydrogel into the center position of the plastic tube or the heat-shrinkable tube through the through hole on the first body or the second body.

2. The gray matter zoned biomimetic spinal scaffold of claim 1, wherein, The biomimetic spinal cord scaffold comprises a small-diameter biomimetic spinal cord scaffold.

3. The gray matter zoned biomimetic spinal scaffold of claim 2, wherein, The preparation process of the small-diameter biomimetic spinal cord scaffold comprises the processes of threading, wire arranging, heat shrinking, glue filling and demolding.

4. The biomimetic spinal cord scaffold of claim 1, wherein, The heat-shrinkable tube is a PVC heat-shrinkable tube, and the diameter of the tube before heat shrinking is 2 mm, and the diameter of the tube after heat shrinking is 1 mm.

5. The gray matter zoned biomimetic spinal scaffold of claim 1, wherein, The injectable hydrogel is selected from hyaluronic acid hydrogel and / or chitosan hydrogel, or modified hyaluronic acid hydrogel, or modified chitosan hydrogel, or hyaluronic acid hydrogel loaded with biological macromolecules, or chitosan hydrogel loaded with biological macromolecules.

6. The gray matter zoned biomimetic spinal scaffold of claim 1, wherein, The directional fiber material is a silk bundle or a directional electrospun fiber.

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